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Sample records for 3-methyladenine dna glycosylase

  1. A model for 3-methyladenine recognition by 3-methyladenine DNA glycosylase I (TAG) from Staphylococcus aureus

    PubMed Central

    Zhu, Xiaofeng; Yan, Xuan; Carter, Lester G.; Liu, Huanting; Graham, Shirley; Coote, Peter J.; Naismith, James

    2012-01-01

    The removal of chemically damaged DNA bases such as 3-methyladenine (3-­MeA) is an essential process in all living organisms and is catalyzed by the enzyme 3-MeA DNA glycosylase I. A key question is how the enzyme selectively recognizes the alkylated 3-MeA over the much more abundant adenine. The crystal structures of native and Y16F-mutant 3-MeA DNA glycosylase I from Staphylococcus aureus in complex with 3-MeA are reported to 1.8 and 2.2 Å resolution, respectively. Isothermal titration calorimetry shows that protonation of 3-MeA decreases its binding affinity, confirming previous fluorescence studies that show that charge–charge recognition is not critical for the selection of 3-MeA over adenine. It is hypothesized that the hydrogen-bonding pattern of Glu38 and Tyr16 of 3-MeA DNA glycosylase I with a particular tautomer unique to 3-MeA contributes to recognition and selection. PMID:22684054

  2. Two DNA glycosylases in Esherichia coli which release primarily 3-methyladenine

    SciTech Connect

    Thomas, L.; Yang, C.; Goldthwait, D.A.

    1982-01-01

    Two enzymes have been partially purified from Escherichia coli and designated 3-methyladenine DNA glycosylases I and II. The apparent molecular weight of glycosylase I is 20,000, and that of II is 27,000. Glycosylase I releases 3-methyladenine (3-MeA) while II releases 3-MeA, 3-methylguanine (3-MeG), 7-methylguanine (7-MeG), and 7-methyladenine (7-MeA). The rate of release of 3-MeA by glycosylase II is 30 times that of 7-MeG. Glycosylase I is missing in mutants tag 1 and tag 2. In crude extracts, the 3-MeA activity of II is approximately 10% of the total 3-MeA activity. A 50% inactivation at 48/sup 0/C required 5 min for I and 65 min for II. The 3-MeA and 7-MeG activities of the glycosylase II preparation could not be separated by isoelectric focusing, by chromatography of DEAE, Sephadex G-100, phosphocellulose, DNA-cellulose, or carboxymethylcellulose, or by heating at 50/sup 0/C.

  3. Repair-deficient 3-methyladenine DNA glycosylase homozygous mutant mouse cells have increased sensitivity to alkylation-induced chromosome damage and cell killing.

    PubMed Central

    Engelward, B P; Dreslin, A; Christensen, J; Huszar, D; Kurahara, C; Samson, L

    1996-01-01

    In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions prevents alkylation-induced cell death because unrepaired 3MeA blocks DNA replication. Whether this lesion is cytotoxic to mammalian cells has been difficult to establish in the absence of 3MeA repair-deficient cell lines. We previously isolated and characterized a mouse 3MeA DNA glycosylase cDNA (Aag) that provides resistance to killing by alkylating agents in E. coli. To determine the in vivo role of Aag, we cloned a large fragment of the Aag gene and used it to create Aag-deficient mouse cells by targeted homologous recombination. Aag null cells have no detectable Aag transcripts or 3MeA DNA glycosylase activity. The loss of Aag renders cells significantly more sensitive to methyl methanesulfonate-induced chromosome damage, and to cell killing induced by two methylating agents, one of which produces almost exclusively 3MeAs. Aag null embryonic stem cells become sensitive to two cancer chemotherapeutic alkylating agents, namely 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C, indicating that Aag status is an important determinant of cellular resistance to these agents. We conclude that this mammalian 3MeA DNA glycosylase plays a pivotal role in preventing alkylation-induced chromosome damage and cytotoxicity. Images PMID:8631315

  4. A new protein superfamily includes two novel 3-methyladenine DNA glycosylases from Bacillus cereus, AlkC and AlkD.

    PubMed

    Alseth, Ingrun; Rognes, Torbjørn; Lindbäck, Toril; Solberg, Inger; Robertsen, Kristin; Kristiansen, Knut Ivan; Mainieri, Davide; Lillehagen, Lucy; Kolstø, Anne-Brit; Bjørås, Magnar

    2006-03-01

    Soil bacteria are heavily exposed to environmental methylating agents such as methylchloride and may have special requirements for repair of alkylation damage on DNA. We have used functional complementation of an Escherichia coli tag alkA mutant to screen for 3-methyladenine DNA glycosylase genes in genomic libraries of the soil bacterium Bacillus cereus. Three genes were recovered: alkC, alkD and alkE. The amino acid sequence of AlkE is homologous to the E. coli AlkA sequence. AlkC and AlkD represent novel proteins without sequence similarity to any protein of known function. However, iterative and indirect sequence similarity searches revealed that AlkC and AlkD are distant homologues of each other within a new protein superfamily that is ubiquitous in the prokaryotic kingdom. Homologues of AlkC and AlkD were also identified in the amoebas Entamoeba histolytica and Dictyostelium discoideum, but no other eukaryotic counterparts of the superfamily were found. The alkC and alkD genes were expressed in E. coli and the proteins were purified to homogeneity. Both proteins were found to be specific for removal of N-alkylated bases, and showed no activity on oxidized or deaminated base lesions in DNA. B. cereus AlkC and AlkD thus define novel families of alkylbase DNA glycosylases within a new protein superfamily. PMID:16468998

  5. A new protein superfamily includes two novel 3-methyladenine DNA glycosylases from Bacillus cereus, AlkC and AlkD

    PubMed Central

    Alseth, Ingrun; Rognes, Torbjørn; Lindbäck, Toril; Solberg, Inger; Robertsen, Kristin; Kristiansen, Knut Ivan; Mainieri, Davide; Lillehagen, Lucy; Kolstø, Anne-Brit; Bjørås, Magnar

    2006-01-01

    Summary Soil bacteria are heavily exposed to environmental methylating agents such as methylchloride and may have special requirements for repair of alkylation damage on DNA. We have used functional complementation of an Escherichia coli tag alkA mutant to screen for 3-methyladenine DNA glycosylase genes in genomic libraries of the soil bacterium Bacillus cereus. Three genes were recovered: alkC, alkD and alkE. The amino acid sequence of AlkE is homologous to the E. coli AlkA sequence. AlkC and AlkD represent novel proteins without sequence similarity to any protein of known function. However, iterative and indirect sequence similarity searches revealed that AlkC and AlkD are distant homologues of each other within a new protein superfamily that is ubiquitous in the prokaryotic kingdom. Homologues of AlkC and AlkD were also identified in the amoebas Entamoeba histolytica and Dictyostelium discoideum, but no other eukaryotic counterparts of the superfamily were found. The alkC and alkD genes were expressed in E. coli and the proteins were purified to homogeneity. Both proteins were found to be specific for removal of N-alkylated bases, and showed no activity on oxidized or deaminated base lesions in DNA. B. cereus AlkC and AlkD thus define novel families of alkylbase DNA glycosylases within a new protein superfamily. PMID:16468998

  6. 3-methyladenine-DNA-glycosylase and O6-alkyl guanine-DNA-alkyltransferase activities and sensitivity to alkylating agents in human cancer cell lines.

    PubMed Central

    Damia, G.; Imperatori, L.; Citti, L.; Mariani, L.; D'Incalci, M.

    1996-01-01

    The activities and the expression of 3-methyladenine glycosylase (3-meAde gly) and O6-alkylguanine-DNA-alkyltransferase (O6 ATase) were investigated in ten human cancer cell lines. Both 3-meAde gly and O6 ATase activities were variable among different cell lines. mRNA levels of the O6 ATase gene, appeared to be related to the content of O6 ATase in different cell lines, whereas no apparent correlation was found between mRNA of 3-meAde gly and the enzyme activity. No correlation was found between the activity of the two enzymes and the sensitivity to alkylating agents of different structures such as CC-1065, tallimustine, dimethylsulphate (DMSO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cis-diamminedichloroplatinum (cDDP) and melphalan (L-PAM). The most striking finding of this study is that a correlation exists between the activity of O6 ATase and 3-meAde gly in the various cell lines investigated (P<0.01), suggesting a common mechanism of regulation of two DNA repair enzymes. Images Figure 2 PMID:8611396

  7. Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid

    PubMed Central

    Terato, Hiroaki; Masaoka, Aya; Asagoshi, Kenjiro; Honsho, Akiko; Ohyama, Yoshihiko; Suzuki, Toshinori; Yamada, Masaki; Makino, Keisuke; Yamamoto, Kazuo; Ide, Hiroshi

    2002-01-01

    Nitrosation of guanine in DNA by nitrogen oxides such as nitric oxide (NO) and nitrous acid leads to formation of xanthine (Xan) and oxanine (Oxa), potentially cytotoxic and mutagenic lesions. In the present study, we have examined the repair capacity of DNA N-glycosylases from Escherichia coli for Xan and Oxa. The nicking assay with the defined substrates containing Xan and Oxa revealed that AlkA [in combination with endonuclease (Endo) IV] and Endo VIII recognized Xan in the tested enzymes. The activity (Vmax/Km) of AlkA for Xan was 5-fold lower than that for 7-methylguanine, and that of Endo VIII was 50-fold lower than that for thymine glycol. The activity of AlkA and Endo VIII for Xan was further substantiated by the release of [3H]Xan from the substrate. The treatment of E.coli with N-methyl-N′-nitro-N-nitrosoguanidine increased the Xan-excising activity in the cell extract from alkA+ but not alkA– strains. The alkA and nei (the Endo VIII gene) double mutant, but not the single mutants, exhibited increased sensitivity to nitrous acid relative to the wild type strain. AlkA and Endo VIII also exhibited excision activity for Oxa, but the activity was much lower than that for Xan. PMID:12434002

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

    PubMed Central

    Engelward, Bevin P.; Weeda, Geert; Wyatt, Michael D.; Broekhof, José L. M.; de Wit, Jan; Donker, Ingrid; Allan, James M.; Gold, Barry; Hoeijmakers, Jan H. J.; Samson, Leona D.

    1997-01-01

    3-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 glycosylase not only for the cytotoxic 3MeA DNA lesion, but also for the mutagenic 1,N6-ethenoadenine (ɛA) and hypoxanthine lesions. Aag appears to be the only 3MeA and hypoxanthine DNA glycosylase in liver, testes, kidney, and lung, and the only ɛA DNA glycosylase in liver, testes, and kidney; another ɛA DNA glycosylase may be expressed in lung. Although alkyladenine DNA glycosylase has the capacity to remove 8-oxoguanine DNA lesions, it does not appear to be the major glycosylase for 8-oxoguanine repair. Fibroblasts derived from Aag −/− mice are alkylation sensitive, indicating that Aag −/− mice may be similarly sensitive. PMID:9371804

  9. Cloning a Eukaryotic DNA Glycosylase Repair Gene by the Suppression of a DNA Repair Defect in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Chen, Jin; Derfler, Bruce; Maskati, Azmat; Samson, Leona

    1989-10-01

    If eukaryotic genes could protect bacteria with defects in DNA repair, this effect could be exploited for the isolation of eukaryotic DNA repair genes. We have thus cloned a DNA repair gene from Saccharomyces cerevisiae that directs the synthesis of a DNA glycosylase that specifically releases 3-methyladenine from alkylated DNA and in so doing protects alkylation-sensitive Escherichia coli from killing by methylating agents. The cloned yeast gene was then used to generate a mutant strain of S. cerevisiae that carries a defect in the glycosylase gene and is extremely sensitive to DNA methylation. This approach may allow the isolation of a large number of eukaryotic DNA repair genes.

  10. Actions of human DNA glycosylases on uracil-containing DNA, methylated DNA and their reconstituted chromatins.

    PubMed

    Ishiwata, K; Oikawa, A

    1979-07-26

    Extracts of human lymphoblastoid cells catalyzed complete release of uracil (Ura) from PBS1 DNA, which contains Ura instead of thymine as a normal component (Ura-DNA), and 3-methyladenine (3-MeAde) from DNA methylated with methyl methanesulfonate (Me-DNA). These two activities, Ura-DNA glycosylase and 3-MeAde-DNA glycosylase, differed in heat stability. Cell extracts released Ura more rapidly and 3-MeAde more slowly from alkali-denatured preparations of Ura- and Me-DNA, respectively, than from native DNA's. On incubation with reconstituted chromatins, prepared from Ura-DNA and Me-DNA, respectively, with calf thymus chromosomal protein by salt gradient dialysis, cell extracts released all the Ura but only about half of the 3-MeAde residues, although both these chromatins were degraded by micrococcal nuclease until about half of the nucleotides became acid soluble. The activities of Ura-DNA and 3-MeAde-DNA glycosylase of xeroderma pigmentosum cells were similar to those of normal cells. PMID:465495

  11. DNA-N-glycosylases process novel O-glycosidic sites in DNA.

    PubMed

    Admiraal, Suzanne J; O'Brien, Patrick J

    2013-06-11

    After the hydrolysis of the N-glycosyl bond between a damaged base and C1' of a deoxyribosyl moiety of DNA, human alkyladenine DNA glycosylase (AAG) and Escherichia coli 3-methyladenine DNA glycosylase II (AlkA) bind tightly to their abasic DNA products, potentially protecting these reactive species. Here we show that both AAG and AlkA catalyze reactions between bound abasic DNA and small, primary alcohols to form novel DNA-O-glycosides. The synthesis reactions are reversible, as the DNA-O-glycosides are converted back into abasic DNA upon being incubated with AAG or AlkA in the absence of alcohol. AAG and AlkA are therefore able to hydrolyze O-glycosidic bonds in addition to N-glycosyl bonds. The newly discovered DNA-O-glycosidase activities of both enzymes compare favorably with their known DNA-N-glycosylase activities: AAG removes both methanol and 1,N(6)-ethenoadenine (εA) from DNA with single-turnover rate constants that are 2.9 × 10(5)-fold greater than the corresponding uncatalyzed rates, whereas the rate enhancement of 3.7 × 10(7) for removal of methanol from DNA by AlkA is 300-fold greater than its rate enhancement for removal of εA from DNA. Although the biological significance of the DNA-O-glycosidase reactions is not known, the evolution of new DNA repair pathways may be aided by enzymes that practice catalytic promiscuity, such as these two unrelated DNA glycosylases. PMID:23688261

  12. Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases.

    PubMed

    Saparbaev, M; Laval, J

    1994-06-21

    The deamination of adenine residues in DNA generates hypoxanthine, which is mutagenic since it gives rise to an A.T to G.C transition. Hypoxanthine is removed by hypoxanthine DNA glycosylase activity present in Escherichia coli and mammalian cells. Using polydeoxyribonucleotides or double-stranded synthetic oligonucleotides that contain dIMP residues, we show that this activity in E. coli is associated with the 3-methyladenine DNA glycosylase II coded for by the alkA gene. This conclusion is based on the following facts: (i) the two enzymatic activities have the same chromatographic behavior on various supports and they have the same molecular weight, (ii) both are induced during the adaptive response, (iii) a multicopy plasmid bearing the alkA gene overproduces both activities, (iv) homogeneous preparation of AlkA has both enzymatic activities, (v) the E. coli alkA- mutant does not show any detectable hypoxanthine DNA glycosylase activity. Under the same experimental conditions, but using different substrates, the same amount of AlkA protein liberates 1 pmol of 3-methyladenine from alkylated DNA and 1.2 fmol of hypoxanthine from dIMP-containing DNA. The Km for the latter substrate is 420 x 10(-9) M as compared to 5 x 10(-9) M for alkylated DNA. Hypoxanthine is released as a free base during the reaction. Duplex oligodeoxynucleotides containing hypoxanthine positioned opposite T, G, C, and A were cleaved efficiently. ANPG protein, APDG protein, and MAG protein--the 3-methyladenine DNA glycosylases of human, rat, and yeast origin, respectively--were also able to release hypoxanthine from various DNA substrates containing dIMP residues. The mammalian enzyme is by far the most efficient hypoxanthine DNA glycosylase of all the enzymes tested. PMID:8016081

  13. Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases.

    PubMed Central

    Saparbaev, M; Laval, J

    1994-01-01

    The deamination of adenine residues in DNA generates hypoxanthine, which is mutagenic since it gives rise to an A.T to G.C transition. Hypoxanthine is removed by hypoxanthine DNA glycosylase activity present in Escherichia coli and mammalian cells. Using polydeoxyribonucleotides or double-stranded synthetic oligonucleotides that contain dIMP residues, we show that this activity in E. coli is associated with the 3-methyladenine DNA glycosylase II coded for by the alkA gene. This conclusion is based on the following facts: (i) the two enzymatic activities have the same chromatographic behavior on various supports and they have the same molecular weight, (ii) both are induced during the adaptive response, (iii) a multicopy plasmid bearing the alkA gene overproduces both activities, (iv) homogeneous preparation of AlkA has both enzymatic activities, (v) the E. coli alkA- mutant does not show any detectable hypoxanthine DNA glycosylase activity. Under the same experimental conditions, but using different substrates, the same amount of AlkA protein liberates 1 pmol of 3-methyladenine from alkylated DNA and 1.2 fmol of hypoxanthine from dIMP-containing DNA. The Km for the latter substrate is 420 x 10(-9) M as compared to 5 x 10(-9) M for alkylated DNA. Hypoxanthine is released as a free base during the reaction. Duplex oligodeoxynucleotides containing hypoxanthine positioned opposite T, G, C, and A were cleaved efficiently. ANPG protein, APDG protein, and MAG protein--the 3-methyladenine DNA glycosylases of human, rat, and yeast origin, respectively--were also able to release hypoxanthine from various DNA substrates containing dIMP residues. The mammalian enzyme is by far the most efficient hypoxanthine DNA glycosylase of all the enzymes tested. Images PMID:8016081

  14. Strandwise translocation of a DNA glycosylase on undamaged DNA

    SciTech Connect

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

    2012-05-14

    Base excision repair of genotoxic nucleobase lesions in the genome is critically dependent upon the ability of DNA glycosylases to locate rare sites of damage embedded in a vast excess of undamaged DNA, using only thermal energy to fuel the search process. Considerable interest surrounds the question of how DNA glycosylases translocate efficiently along DNA while maintaining their vigilance for target damaged sites. Here, we report the observation of strandwise translocation of 8-oxoguanine DNA glycosylase, MutM, along undamaged DNA. In these complexes, the protein is observed to translocate by one nucleotide on one strand while remaining untranslocated on the complementary strand. We further report that alterations of single base-pairs or a single amino acid substitution (R112A) can induce strandwise translocation. Molecular dynamics simulations confirm that MutM can translocate along DNA in a strandwise fashion. These observations reveal a previously unobserved mode of movement for a DNA-binding protein along the surface of DNA.

  15. Strandwise translocation of a DNA glycosylase on undamaged DNA.

    PubMed

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

    2012-01-24

    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. PMID:22219368

  16. Recent Advances in the Structural Mechanisms of DNA Glycosylases

    PubMed Central

    Brooks, Sonja C.; Adhikary, Suraj; Rubinson, Emily H.; Eichman, Brandt F.

    2012-01-01

    DNA glycosylases safeguard the genome by locating and excising a diverse array of aberrant nucleobases created from oxidation, alkylation, and deamination of DNA. Since the discovery 28 years ago that these enzymes employ a base flipping mechanism to trap their substrates, six different protein architectures have been identified to perform the same basic task. Work over the past several years has unraveled details for how the various DNA glycosylases survey DNA, detect damage within the duplex, select for the correct modification, and catalyze base excision. Here, we provide a broad overview of these latest advances in glycosylase mechanisms gleaned from structural enzymology, highlighting features common to all glycosylases as well as key differences that define their particular substrate specificities. PMID:23076011

  17. Direct visualization of a DNA glycosylase searching for damage.

    PubMed

    Chen, Liwei; Haushalter, Karl A; Lieber, Charles M; Verdine, Gregory L

    2002-03-01

    DNA glycosylases preserve the integrity of genetic information by recognizing damaged bases in the genome and catalyzing their excision. It is unknown how DNA glycosylases locate covalently modified bases hidden in the DNA helix amongst vast numbers of normal bases. Here we employ atomic-force microscopy (AFM) with carbon nanotube probes to image search intermediates of human 8-oxoguanine DNA glycosylase (hOGG1) scanning DNA. We show that hOGG1 interrogates DNA at undamaged sites by inducing drastic kinks. The sharp DNA bending angle of these non-lesion-specific search intermediates closely matches that observed in the specific complex of 8-oxoguanine-containing DNA bound to hOGG1. These findings indicate that hOGG1 actively distorts DNA while searching for damaged bases. PMID:11927259

  18. Structure of a DNA glycosylase searching for lesions.

    PubMed

    Banerjee, Anirban; Santos, Webster L; Verdine, Gregory L

    2006-02-24

    DNA glycosylases must interrogate millions of base pairs of undamaged DNA in order to locate and then excise one damaged nucleobase. The nature of this search process remains poorly understood. Here we report the use of disulfide cross-linking (DXL) technology to obtain structures of a bacterial DNA glycosylase, MutM, interrogating undamaged DNA. These structures, solved to 2.0 angstrom resolution, reveal the nature of the search process: The protein inserts a probe residue into the helical stack and severely buckles the target base pair, which remains intrahelical. MutM therefore actively interrogates the intact DNA helix while searching for damage. PMID:16497933

  19. Expansion Mechanisms and Evolutionary History on Genes Encoding DNA Glycosylases and Their Involvement in Stress and Hormone Signaling.

    PubMed

    Jiang, Shu-Ye; Ramachandran, Srinivasan

    2016-01-01

    DNA glycosylases catalyze the release of methylated bases. They play vital roles in the base excision repair pathway and might also function in DNA demethylation. At least three families of DNA glycosylases have been identified, which included 3'-methyladenine DNA glycosylase (MDG) I, MDG II, and HhH-GPD (Helix-hairpin-Helix and Glycine/Proline/aspartate (D)). However, little is known on their genome-wide identification, expansion, and evolutionary history as well as their expression profiling and biological functions. In this study, we have genome-widely identified and evolutionarily characterized these family members. Generally, a genome encodes only one MDG II gene in most of organisms. No MDG I or MDG II gene was detected in green algae. However, HhH-GPD genes were detectable in all available organisms. The ancestor species contain small size of MDG I and HhH-GPD families. These two families were mainly expanded through the whole-genome duplication and segmental duplication. They were evolutionarily conserved and were generally under purifying selection. However, we have detected recent positive selection among the Oryza genus, which might play roles in species divergence. Further investigation showed that expression divergence played important roles in gene survival after expansion. All of these family genes were expressed in most of developmental stages and tissues in rice plants. High ratios of family genes were downregulated by drought and fungus pathogen as well as abscisic acid (ABA) and jasmonic acid (JA) treatments, suggesting a negative regulation in response to drought stress and pathogen infection through ABA- and/or JA-dependent hormone signaling pathway. PMID:27026054

  20. Detection of Damaged DNA Bases by DNA Glycosylase Enzymes†

    PubMed Central

    Friedman, Joshua I.; Stivers, James T.

    2010-01-01

    A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly-ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we refer to as the search complex (SC). Sliding is frequently punctuated by the formation of a transient “interrogation” complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome, and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location. PMID:20469926

  1. Interaction of the recombinant human methylpurine-DNA glycosylase (MPG protein) with oligodeoxyribonucleotides containing either hypoxanthine or abasic sites.

    PubMed Central

    Miao, F; Bouziane, M; O'Connor, T R

    1998-01-01

    Methylpurine-DNA glycosylases (MPG proteins, 3-methyladenine-DNA glycosylases) excise numerous damaged bases from DNA during the first step of base excision repair. The damaged bases removed by these proteins include those induced by both alkylating agents and/or oxidizing agents. The intrinsic kinetic parameters (k(cat) and K(m)) for the excision of hypoxanthine by the recombinant human MPG protein from a 39 bp oligodeoxyribonucleotide harboring a unique hypoxanthine were determined. Comparison with other reactions catalyzed by the human MPG protein suggests that the differences in specificity are primarily in product release and not binding. Analysis of MPG protein binding to the 39 bp oligodeoxyribonucleotide revealed that the apparent dissociation constant is of the same order of magnitude as the K(m) and that a 1:1 complex is formed. The MPG protein also forms a strong complex with the product of excision, an abasic site, as well as with a reduced abasic site. DNase I footprinting experiments with the MPG protein on an oligodeoxyribonucleotide with a unique hypoxanthine at a defined position indicate that the protein protects 11 bases on the strand with the hypoxanthine and 12 bases on the complementary strand. Competition experiments with different length, double-stranded, hypoxanthine-containing oligodeoxyribonucleotides show that the footprinted region is relatively small. Despite the small footprint, however, oligodeoxyribonucleotides comprising <15 bp with a hypoxanthine have a 10-fold reduced binding capacity compared with hypoxanthine-containing oligodeoxyribonucleotides >20 bp in length. These results provide a basis for other structural studies of the MPG protein with its targets. PMID:9705516

  2. DNA glycosylase enzymes induced during chemical adaptation of M. luteus.

    PubMed Central

    Riazuddin, S; Athar, A; Ahmed, Z; Lali, S M; Sohail, A

    1987-01-01

    Five peaks of DNA glycosylase activity showing a preference for MNNG alkylated DNA have been identified from extracts of adapted M. luteus. They are numerically designated as GI to GV in order of their decreasing molecular weights. The first two of these peaks have been highly purified. GI, is a constitutive heat labile protein, 35% stimulated by the presence of 50 mM NaCl, acts exclusively on 3 MeA residues in alkylated DNA, 60-70% inhibited by the presence of 2 mM free 3MeA and has been designated as 3MeA DNA glycosylase enzyme. GII, which is an inducible protein, is heat stable, 28% inhibited by the presence of 50 mM NaCl, removes 3MeA, 3MeG, 7MeA & 7MeG with different efficiency, and has been designated as 3,7 methylpurine DNA glycosylase enzyme. The rate of release of 3 methylpurines is 30 times that of 7MeG. There is no activity of either enzyme on O2-MeC, O2-MeT, O4-MeT or O6-MeG. The apparent molecular weights of GI and GII proteins are 28 Kd and 22 Kd respectively. PMID:3628000

  3. Human polymorphic variants of the NEIL1 DNA glycosylase.

    PubMed

    Roy, Laura M; Jaruga, Pawel; Wood, Thomas G; McCullough, Amanda K; Dizdaroglu, Miral; Lloyd, R Stephen

    2007-05-25

    In mammalian cells, the repair of DNA bases that have been damaged by reactive oxygen species is primarily initiated by a series of DNA glycosylases that include OGG1, NTH1, NEIL1, and NEIL2. To explore the functional significance of NEIL1, we recently reported that neil1 knock-out and heterozygotic mice develop the majority of symptoms of metabolic syndrome (Vartanian, V., Lowell, B., Minko, I. G., Wood, T. G., Ceci, J. D., George, S., Ballinger, S. W., Corless, C. L., McCullough, A. K., and Lloyd, R. S. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1864-1869). To determine whether this phenotype could be causally related to human disease susceptibility, we have characterized four polymorphic variants of human NEIL1. Although three of the variants (S82C, G83D, and D252N) retained near wild type levels of nicking activity on abasic (AP) site-containing DNA, G83D did not catalyze the wild type beta,delta-elimination reaction but primarily yielded the beta-elimination product. The AP nicking activity of the C136R variant was significantly reduced. Glycosylase nicking activities were measured on both thymine glycol-containing oligonucleotides and gamma-irradiated genomic DNA using gas chromatography/mass spectrometry. Two of the polymorphic variants (S82C and D252N) showed near wild type enzyme specificity and kinetics, whereas G83D was devoid of glycosylase activity. Although insufficient quantities of C136R could be obtained to carry out gas chromatography/mass spectrometry analyses, this variant was also devoid of the ability to incise thymine glycol-containing oligonucleotide, suggesting that it may also be glycosylase-deficient. Extrapolation of these data suggests that individuals who are heterozygous for these inactive variant neil1 alleles may be at increased risk for metabolic syndrome. PMID:17389588

  4. Molecular basis for discriminating between normal and damaged bases by the human alkyladenine glycosylase, AAG

    PubMed Central

    Lau, Albert Y.; Wyatt, Michael D.; Glassner, Brian J.; Samson, Leona D.; Ellenberger, Tom

    2000-01-01

    The human 3-methyladenine DNA glycosylase [alkyladenine DNA glycosylase (AAG)] catalyzes the first step of base excision repair by cleaving damaged bases from DNA. Unlike other DNA glycosylases that are specific for a particular type of damaged base, AAG excises a chemically diverse selection of substrate bases damaged by alkylation or deamination. The 2.1-Å crystal structure of AAG complexed to DNA containing 1,N6-ethenoadenine suggests how modified bases can be distinguished from normal DNA bases in the enzyme active site. Mutational analyses of residues contacting the alkylated base in the crystal structures suggest that the shape of the damaged base, its hydrogen-bonding characteristics, and its aromaticity all contribute to the selective recognition of damage by AAG. PMID:11106395

  5. A DNA enzyme with N-glycosylase activity

    NASA Technical Reports Server (NTRS)

    Sheppard, T. L.; Ordoukhanian, P.; Joyce, G. F.

    2000-01-01

    In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10(6)-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an N-glycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.

  6. Molecular crowding enhances facilitated diffusion of two human DNA glycosylases

    PubMed Central

    Cravens, Shannen L.; Schonhoft, Joseph D.; Rowland, Meng M.; Rodriguez, Alyssa A.; Anderson, Breeana G.; Stivers, James T.

    2015-01-01

    Intracellular space is at a premium due to the high concentrations of biomolecules and is expected to have a fundamental effect on how large macromolecules move in the cell. Here, we report that crowded solutions promote intramolecular DNA translocation by two human DNA repair glycosylases. The crowding effect increases both the efficiency and average distance of DNA chain translocation by hindering escape of the enzymes to bulk solution. The increased contact time with the DNA chain provides for redundant damage patrolling within individual DNA chains at the expense of slowing the overall rate of damaged base removal from a population of molecules. The significant biological implication is that a crowded cellular environment could influence the mechanism of damage recognition as much as any property of the enzyme or DNA. PMID:25845592

  7. Molecular crowding enhances facilitated diffusion of two human DNA glycosylases.

    PubMed

    Cravens, Shannen L; Schonhoft, Joseph D; Rowland, Meng M; Rodriguez, Alyssa A; Anderson, Breeana G; Stivers, James T

    2015-04-30

    Intracellular space is at a premium due to the high concentrations of biomolecules and is expected to have a fundamental effect on how large macromolecules move in the cell. Here, we report that crowded solutions promote intramolecular DNA translocation by two human DNA repair glycosylases. The crowding effect increases both the efficiency and average distance of DNA chain translocation by hindering escape of the enzymes to bulk solution. The increased contact time with the DNA chain provides for redundant damage patrolling within individual DNA chains at the expense of slowing the overall rate of damaged base removal from a population of molecules. The significant biological implication is that a crowded cellular environment could influence the mechanism of damage recognition as much as any property of the enzyme or DNA. PMID:25845592

  8. Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

    SciTech Connect

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; Chattopadhyay, Debasish

    2015-06-02

    Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This also represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.

  9. Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

    DOE PAGESBeta

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; Chattopadhyay, Debasish

    2015-06-02

    Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This alsomore » represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.« less

  10. Glycosylases utilize ``stop and go'' motion to locate DNA damage

    NASA Astrophysics Data System (ADS)

    Nelson, Shane

    2015-03-01

    Oxidative damage to DNA results in alterations that are mutagenic or even cytotoxic. Base excision repair is a mechanism that functions to identify and correct these lesions, and is present in organisms ranging from bacteria to humans. DNA glycosylases are the first enzymes in this pathway and function to locate and remove oxidatively damaged bases, and do so utilizing only thermal energy. However, the question remains of how these enzymes locate and recognize a damaged base among millions of undamaged bases. Utilizing fluorescence video microscopy with high spatial and temporal resolution, we have observed a number of different fluorescently labeled glycosylases (including bacterial FPG, NEI, and NTH as well as mammalian MutyH and OGG). These enzymes diffuse along DNA tightropes at approximately 0.01 +/- 0.005 μm2/s with binding lifetimes ranging from one second to several minutes. Chemically induced damage to the DNA substrate causes a ~ 50% reduction in diffusion coefficients and a ~ 400% increase in binding lifetimes, while mutation of the key ``wedge residue'' - which has been shown to be responsible for damage detection - results in a 200% increase in the diffusion coefficient. Utilizing a sliding window approach to measure diffusion coefficients within individual trajectories, we observe that distributions of diffusion coefficients are bimodal, consistent with periods of diffusive motion interspersed with immobile periods. Utilizing a unique chemo-mechanical simulation approach, we demonstrate that the motion of these glycosylases can be explained as free diffusion along the helical pitch of the DNA, punctuated with two different types of pauses: 1) rapid, short-lived pauses as the enzyme rapidly probes DNA bases to interrogate for damage and, 2) less frequent, longer lived pauses that reflect the enzyme bound to and catalytically removing a damaged base. These simulations also indicate that the wedge residue is critical for interrogation and recognition of

  11. Functional Characterization of 8-Oxoguanine DNA Glycosylase of Trypanosoma cruzi

    PubMed Central

    Mendes, Isabela Cecília; de Moura, Michelle Barbi; Campos, Priscila Carneiro; Macedo, Andrea Mara; Franco, Glória Regina; Pena, Sérgio Danilo Junho; Teixeira, Santuza Maria Ribeiro; Van Houten, Bennett; Machado, Carlos Renato

    2012-01-01

    The oxidative lesion 8-oxoguanine (8-oxoG) is removed during base excision repair by the 8-oxoguanine DNA glycosylase 1 (Ogg1). This lesion can erroneously pair with adenine, and the excision of this damaged base by Ogg1 enables the insertion of a guanine and prevents DNA mutation. In this report, we identified and characterized Ogg1 from the protozoan parasite Trypanosoma cruzi (TcOgg1), the causative agent of Chagas disease. Like most living organisms, T. cruzi is susceptible to oxidative stress, hence DNA repair is essential for its survival and improvement of infection. We verified that the TcOGG1 gene encodes an 8-oxoG DNA glycosylase by complementing an Ogg1-defective Saccharomyces cerevisiae strain. Heterologous expression of TcOGG1 reestablished the mutation frequency of the yeast mutant ogg1−/− (CD138) to wild type levels. We also demonstrate that the overexpression of TcOGG1 increases T. cruzi sensitivity to hydrogen peroxide (H2O2). Analysis of DNA lesions using quantitative PCR suggests that the increased susceptibility to H2O2 of TcOGG1-overexpressor could be a consequence of uncoupled BER in abasic sites and/or strand breaks generated after TcOgg1 removes 8-oxoG, which are not rapidly repaired by the subsequent BER enzymes. This hypothesis is supported by the observation that TcOGG1-overexpressors have reduced levels of 8-oxoG both in the nucleus and in the parasite mitochondrion. The localization of TcOgg1 was examined in parasite transfected with a TcOgg1-GFP fusion, which confirmed that this enzyme is in both organelles. Taken together, our data indicate that T. cruzi has a functional Ogg1 ortholog that participates in nuclear and mitochondrial BER. PMID:22876325

  12. The role of 8-oxoguanine DNA glycosylase-1 in inflammation.

    PubMed

    Ba, Xueqing; Aguilera-Aguirre, Leopoldo; Rashid, Qura Tul Ain Nmi; Bacsi, Attila; Radak, Zsolt; Sur, Sanjiv; Hosoki, Koa; Hegde, Muralidhar L; Boldogh, Istvan

    2014-01-01

    Many, if not all, environmental pollutants/chemicals and infectious agents increase intracellular levels of reactive oxygen species (ROS) at the site of exposure. ROS not only function as intracellular signaling entities, but also induce damage to cellular molecules including DNA. Among the several dozen ROS-induced DNA base lesions generated in the genome, 8-oxo-7,8-dihydroguanine (8-oxoG) is one of the most abundant because of guanine's lowest redox potential among DNA bases. In mammalian cells, 8-oxoG is repaired by the 8-oxoguanine DNA glycosylase-1 (OGG1)-initiated DNA base excision repair pathway (OGG1-BER). Accumulation of 8-oxoG in DNA has traditionally been associated with mutagenesis, as well as various human diseases and aging processes, while the free 8-oxoG base in body fluids is one of the best biomarkers of ongoing pathophysiological processes. In this review, we discuss the biological significance of the 8-oxoG base and particularly the role of OGG1-BER in the activation of small GTPases and changes in gene expression, including those that regulate pro-inflammatory chemokines/cytokines and cause inflammation. PMID:25250913

  13. Immunological lesions in human uracil DNA glycosylase: association with Bloom syndrome.

    PubMed Central

    Seal, G; Brech, K; Karp, S J; Cool, B L; Sirover, M A

    1988-01-01

    Three monoclonal antibodies that react with uracil DNA glycosylase of normal human placenta were tested to determine whether one of the antibodies could be used as a negative marker for Bloom syndrome. As defined by enzyme-linked immunosorbent assay, monoclonal antibody 40.10.09, which reacts with normal human glycosylase, neither recognized nor inhibited native uracil DNA glycosylase from any of five separate Bloom syndrome cell strains. Immunoblot analyses demonstrated that the denatured glycosylase protein from all five Bloom syndrome cell strains was immunoreactive with the 40.10.09 antibody. Further, each native enzyme was immunoreactive with two other anti-human placental uracil DNA glycosylase monoclonal antibodies. In contrast, ELISA reactivity was observed with all three monoclonal antibodies in reactions of glycosylases from 5 normal human cell types and 13 abnormal human cell strains. These results experimentally verify the specificity of the aberrant reactivity of the Bloom syndrome uracil DNA glycosylase. The possibility arises that determination of the lack of immunoreactivity with antibody 40.10.09 may have value in the early diagnosis of Bloom syndrome. Images PMID:3353381

  14. Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation

    PubMed Central

    Arab, Khelifa; Kienhöfer, Sabine; von Seggern, Annika; Niehrs, Christof

    2016-01-01

    DNA 5-methylcytosine is a dynamic epigenetic mark which plays important roles in development and disease. In the Tet-Tdg demethylation pathway, methylated cytosine is iteratively oxidized by Tet dioxygenases and unmodified cytosine is restored via thymine DNA glycosylase (Tdg). Here we show that human NEIL1 and NEIL2 DNA glycosylases coordinate abasic site processing during TET–TDG DNA demethylation. NEIL1 and NEIL2 cooperate with TDG during base excision: TDG occupies the abasic site and is displaced by NEILs, which further process the baseless sugar, thereby stimulating TDG substrate turnover. In early Xenopus embryos Neil2 cooperates with Tdg to remove oxidized methylcytosines and to specify neural crest development together with Tet3. Thus, Neils function as AP lyases in the coordinated AP site hand-over during oxidative DNA demethylation. PMID:26751644

  15. Lesion search and recognition by thymine DNA glycosylase revealed by single molecule imaging.

    PubMed

    Buechner, Claudia N; Maiti, Atanu; Drohat, Alexander C; Tessmer, Ingrid

    2015-03-11

    The ability of DNA glycosylases to rapidly and efficiently detect lesions among a vast excess of nondamaged DNA bases is vitally important in base excision repair (BER). Here, we use single molecule imaging by atomic force microscopy (AFM) supported by a 2-aminopurine fluorescence base flipping assay to study damage search by human thymine DNA glycosylase (hTDG), which initiates BER of mutagenic and cytotoxic G:T and G:U mispairs in DNA. Our data reveal an equilibrium between two conformational states of hTDG-DNA complexes, assigned as search complex (SC) and interrogation complex (IC), both at target lesions and undamaged DNA sites. Notably, for both hTDG and a second glycosylase, hOGG1, which recognizes structurally different 8-oxoguanine lesions, the conformation of the DNA in the SC mirrors innate structural properties of their respective target sites. In the IC, the DNA is sharply bent, as seen in crystal structures of hTDG lesion recognition complexes, which likely supports the base flipping required for lesion identification. Our results support a potentially general concept of sculpting of glycosylases to their targets, allowing them to exploit the energetic cost of DNA bending for initial lesion sensing, coupled with continuous (extrahelical) base interrogation during lesion search by DNA glycosylases. PMID:25712093

  16. Uracil-DNA Glycosylase UNG Promotes Tet-mediated DNA Demethylation.

    PubMed

    Xue, Jian-Huang; Xu, Gui-Fang; Gu, Tian-Peng; Chen, Guo-Dong; Han, Bin-Bin; Xu, Zhi-Mei; Bjørås, Magnar; Krokan, Hans E; Xu, Guo-Liang; Du, Ya-Rui

    2016-01-01

    In mammals, active DNA demethylation involves oxidation of 5-methylcytosine (5mC) into 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by Tet dioxygenases and excision of these two oxidized bases by thymine DNA glycosylase (TDG). Although TDG is essential for active demethylation in embryonic stem cells and induced pluripotent stem cells, it is hardly expressed in mouse zygotes and dispensable in pronuclear DNA demethylation. To search for other factors that might contribute to demethylation in mammalian cells, we performed a functional genomics screen based on a methylated luciferase reporter assay. UNG2, one of the glycosylases known to excise uracil residues from DNA, was found to reduce DNA methylation, thus activating transcription of a methylation-silenced reporter gene when co-transfected with Tet2 into HEK293T cells. Interestingly, UNG2 could decrease 5caC from the genomic DNA and a reporter plasmid in transfected cells, like TDG. Furthermore, deficiency in Ung partially impaired DNA demethylation in mouse zygotes. Our results suggest that UNG might be involved in Tet-mediated DNA demethylation. PMID:26620559

  17. Structural and mutation studies of two DNA demethylation related glycosylases: MBD4 and TDG

    PubMed Central

    Hashimoto, Hideharu

    2014-01-01

    Two mammalian DNA glycosylases, methyl-CpG binding domain protein 4 (MBD4) and thymine DNA glycosylase (TDG), are involved in active DNA demethylation via the base excision repair pathway. Both MBD4 and TDG excise the mismatch base from G:X, where X is uracil, thymine, and 5-hydroxymethyluracil (5hmU). In addition, TDG excises 5mC oxidized bases i.e. when X is 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) not 5-hydroxymethylcytosine (5hmC). A MBD4 inactive mutant and substrate crystal structure clearly explains how MBD4 glycosylase discriminates substrates: 5mC are not able to be directly excised, but a deamination process from 5mC to thymine is required. On the other hand, TDG is much more complicated; in this instance, crystal structures show that TDG recognizes G:X mismatch DNA containing DNA and G:5caC containing DNA from the minor groove of DNA, which suggested that TDG might recognize 5mC oxidized product 5caC like mismatch DNA. In mutation studies, a N157D mutation results in a more 5caC specific glycosylase, and a N191A mutation inhibits 5caC activity while that when X=5fC or T remains. Here I revisit the recent MBD4 glycos ylase domain co-crystal structures with DNA, as well as TDG glycosylase domain co-crystal structures with DNA in conjunction with its mutation studies.

  18. Structural Investigation of a Viral Ortholog of Human NEIL2/3 DNA Glycosylases

    PubMed Central

    Prakash, Aishwarya; Eckenroth, Brian E.; Averill, April M.; Imamura, Kayo; Wallace, Susan S.; Doublié, Sylvie

    2013-01-01

    Assault to DNA that leads to oxidative base damage is repaired by the base excision repair (BER) pathway with specialized enzymes called DNA glycosylases catalyzing the first step of this pathway. These glycosylases can be categorized into two families: the HhH superfamily, which includes endonuclease III (or Nth), and the Fpg/Nei family, which comprises formamidopyrimidine DNA glycosylase (or Fpg) and endonuclease VIII (or Nei). In humans there are three Nei-like (NEIL) glycosylases: NEIL1, 2, and 3. Here we present the first crystal structure of a viral ortholog of the human NEIL2/NEIL3 proteins, Mimivirus Nei2 (MvNei2), determined at 2.04 Å resolution. The C-terminal region of the MvNei2 enzyme comprises two conserved DNA binding motifs: the helix-two-turns-helix (H2TH) motif and a C-H-C-C type zinc-finger similar to that of human NEIL2. The N-terminal region of MvNei2 is most closely related to NEIL3. Like NEIL3, MvNei2 bears a valine at position 2 instead of the usual proline and it lacks two of the three conserved void-filling residues present in other members of the Fpg/Nei family. Mutational analysis of the only conserved void-filling residue methionine 72 to alanine yields an MvNei2 variant with impaired glycosylase activity. Mutation of the adjacent His73 causes the enzyme to be more productive thereby suggesting a plausible role for this residue in the DNA lesion search process. PMID:24120312

  19. Mutations at Arginine 276 transform human uracil-DNA glycosylase into a single-stranded DNA-specific uracil-DNA glycosylase

    PubMed Central

    Chen, Cheng-Yao; Mosbaugh, Dale W.; Bennett, Samuel E.

    2011-01-01

    To investigate the role of Arginine 276 in the conserved leucine-loop of human uracil-DNA glycosylase (UNG), the effects of six R276 amino acid substitutions (C, E, H, L, W, and Y) on nucleotide flipping and enzyme conformational change were determined using transient and steady state, fluorescence-based, kinetic analysis. Relative to UNG, the mutant proteins exhibited a 2.6- to 7.7-fold reduction in affinity for a doubled-stranded oligonucleotide containing a pseudouracil residue opposite 2-aminopurine, as judged by steady-state DNA binding-base flipping assays. An anisotropy binding assay was utilized to determine the Kd of UNG and the R276 mutants for carboxyfluorescein-labeled uracil-containing single- and double-stranded oligonucleotides; the binding affinities varied 11-fold for single-stranded uracil-DNA, and 43-fold for double-stranded uracil-DNA. Productive uracil-DNA binding was monitored by rapid quenching of UNG intrinsic protein fluorescence. Relative to UNG, the rate of intrinsic fluorescence quenching of five mutant proteins for binding double-stranded uracil-DNA was reduced approximately 50%; the R276E mutant exhibited 1% of the rate of fluorescence quenching of UNG. When reacted with single-stranded uracil-DNA, the rate of UNG fluorescence quenching increased. Moreover, the rate of fluorescence quenching for all the mutant proteins, except R276E, was slightly faster than UNG. The kcat of the R276 mutants was comparable to UNG on single-stranded DNA and differentially affected by NaCl; however, kcat on double-stranded DNA substrate was reduced 4–12-fold and decreased sharply at NaCl concentrations as low as 20 mM. Taken together, these results indicate that the effects of mutations at Arg276 were largely limited to enzyme interactions with double-stranded uracil-containing DNA, and suggested that mutations at Arg276 effectively transformed UNG into a single-stranded DNA-specific uracil-DNA glycosylase. PMID:15970468

  20. Analysis of nuclear uracil-DNA glycosylase (nUDG) turnover during the cell cycle.

    PubMed

    Fischer, Jennifer A; Caradonna, Salvatore

    2011-01-01

    Uracil-DNA glycosylases (UDG/UNG) are enzymes that remove uracil from DNA and initiate base-excision repair. These enzymes play a key role in maintaining genomic integrity by reducing the mutagenic events caused by G:C to A:T transition mutations. The recent finding that a family of RNA editing enzymes (AID/APOBECs) can deaminate cytosine in DNA has raised the interest in these base-excision repair enzymes. The methodology presented here focuses on determining the regulation of the nuclear isoform of uracil-DNA glycosylase (nUDG), a 36,000 Da protein. In synchronized HeLa cells, nUDG protein levels decrease to barely detectable levels during the S phase of the cell cycle. Immunoblot analysis of immunoprecipitated or affinity-isolated nUDG reveals ubiquitin-conjugated nUDG when proteolysis is inhibited by agents that block proteasomal-dependent protein degradation. PMID:21755446

  1. Atomic substitution reveals the structural basis for substrate adenine recognition and removal by adenine DNA glycosylase

    SciTech Connect

    Lee, Seongmin; Verdine, Gregory L.

    2010-01-14

    Adenine DNA glycosylase catalyzes the glycolytic removal of adenine from the promutagenic A {center_dot} oxoG base pair in DNA. The general features of DNA recognition by an adenine DNA glycosylase, Bacillus stearothermophilus MutY, have previously been revealed via the X-ray structure of a catalytically inactive mutant protein bound to an A:oxoG-containing DNA duplex. Although the structure revealed the substrate adenine to be, as expected, extruded from the DNA helix and inserted into an extrahelical active site pocket on the enzyme, the substrate adenine engaged in no direct contacts with active site residues. This feature was paradoxical, because other glycosylases have been observed to engage their substrates primarily through direct contacts. The lack of direct contacts in the case of MutY suggested that either MutY uses a distinctive logic for substrate recognition or that the X-ray structure had captured a noncatalytically competent state in lesion recognition. To gain further insight into this issue, we crystallized wild-type MutY bound to DNA containing a catalytically inactive analog of 2'-deoxyadenosine in which a single 2'-H atom was replaced by fluorine. The structure of this fluorinated lesion-recognition complex (FLRC) reveals the substrate adenine buried more deeply into the active site pocket than in the prior structure and now engaged in multiple direct hydrogen bonding and hydrophobic interactions. This structure appears to capture the catalytically competent state of adenine DNA glycosylases, and it suggests a catalytic mechanism for this class of enzymes, one in which general acid-catalyzed protonation of the nucleobase promotes glycosidic bond cleavage.

  2. A poxvirus-encoded uracil DNA glycosylase is essential for virus viability.

    PubMed Central

    Stuart, D T; Upton, C; Higman, M A; Niles, E G; McFadden, G

    1993-01-01

    Infection of cultured mammalian cells with the Leporipoxvirus Shope fibroma virus (SFV) causes the induction of a novel uracil DNA glycosylase activity in the cytoplasms of the infected cells. The induction of this activity, early in infection, correlates with the early expression of the SFV BamHI D6R open reading frame which possesses significant protein sequence similarity to eukaryotic and prokaryotic uracil DNA glycosylases. The SFV BamHI D6R open reading frame and the homologous HindIII D4R open reading frame from the Orthopoxvirus vaccinia virus were cloned under the regulation of a phage T7 promoter and expressed in Escherichia coli as insoluble high-molecular-weight aggregates. During electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the E. coli-expressed proteins migrate with an apparent molecular mass of 25 kDa. The insoluble protein aggregate generated by expression in E. coli was solubilized in urea and, following a subsequent refolding step, displayed the ability to excise uracil residues from double-stranded plasmid DNA substrates, with the subsequent formation of apyrimidinic sites. The viral enzyme, like all other characterized uracil DNA glycosylases, is active in the presence of high concentrations of EDTA, is substrate inhibited by uracil, and does not display any endonuclease activity. Attempts to inactivate the HindIII D4R gene of vaccinia virus by targeted insertion of a dominant xanthine-guanine phosphoribosyltransferase selection marker or direct insertion of a frame-shifted oligonucleotide were uniformly unsuccessful demonstrating that, unlike the uracil DNA glycosylase described for herpesviruses, the poxvirus enzyme is essential for virus viability. Images PMID:8474156

  3. Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA.

    PubMed

    Burmeister, Wim P; Tarbouriech, Nicolas; Fender, Pascal; Contesto-Richefeu, Céline; Peyrefitte, Christophe N; Iseni, Frédéric

    2015-07-17

    Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A201-50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A201-50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a Kd of <1.4 μm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action. PMID:26045555

  4. Role of a MutY DNA Glycosylase in Combating Oxidative DNA damage in Helicobacter pylori

    PubMed Central

    Eutsey, Rory; Wang, Ge; Maier, Robert J.

    2007-01-01

    MutY is an adenine glycosylase that has the ability to efficiently remove adenines from adenine/7,8-dihydro-8-oxoguanine (8-oxo-G) or adenine/guanine mismatches, and plays an important role in oxidative DNA damage repair. The human gastric pathogen Helicobacter pylori has a homolog of the MutY enzyme. To investigate the physiological roles of MutY in H. pylori, we constructed and characterized a mutY mutant. H. pylori mutY mutants incubated at 5% O2 have a 325 fold higher spontaneous mutation rate than its parent. The mutation rate is further increased by exposing the mutant to atmospheric levels of oxygen, an effect that is not seen in an E. coli mutY mutant. Most of the mutations that occurred in H. pylori mutY mutants, as examined by rpoB sequence changes that confer rifampicin resistance, are GC to TA transversions. The H. pylori enzyme has the ability to complement an E. coli mutY mutant, restoring its mutation frequency to the wild-type level. Pure H. pylori MutY has the ability to remove adenines from A/8-oxo-G mismatches, but strikingly no ability to cleave A/G mismatches. This is surprising because E. coli MutY can more rapidly turnover A/G than A/8-oxo-G. Thus, H. pylori MutY is an adenine glycosylase involved in the repair of oxidative DNA damage with a specificity for detecting 8-oxo-G. In addition, H. pylori mutY mutants are only 30% as efficient as wild-type in colonizing the stomach of mice, indicating that H. pylori MutY plays a significant role in oxidative DNA damage repair in vivo. PMID:16996809

  5. Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase

    PubMed Central

    Ormeño, Fernando; Barrientos, Camila; Ramirez, Santiago; Ponce, Iván; Valenzuela, Lucía; Sepúlveda, Sofía; Bitar, Mainá; Kemmerling, Ulrike; Machado, Carlos Renato; Cabrera, Gonzalo; Galanti, Norbel

    2016-01-01

    Trypanosoma cruzi, the etiological agent of Chagas’ disease, presents three cellular forms (trypomastigotes, epimastigotes and amastigotes), all of which are submitted to oxidative species in its hosts. However, T. cruzi is able to resist oxidative stress suggesting a high efficiency of its DNA repair machinery.The Base Excision Repair (BER) pathway is one of the main DNA repair mechanisms in other eukaryotes and in T. cruzi as well. DNA glycosylases are enzymes involved in the recognition of oxidative DNA damage and in the removal of oxidized bases, constituting the first step of the BER pathway. Here, we describe the presence and activity of TcNTH1, a nuclear T. cruzi DNA glycosylase. Surprisingly, purified recombinant TcNTH1 does not remove the thymine glycol base, but catalyzes the cleavage of a probe showing an AP site. The same activity was found in epimastigote and trypomastigote homogenates suggesting that the BER pathway is not involved in thymine glycol DNA repair. TcNTH1 DNA-binding properties assayed in silico are in agreement with the absence of a thymine glycol removing function of that parasite enzyme. Over expression of TcNTH1 decrease parasite viability when transfected epimastigotes are submitted to a sustained production of H2O2.Therefore, TcNTH1 is the only known NTH1 orthologous unable to eliminate thymine glycol derivatives but that recognizes and cuts an AP site, most probably by a beta-elimination mechanism. We cannot discard that TcNTH1 presents DNA glycosylase activity on other DNA base lesions. Accordingly, a different DNA repair mechanism should be expected leading to eliminate thymine glycol from oxidized parasite DNA. Furthermore, TcNTH1 may play a role in the AP site recognition and processing. PMID:27284968

  6. Synthesis and Characterization of DNA Minor Groove Binding Alkylating Agents

    PubMed Central

    Iyer, Prema; Srinivasan, Ajay; Singh, Sreelekha K.; Mascara, Gerard P.; Zayitova, Sevara; Sidone, Brian; Fouquerel, Elise; Svilar, David; Sobol, Robert W.; Bobola, Michael S.; Silber, John R.; Gold, Barry

    2012-01-01

    Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases the N-terminus was appended with a O-methyl sulfonate ester while the C-terminus group was varied with non-polar and polar sidechains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) vs. major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is > 10-fold higher than the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells over-expressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization. PMID:23234400

  7. Synthesis and characterization of DNA minor groove binding alkylating agents.

    PubMed

    Iyer, Prema; Srinivasan, Ajay; Singh, Sreelekha K; Mascara, Gerard P; Zayitova, Sevara; Sidone, Brian; Fouquerel, Elise; Svilar, David; Sobol, Robert W; Bobola, Michael S; Silber, John R; Gold, Barry

    2013-01-18

    Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization. PMID:23234400

  8. A 5-methylcytosine DNA glycosylase/lyase demethylates the retrotransposon Tos17 and promotes its transposition in rice

    PubMed Central

    La, Honggui; Ding, Bo; Mishra, Gyan P.; Zhou, Bo; Yang, Hongmei; Bellizzi, Maria del Rosario; Chen, Songbiao; Meyers, Blake C.; Peng, Zhaohua; Zhu, Jian-Kang; Wang, Guo-Liang

    2011-01-01

    DNA 5-methylcytosine (5-meC) is an important epigenetic mark for transcriptional gene silencing in many eukaryotes. In Arabidopsis, 5-meC DNA glycosylase/lyases actively remove 5-meC to counteract transcriptional gene silencing in a locus-specific manner, and have been suggested to maintain the expression of transposons. However, it is unclear whether plant DNA demethylases can promote the transposition of transposons. Here we report the functional characterization of the DNA glycosylase/lyase DNG701 in rice. DNG701 encodes a large (1,812 amino acid residues) DNA glycosylase domain protein. Recombinant DNG701 protein showed 5-meC DNA glycosylase and lyase activities in vitro. Knockout or knockdown of DNG701 in rice plants led to DNA hypermethylation and reduced expression of the retrotransposon Tos17. Tos17 showed less transposition in calli derived from dng701 knockout mutant seeds compared with that in wild-type calli. Overexpression of DNG701 in both rice calli and transgenic plants substantially reduced DNA methylation levels of Tos17 and enhanced its expression. The overexpression also led to more frequent transposition of Tos17 in calli. Our results demonstrate that rice DNG701 is a 5-meC DNA glycosylase/lyase responsible for the demethylation of Tos17 and this DNA demethylase plays a critical role in promoting Tos17 transposition in rice calli. PMID:21896764

  9. 8-oxoguanine DNA glycosylase-1 driven DNA base excision repair: role in asthma pathogenesis

    PubMed Central

    Ba, Xueqing; Aguilera Aguirre, Leopoldo; Sur, Sanjiv; Boldogh, Istvan

    2015-01-01

    Purpose of review To provide both an overview and evidence of the potential etiology of oxidative DNA base damage and repair-signaling in chronic inflammation and histological changes associated with asthma. Recent findings Asthma is initiated/maintained by immunological, genetic/epigenetic and environmental factors. It is a world-wide health problem, as current therapies suppress symptoms rather than prevent/reverse the disease, largely due to gaps in understanding its molecular mechanisms. Inflammation, oxidative stress and DNA damage are inseparable phenomena, but their molecular roles in asthma pathogenesis are unclear. It was found that among oxidatively modified DNA bases, 8-oxoguanine (8-oxoG) is one of the most abundant, and its levels in DNA and body fluids are considered a biomarker of ongoing asthmatic processes. Free 8-oxoG forms a complex with 8-oxoguanine DNA glycosylase-1 (OGG1) and activates RAS-family GTPases that induce gene expression to mobilize innate and adaptive immune systems, along with genes regulating airway hyperplasia, hyper-responsiveness and lung remodeling in atopic and non-atopic asthma. Summary DNA’s integrity must be maintained to prevent mutation, so its continuous repair and downstream signaling “fuels” chronic inflammatory processes in asthma, and forms the basic mechanism whose elucidation will allow the development of new drug targets for the prevention/reversal of lung diseases. PMID:25486379

  10. A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily

    PubMed Central

    Sang, Pau Biak; Srinath, Thiruneelakantan; Patil, Aravind Goud; Woo, Eui-Jeon; Varshney, Umesh

    2015-01-01

    Uracil DNA glycosylases (UDGs) are an important group of DNA repair enzymes, which pioneer the base excision repair pathway by recognizing and excising uracil from DNA. Based on two short conserved sequences (motifs A and B), UDGs have been classified into six families. Here we report a novel UDG, UdgX, from Mycobacterium smegmatis and other organisms. UdgX specifically recognizes uracil in DNA, forms a tight complex stable to sodium dodecyl sulphate, 2-mercaptoethanol, urea and heat treatment, and shows no detectable uracil excision. UdgX shares highest homology to family 4 UDGs possessing Fe-S cluster. UdgX possesses a conserved sequence, KRRIH, which forms a flexible loop playing an important role in its activity. Mutations of H in the KRRIH sequence to S, G, A or Q lead to gain of uracil excision activity in MsmUdgX, establishing it as a novel member of the UDG superfamily. Our observations suggest that UdgX marks the uracil-DNA for its repair by a RecA dependent process. Finally, we observed that the tight binding activity of UdgX is useful in detecting uracils in the genomes. PMID:26304551

  11. MUTYH DNA glycosylase: the rationale for removing undamaged bases from the DNA

    PubMed Central

    Markkanen, Enni; Dorn, Julia; Hübscher, Ulrich

    2013-01-01

    Maintenance of genetic stability is crucial for all organisms in order to avoid the onset of deleterious diseases such as cancer. One of the many proveniences of DNA base damage in mammalian cells is oxidative stress, arising from a variety of endogenous and exogenous sources, generating highly mutagenic oxidative DNA lesions. One of the best characterized oxidative DNA lesion is 7,8-dihydro-8-oxoguanine (8-oxo-G), which can give rise to base substitution mutations (also known as point mutations). This mutagenicity is due to the miscoding potential of 8-oxo-G that instructs most DNA polymerases (pols) to preferentially insert an Adenine (A) opposite 8-oxo-G instead of the appropriate Cytosine (C). If left unrepaired, such A:8-oxo-G mispairs can give rise to CG→AT transversion mutations. A:8-oxo-G mispairs are proficiently recognized by the MutY glycosylase homologue (MUTYH). MUTYH can remove the mispaired A from an A:8-oxo-G, giving way to the canonical base-excision repair (BER) that ultimately restores undamaged Guanine (G). The importance of this MUTYH-initiated pathway is illustrated by the fact that biallelic mutations in the MUTYH gene are associated with a hereditary colorectal cancer syndrome termed MUTYH-associated polyposis (MAP). In this review, we will focus on MUTYH, from its discovery to the most recent data regarding its cellular roles and interaction partners. We discuss the involvement of the MUTYH protein in the A:8-oxo-G BER pathway acting together with pol λ, the pol that can faithfully incorporate C opposite 8-oxo-G and thus bypass this lesion in a correct manner. We also outline the current knowledge about the regulation of MUTYH itself and the A:8-oxo-G repair pathway by posttranslational modifications (PTM). Finally, to achieve a clearer overview of the literature, we will briefly touch on the rather confusing MUTYH nomenclature. In short, MUTYH is a unique DNA glycosylase that catalyzes the excision of an undamaged base from DNA. PMID

  12. Thymine DNA glycosylase exhibits negligible affinity for nucleobases that it removes from DNA

    PubMed Central

    Malik, Shuja S.; Coey, Christopher T.; Varney, Kristen M.; Pozharski, Edwin; Drohat, Alexander C.

    2015-01-01

    Thymine DNA Glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation. Initiating base excision repair, TDG removes thymine from mutagenic G·T mispairs caused by 5-methylcytosine (mC) deamination and other lesions including uracil (U) and 5-hydroxymethyluracil (hmU). In DNA demethylation, TDG excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), which are generated from mC by Tet (ten–eleven translocation) enzymes. Using improved crystallization conditions, we solved high-resolution (up to 1.45 Å) structures of TDG enzyme–product complexes generated from substrates including G·U, G·T, G·hmU, G·fC and G·caC. The structures reveal many new features, including key water-mediated enzyme–substrate interactions. Together with nuclear magnetic resonance experiments, the structures demonstrate that TDG releases the excised base from its tight product complex with abasic DNA, contrary to previous reports. Moreover, DNA-free TDG exhibits no significant binding to free nucleobases (U, T, hmU), indicating a Kd >> 10 mM. The structures reveal a solvent-filled channel to the active site, which might facilitate dissociation of the excised base and enable caC excision, which involves solvent-mediated acid catalysis. Dissociation of the excised base allows TDG to bind the beta rather than the alpha anomer of the abasic sugar, which might stabilize the enzyme–product complex. PMID:26358812

  13. A New Family of HEAT-Like Repeat Proteins Lacking a Critical Substrate Recognition Motif Present in Related DNA Glycosylases.

    PubMed

    Mullins, Elwood A; Shi, Rongxin; Kotsch, Lyle A; Eichman, Brandt F

    2015-01-01

    DNA glycosylases are important repair enzymes that eliminate a diverse array of aberrant nucleobases from the genomes of all organisms. Individual bacterial species often contain multiple paralogs of a particular glycosylase, yet the molecular and functional distinctions between these paralogs are not well understood. The recently discovered HEAT-like repeat (HLR) DNA glycosylases are distributed across all domains of life and are distinct in their specificity for cationic alkylpurines and mechanism of damage recognition. Here, we describe a number of phylogenetically diverse bacterial species with two orthologs of the HLR DNA glycosylase AlkD. One ortholog, which we designate AlkD2, is substantially less conserved. The crystal structure of Streptococcus mutans AlkD2 is remarkably similar to AlkD but lacks the only helix present in AlkD that penetrates the DNA minor groove. We show that AlkD2 possesses only weak DNA binding affinity and lacks alkylpurine excision activity. Mutational analysis of residues along this DNA binding helix in AlkD substantially reduced binding affinity for damaged DNA, for the first time revealing the importance of this structural motif for damage recognition by HLR glycosylases. PMID:25978435

  14. Base excision repair: NMR backbone assignments of Escherichia coli formamidopyrimidine-DNA glycosylase

    SciTech Connect

    Buchko, Garry W.; Wallace, Susan S.; Kennedy, Michael A.

    2002-03-01

    Oxidative damage is emerging as one of the most important mechanisms responsible for mutagenesis, carcinogenesis, aging, and various diseases (Farr and Kogma, 1991). One of the potential targets for oxidation is cellular DNA. While exposure to exogenous agents, such as ionizing radiation and chemicals, contributes to damaging DNA, the most important oxidative agents are endogenous, such as the reactive free radicals produced during normal oxidative metabolism (Adelman et., 1988). To mitigate the potentially deleterious effects of oxidative DNA damage virtually all aerobic organisms have developed complex repair mechanisms (Petit and Sancar, 1999). One repair mechanism, base excision repair (BER), appears to be responsible for replacing most oxidative DNA damage (David and Williams, 1998). Formamidopyrimidine-DNA glycosylase (Fpg), a 269-residue metalloprotein with a molecular weight of 30.2 kDa, is a key BER enzyme in prokaryotes (Boiteaux et al., 1987). Substrates recognized and released by Fpg include 7,8-dihydro-8-oxoguanine (8-oxoG), 2,6 diamino-4-hydroxy-5-formamido pyrimidine (Fapy-G), the adenine equivalents 8-oxoA and Fapy-A, 5-hydroxycytosine, 5-hydroxyuracil, B ureidoisobutiric acid, and a-R-hydroxy-B-ureidoisobutiric acid (Freidberg et al., 1995). In vitro Fpg bind double-stranded DNA and performs three catalytic activities: (i) DNA glycosylase, (ii) AP lyase, and (iii) deoxyribophosphodiesterase.

  15. Induction of NEIL1 and NEIL2 DNA glycosylases in aniline-induced splenic toxicity

    SciTech Connect

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

    2011-02-15

    The mechanisms by which aniline exposure elicits splenotoxic response, especially the tumorigenic response, are not well-understood. Earlier, we have shown that aniline-induced oxidative stress is associated with increased oxidative DNA damage in rat spleen. The base excision repair (BER) pathway is the major mechanism for the repair of oxidative DNA base lesions, and we have shown an up-regulation of 8-oxoguanine glycosylase 1 (OGG1), a specific DNA glycosylase involved in the removal of 8-hydroxy-2'-deoxyguanosine (8-OHdG) adducts, following aniline exposure. Nei-like DNA glycosylases (NEIL1/2) belong to a family of BER proteins that are distinct from other DNA glycosylases, including OGG1. However, contribution of NEIL1/2 in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on evaluating if NEILs also contribute to the repair of oxidative DNA lesions in the spleen following aniline exposure. To achieve that, male SD rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. The BER activity of NEIL1/2 was assayed using a bubble structure substrate containing 5-OHU (preferred substrates for NEIL1 and NEIL2) and by quantitating the cleavage products. Aniline treatment led to a 1.25-fold increase in the NEIL1/2-associated BER activity in the nuclear extracts of spleen compared to the controls. Real-time PCR analysis for NEIL1 and NEIL2 mRNA expression in the spleen revealed 2.7- and 3.9-fold increases, respectively, in aniline-treated rats compared to controls. Likewise, Western blot analysis showed that protein expression of NEIL1 and NEIL2 in the nuclear extract of spleens from aniline-treated rats was 2.0- and 3.8-fold higher than controls, respectively. Aniline treatment also led to stronger immunoreactivity for NEIL1 and NEIL2 in the spleens, confined to the red pulp areas. These studies, thus, show that aniline

  16. DNA damage processing by human 8-oxoguanine-DNA glycosylase mutants with the occluded active site.

    PubMed

    Lukina, Maria V; Popov, Alexander V; Koval, Vladimir V; Vorobjev, Yuri N; Fedorova, Olga S; Zharkov, Dmitry O

    2013-10-01

    8-Oxoguanine-DNA glycosylase (OGG1) removes premutagenic lesion 8-oxoguanine (8-oxo-G) from DNA and then nicks the nascent abasic (apurinic/apyrimidinic) site by β-elimination. Although the structure of OGG1 bound to damaged DNA is known, the dynamic aspects of 8-oxo-G recognition are not well understood. To comprehend the mechanisms of substrate recognition and processing, we have constructed OGG1 mutants with the active site occluded by replacement of Cys-253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. The conformational dynamics of OGG1 mutants were characterized by single-turnover kinetics and stopped-flow kinetics with fluorescent detection. Additionally, the conformational mobility of wild type and the mutant OGG1 substrate complex was assessed using molecular dynamics simulations. Although pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of 8-oxo-G and apurinic/apyrimidinic sites. Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate β-elimination. The results agree with the concept of enzyme plasticity, suggesting that the active site of OGG1 is flexible enough to compensate partially for distortions caused by mutation. PMID:23955443

  17. Structural Basis for Avoidance of Promutagenic DNA Repair by MutY Adenine DNA Glycosylase*

    PubMed Central

    Wang, Lan; Lee, Seung-Joo; Verdine, Gregory L.

    2015-01-01

    The highly mutagenic A:oxoG (8-oxoguanine) base pair in DNA most frequently arises by aberrant replication of the primary oxidative lesion C:oxoG. This lesion is particularly insidious because neither of its constituent nucleobases faithfully transmit genetic information from the original C:G base pair. Repair of A:oxoG is initiated by adenine DNA glycosylase, which catalyzes hydrolytic cleavage of the aberrant A nucleobase from the DNA backbone. These enzymes, MutY in bacteria and MUTYH in humans, scrupulously avoid processing of C:oxoG because cleavage of the C residue in C:oxoG would actually promote mutagenic conversion to A:oxoG. Here we analyze the structural basis for rejection of C:oxoG by MutY, using a synthetic crystallography approach to capture the enzyme in the process of inspecting the C:oxoG anti-substrate, with which it ordinarily binds only fleetingly. We find that MutY uses two distinct strategies to avoid presentation of C to the enzyme active site. Firstly, MutY possesses an exo-site that serves as a decoy for C, and secondly, repulsive forces with a key active site residue prevent stable insertion of C into the nucleobase recognition pocket within the enzyme active site. PMID:25995449

  18. Structural Basis for Avoidance of Promutagenic DNA Repair by MutY Adenine DNA Glycosylase.

    PubMed

    Wang, Lan; Lee, Seung-Joo; Verdine, Gregory L

    2015-07-10

    The highly mutagenic A:oxoG (8-oxoguanine) base pair in DNA most frequently arises by aberrant replication of the primary oxidative lesion C:oxoG. This lesion is particularly insidious because neither of its constituent nucleobases faithfully transmit genetic information from the original C:G base pair. Repair of A:oxoG is initiated by adenine DNA glycosylase, which catalyzes hydrolytic cleavage of the aberrant A nucleobase from the DNA backbone. These enzymes, MutY in bacteria and MUTYH in humans, scrupulously avoid processing of C:oxoG because cleavage of the C residue in C:oxoG would actually promote mutagenic conversion to A:oxoG. Here we analyze the structural basis for rejection of C:oxoG by MutY, using a synthetic crystallography approach to capture the enzyme in the process of inspecting the C:oxoG anti-substrate, with which it ordinarily binds only fleetingly. We find that MutY uses two distinct strategies to avoid presentation of C to the enzyme active site. Firstly, MutY possesses an exo-site that serves as a decoy for C, and secondly, repulsive forces with a key active site residue prevent stable insertion of C into the nucleobase recognition pocket within the enzyme active site. PMID:25995449

  19. Thermodynamics of the DNA Damage Repair Steps of Human 8-Oxoguanine DNA Glycosylase

    PubMed Central

    Kuznetsov, Nikita A.; Kuznetsova, Alexandra A.; Vorobjev, Yuri N.; Krasnoperov, Lev N.; Fedorova, Olga S.

    2014-01-01

    Human 8-oxoguanine DNA glycosylase (hOGG1) is a key enzyme responsible for initiating the base excision repair of 7,8-dihydro-8-oxoguanosine (oxoG). In this study a thermodynamic analysis of the interaction of hOGG1 with specific and non-specific DNA-substrates is performed based on stopped-flow kinetic data. The standard Gibbs energies, enthalpies and entropies of specific stages of the repair process were determined via kinetic measurements over a temperature range using the van’t Hoff approach. The three steps which are accompanied with changes in the DNA conformations were detected via 2-aminopurine fluorescence in the process of binding and recognition of damaged oxoG base by hOGG1. The thermodynamic analysis has demonstrated that the initial step of the DNA substrates binding is mainly governed by energy due to favorable interactions in the process of formation of the recognition contacts, which results in negative enthalpy change, as well as due to partial desolvation of the surface between the DNA and enzyme, which results in positive entropy change. Discrimination of non-specific G base versus specific oxoG base is occurring in the second step of the oxoG-substrate binding. This step requires energy consumption which is compensated by the positive entropy contribution. The third binding step is the final adjustment of the enzyme/substrate complex to achieve the catalytically competent state which is characterized by large endothermicity compensated by a significant increase of entropy originated from the dehydration of the DNA grooves. PMID:24911585

  20. Listeria monocytogenes DNA Glycosylase AdlP Affects Flagellar Motility, Biofilm Formation, Virulence, and Stress Responses

    PubMed Central

    Zhang, Ting; Bae, Dongryeoul

    2016-01-01

    ABSTRACT The temperature-dependent alteration of flagellar motility gene expression is critical for the foodborne pathogen Listeria monocytogenes to respond to a changing environment. In this study, a genetic determinant, L. monocytogenes f2365_0220 (lmof2365_0220), encoding a putative protein that is structurally similar to the Bacillus cereus alkyl base DNA glycosylase (AlkD), was identified. This determinant was involved in the transcriptional repression of flagellar motility genes and was named adlP (encoding an AlkD-like protein [AdlP]). Deletion of adlP activated the expression of flagellar motility genes at 37°C and disrupted the temperature-dependent inhibition of L. monocytogenes motility. The adlP null strains demonstrated decreased survival in murine macrophage-like RAW264.7 cells and less virulence in mice. Furthermore, the deletion of adlP significantly decreased biofilm formation and impaired the survival of bacteria under several stress conditions, including the presence of a DNA alkylation compound (methyl methanesulfonate), an oxidative agent (H2O2), and aminoglycoside antibiotics. Our findings strongly suggest that adlP may encode a bifunctional protein that transcriptionally represses the expression of flagellar motility genes and influences stress responses through its DNA glycosylase activity. IMPORTANCE We discovered a novel protein that we named AlkD-like protein (AdlP). This protein affected flagellar motility, biofilm formation, and virulence. Our data suggest that AdlP may be a bifunctional protein that represses flagellar motility genes and influences stress responses through its DNA glycosylase activity. PMID:27316964

  1. Single Qdot-labeled glycosylase molecules use a wedge amino acid to probe for lesions while scanning along DNA

    PubMed Central

    Dunn, Andrew R.; Kad, Neil M.; Nelson, Shane R.; Warshaw, David M.; Wallace, Susan S.

    2011-01-01

    Within the base excision repair (BER) pathway, the DNA N-glycosylases are responsible for locating and removing the majority of oxidative base damages. Endonuclease III (Nth), formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) are members of two glycosylase families: the helix–hairpin–helix (HhH) superfamily and the Fpg/Nei family. The search mechanisms employed by these two families of glycosylases were examined using a single molecule assay to image quantum dot (Qdot)-labeled glycosylases interacting with YOYO-1 stained λ-DNA molecules suspended between 5 µm silica beads. The HhH and Fpg/Nei families were found to have a similar diffusive search mechanism described as a continuum of motion, in keeping with rotational diffusion along the DNA molecule ranging from slow, sub-diffusive to faster, unrestricted diffusion. The search mechanism for an Fpg variant, F111A, lacking a phenylalanine wedge residue no longer displayed slow, sub-diffusive motion compared to wild type, suggesting that Fpg base interrogation may be accomplished by Phe111 insertion. PMID:21666255

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

    PubMed Central

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

    2015-01-01

    Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal guanine. We combined molecular dynamics simulation and stopped-flow kinetics with fluorescence detection to track the events in the recognition of oxoG by Fpg and its mutants with a key phenylalanine residue, which intercalates next to the damaged base, changed to either alanine (F110A) or fluorescent reporter tryptophan (F110W). Guanine was sampled by Fpg, as evident from the F110W stopped-flow traces, but less extensively than oxoG. The wedgeless F110A enzyme could bend DNA but failed to proceed further in oxoG recognition. Modeling of the base eversion with energy decomposition suggested that the wedge destabilizes the intrahelical base primarily through buckling both surrounding base pairs. Replacement of oxoG with abasic (AP) site rescued the activity, and calculations suggested that wedge insertion is not required for AP site destabilization and eversion. Our results suggest that Fpg, and possibly other DNA glycosylases, convert part of the binding energy into active destabilization of their substrates, using the energy differences between normal and damaged bases for fast substrate discrimination. PMID:25520195

  3. Crystal Structure of Human Thymine DNA Glycosylase Bound to DNA Elucidates Sequence-Specific Mismatch Recognition

    SciTech Connect

    Maiti, A.; Morgan, M.T.; Pozharski, E.; Drohat, A.C.

    2009-05-19

    Cytosine methylation at CpG dinucleotides produces m{sup 5}CpG, an epigenetic modification that is important for transcriptional regulation and genomic stability in vertebrate cells. However, m{sup 5}C deamination yields mutagenic G{center_dot}T mispairs, which are implicated in genetic disease, cancer, and aging. Human thymine DNA glycosylase (hTDG) removes T from G{center_dot}T mispairs, producing an abasic (or AP) site, and follow-on base excision repair proteins restore the G{center_dot}C pair. hTDG is inactive against normal A{center_dot}T pairs, and is most effective for G{center_dot}T mispairs and other damage located in a CpG context. The molecular basis of these important catalytic properties has remained unknown. Here, we report a crystal structure of hTDG (catalytic domain, hTDG{sup cat}) in complex with abasic DNA, at 2.8 {angstrom} resolution. Surprisingly, the enzyme crystallized in a 2:1 complex with DNA, one subunit bound at the abasic site, as anticipated, and the other at an undamaged (nonspecific) site. Isothermal titration calorimetry and electrophoretic mobility-shift experiments indicate that hTDG and hTDG{sup cat} can bind abasic DNA with 1:1 or 2:1 stoichiometry. Kinetics experiments show that the 1:1 complex is sufficient for full catalytic (base excision) activity, suggesting that the 2:1 complex, if adopted in vivo, might be important for some other activity of hTDG, perhaps binding interactions with other proteins. Our structure reveals interactions that promote the stringent specificity for guanine versus adenine as the pairing partner of the target base and interactions that likely confer CpG sequence specificity. We find striking differences between hTDG and its prokaryotic ortholog (MUG), despite the relatively high (32%) sequence identity.

  4. Transcriptional regulation of thymine DNA glycosylase (TDG) by the tumor suppressor protein p53.

    PubMed

    da Costa, Nathalia Meireles; Hautefeuille, Agnès; Cros, Marie-Pierre; Melendez, Matias Eliseo; Waters, Timothy; Swann, Peter; Hainaut, Pierre; Pinto, Luis Felipe Ribeiro

    2012-12-15

    Thymine DNA glycosylase (TDG) belongs to the superfamily of uracil DNA glycosylases (UDG) and is the first enzyme in the base-excision repair pathway (BER) that removes thymine from G:T mismatches at CpG sites. This glycosylase activity has also been found to be critical for active demethylation of genes involved in embryonic development. Here we show that wild-type p53 transcriptionally regulates TDG expression. Chromatin immunoprecipitation (ChIP) and luciferase assays indicate that wild-type p53 binds to a domain of TDG promoter containing two p53 consensus response elements (p53RE) and activates its transcription. Next, we have used a panel of cell lines with different p53 status to demonstrate that TDG mRNA and protein expression levels are induced in a p53-dependent manner under different conditions. This panel includes isogenic breast and colorectal cancer cell lines with wild-type or inactive p53, esophageal squamous cell carcinoma cell lines lacking p53 or expressing a temperature-sensitive p53 mutant and normal human bronchial epithelial cells. Induction of TDG mRNA expression is accompanied by accumulation of TDG protein in both nucleus and cytoplasm, with nuclear re-localization occurring upon DNA damage in p53-competent, but not -incompetent, cells. These observations suggest a role for p53 activity in TDG nuclear translocation. Overall, our results show that TDG expression is directly regulated by p53, suggesting that loss of p53 function may affect processes mediated by TDG, thus negatively impacting on genetic and epigenetic stability. PMID:23165212

  5. Transcriptional regulation of thymine DNA glycosylase (TDG) by the tumor suppressor protein p53

    PubMed Central

    da Costa, Nathalia Meireles; Hautefeuille, Agnès; Cros, Marie-Pierre; Melendez, Matias Eliseo; Waters, Timothy; Swann, Peter; Hainaut, Pierre; Pinto, Luis Felipe Ribeiro

    2012-01-01

    Thymine DNA glycosylase (TDG) belongs to the superfamily of uracil DNA glycosylases (UDG) and is the first enzyme in the base-excision repair pathway (BER) that removes thymine from G:T mismatches at CpG sites. This glycosylase activity has also been found to be critical for active demethylation of genes involved in embryonic development. Here we show that wild-type p53 transcriptionally regulates TDG expression. Chromatin immunoprecipitation (ChIP) and luciferase assays indicate that wild-type p53 binds to a domain of TDG promoter containing two p53 consensus response elements (p53RE) and activates its transcription. Next, we have used a panel of cell lines with different p53 status to demonstrate that TDG mRNA and protein expression levels are induced in a p53-dependent manner under different conditions. This panel includes isogenic breast and colorectal cancer cell lines with wild-type or inactive p53, esophageal squamous cell carcinoma cell lines lacking p53 or expressing a temperature-sensitive p53 mutant and normal human bronchial epithelial cells. Induction of TDG mRNA expression is accompanied by accumulation of TDG protein in both nucleus and cytoplasm, with nuclear re-localization occurring upon DNA damage in p53-competent, but not -incompetent, cells. These observations suggest a role for p53 activity in TDG nuclear translocation. Overall, our results show that TDG expression is directly regulated by p53, suggesting that loss of p53 function may affect processes mediated by TDG, thus negatively impacting on genetic and epigenetic stability. PMID:23165212

  6. Formamidopyrimidine-DNA glycosylase of Escherichia coli: cloning and sequencing of the fpg structural gene and overproduction of the protein.

    PubMed Central

    Boiteux, S; O'Connor, T R; Laval, J

    1987-01-01

    An Escherichia coli genomic library composed of large DNA fragments (10-15 kb) was constructed using the plasmid pBR322 as vector. From it 700 clones were individually screened for increased excision of the ring-opened form of N7-methylguanine (2-6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine) or Fapy. One clone overproduced the Fapy-DNA glycosylase activity by a factor of 10-fold as compared with the wild-type strain. The Fapy-DNA glycosylase overproducer character was associated with a 15-kb recombinant plasmid (pFPG10). After subcloning a 1.4-kb fragment which contained the Fapy-DNA glycosylase gene (fpg+) was inserted in the plasmids pUC18 and pUC19 yielding pFPG50 and pFPG60 respectively. The cells harbouring pFPG60 displayed a 50- to 100-fold increase in glycosylase activity and overexpressed a 31-kd protein. From these cells the Fapy-DNA glycosylase was purified to apparent physical homogeneity as evidenced by a single protein band at 31 kd on SDS-polyacrylamide gels. The amino acid composition of the protein and the amino acid sequence deduced from the nucleotide sequence demonstrate that the cloned fragment contains the structural gene coding for the Fapy-DNA glycosylase. The nucleotide sequence of the fpg gene is composed of 809 base pairs and codes for a protein of 269 amino acids with a calculated mol. wt of 30.2 kd. Images Fig. 2. PMID:3319582

  7. The effect of sequence context on the activity of cytosine DNA glycosylases.

    PubMed

    Kimber, Scott T; Brown, Tom; Fox, Keith R

    2015-12-01

    We have prepared single (N204D) and double (N204D:L272A) mutants of human uracil DNA glycosylase (hUDG), generating two cytosine DNA glycosylases (hCDG and hCYDG). Both these enzymes are able to excise cytosine (but not 5-methylcytosine), when this base is part of a mismatched base pair. hCDG is more active than the equivalent E. coli enzyme (eCYDG) and also has some activity when the cytosine is paired with guanine, unlike eCYDG. hCDG also has some activity against single stranded DNA, while having poor activity towards an unnatural base pair that forces the cytosine into an extrahelical conformation (in contrast to eCYDG for which a bulky base enhances the enzyme's activity). We also examined how sequence context affects the activity of these enzymes, determining the effect of flanking base pairs on cleavage efficiency. An abasic site or a hexaethylene glycol linker placed opposite the target cytosine, also causes an increase in activity compared with an AC mismatch. Flanking an AC mismatch with GC base pairs resulted in a 100-fold decrease in excision activity relative to flanking AT base pairs and the 5'-flanking base pair had a greater effect on the rate of cleavage. However, this effect is not simply due to the stability of the flanking base pairs as adjacent GT mismatches also produce low cleavage efficiency. PMID:26463365

  8. Regulation of DNA glycosylases and their role in limiting disease

    PubMed Central

    SAMPATH, HARINI; MCCULLOUGH, AMANDA K.; LLOYD, R. STEPHEN

    2016-01-01

    This review will present a current understanding of mechanisms for the initiation of base excision repair (BER) of oxidatively-induced DNA damage and the biological consequences of deficiencies in these enzymes in mouse model systems and human populations. PMID:22300253

  9. Coordination of MYH DNA glycosylase and APE1 endonuclease activities via physical interactions.

    PubMed

    Luncsford, Paz J; Manvilla, Brittney A; Patterson, Dimeka N; Malik, Shuja S; Jin, Jin; Hwang, Bor-Jang; Gunther, Randall; Kalvakolanu, Snigdha; Lipinski, Leonora J; Yuan, Weirong; Lu, Wuyuan; Drohat, Alexander C; Lu, A-Lien; Toth, Eric A

    2013-12-01

    MutY homologue (MYH) is a DNA glycosylase which excises adenine paired with the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG, or G(o)) during base excision repair (BER). Base excision by MYH results in an apurinic/apyrimidinic (AP) site in the DNA where the DNA sugar-phosphate backbone remains intact. A key feature of MYH activity is its physical interaction and coordination with AP endonuclease I (APE1), which subsequently nicks DNA 5' to the AP site. Because AP sites are mutagenic and cytotoxic, they must be processed by APE1 immediately after the action of MYH glycosylase. Our recent reports show that the interdomain connector (IDC) of human MYH (hMYH) maintains interactions with hAPE1 and the human checkpoint clamp Rad9-Rad1-Hus1 (9-1-1) complex. In this study, we used NMR chemical shift perturbation experiments to determine hMYH-binding site on hAPE1. Chemical shift perturbations indicate that the hMYH IDC peptide binds to the DNA-binding site of hAPE1 and an additional site which is distal to the APE1 DNA-binding interface. In these two binding sites, N212 and Q137 of hAPE1 are key mediators of the MYH/APE1 interaction. Intriguingly, despite the fact that hHus1 and hAPE1 both interact with the MYH IDC, hHus1 does not compete with hAPE1 for binding to hMYH. Rather, hHus1 stabilizes the hMYH/hAPE1 complex both in vitro and in cells. This is consistent with a common theme in BER, namely that the assembly of protein-DNA complexes enhances repair by efficiently coordinating multiple enzymatic steps while simultaneously minimizing the release of harmful repair intermediates. PMID:24209961

  10. Coordination of MYH DNA glycosylase and APE1 endonuclease activities via physical interactions

    PubMed Central

    Luncsford, Paz J.; Manvilla, Brittney A.; Patterson, Dimeka N.; Malik, Shuja S.; Jin, Jin; Hwang, Bor-Jang; Gunther, Randall; Kalvakolanu, Snigdha; Lipinski, Leonora J.; Yuan, Weirong; Lu, Wuyuan; Drohat, Alexander C.; Lu-Chang, A-Lien; Toth, Eric A.

    2013-01-01

    MutY homologue (MYH) is a DNA glycosylase which excises adenine paired with the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG, or G°) during base excision repair (BER). Base excision by MYH results in an apurinic/apyrimidinic (AP) site in the DNA where the DNA sugar-phosphate backbone remains intact. A key feature of MYH activity is its physical interaction and coordination with AP endonuclease I (APE1), which subsequently nicks DNA 5' to the AP site. Because AP sites are mutagenic and cytotoxic, they must be processed by APE1 immediately after the action of MYH glycosylase. Our recent reports show that the interdomain connector (IDC) of human MYH (hMYH) maintains interactions with hAPE1 and the human checkpoint clamp Rad9-Rad1-Hus1 (9-1-1) complex. In this study, we used NMR chemical shift perturbation experiments to determine hMYH-binding site on hAPE1. Chemical shift perturbations indicate that the hMYH IDC peptide binds to the DNA-binding site of hAPE1 and an additional site which is distal to the APE1 DNA-binding interface. In these two binding sites, N212 and Q137 of hAPE1 are key mediators of the MYH/APE1 interaction. Intriguingly, despite the fact that hHus1 and hAPE1 both interact with the MYH IDC, hHus1 does not compete with hAPE1 for binding to hMYH. Rather, hHus1 stabilizes the hMYH/hAPE1 complex both in vitro and in cells. This is consistent with a common theme in BER, namely that the assembly of protein-DNA complexes enhances repair by efficiently coordinating multiple enzymatic steps while simultaneously minimizing the release of harmful repair intermediates. PMID:24209961

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

    SciTech Connect

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

    2004-05-05

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

  12. Structural Characterization of Human 8-Oxoguanine DNA Glycosylase Variants Bearing Active Site Mutations

    SciTech Connect

    Radom,C.; Banerjee, A.; Verdine, G.

    2007-01-01

    The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and inserts it into an extrahelical enzyme active site pocket lined with residues that participate in lesion recognition and catalysis. Structural analysis has been performed on mutant versions of hOGG1 having changes in catalytic residues but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact residues. Here we report high resolution structural analysis of such recognition variants. We found that Ala substitution at residues that contact the phosphate 5 to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation) simply removed key functionality from the contact interface but otherwise had no effect on structure. Ala substitution at the only residue making an oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA contact surface of hOGG1, but this was overcome by local interactions within the folded protein, indicating that this oxoG recognition motif is 'hardwired'. Introduction of a side chain intended to sterically obstruct the active site pocket (Q315F mutation) led to two different structures, one of which (Q315F{sup *149}) has the oxoG lesion in an exosite flanking the active site and the other of which (Q315F{sup *292}) has the oxoG inserted nearly completely into the lesion recognition pocket. The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding.

  13. Repair of Alkylation Damage in Eukaryotic Chromatin Depends on Searching Ability of Alkyladenine DNA Glycosylase.

    PubMed

    Zhang, Yaru; O'Brien, Patrick J

    2015-11-20

    Human alkyladenine DNA glycosylase (AAG) initiates the base excision repair pathway by excising alkylated and deaminated purine lesions. In vitro biochemical experiments demonstrate that AAG uses facilitated diffusion to efficiently search DNA to find rare sites of damage and suggest that electrostatic interactions are critical to the searching process. However, it remains an open question whether DNA searching limits the rate of DNA repair in vivo. We constructed AAG mutants with altered searching ability and measured their ability to protect yeast from alkylation damage in order to address this question. Each of the conserved arginine and lysine residues that are near the DNA binding interface were mutated, and the functional impacts were evaluated using kinetic and thermodynamic analysis. These mutations do not perturb catalysis of N-glycosidic bond cleavage, but they decrease the ability to capture rare lesion sites. Nonspecific and specific DNA binding properties are closely correlated, suggesting that the electrostatic interactions observed in the specific recognition complex are similarly important for DNA searching complexes. The ability of the mutant proteins to complement repair-deficient yeast cells is positively correlated with the ability of the proteins to search DNA in vitro, suggesting that cellular resistance to DNA alkylation is governed by the ability to find and efficiently capture cytotoxic lesions. It appears that chromosomal access is not restricted and toxic sites of alkylation damage are readily accessible to a searching protein. PMID:26317160

  14. Kinetics and binding of the thymine-DNA mismatch glycosylase, Mig-Mth, with mismatch-containing DNA substrates.

    PubMed

    Begley, Thomas J; Haas, Brian J; Morales, Juan C; Kool, Eric T; Cunningham, Richard P

    2003-01-01

    We have examined the removal of thymine residues from T-G mismatches in DNA by the thymine-DNA mismatch glycosylase from Methanobacterium thermoautrophicum (Mig-Mth), within the context of the base excision repair (BER) pathway, to investigate why this glycosylase has such low activity in vitro. Using single-turnover kinetics and steady-state kinetics, we calculated the catalytic and product dissociation rate constants for Mig-Mth, and determined that Mig-Mth is inhibited by product apyrimidinic (AP) sites in DNA. Electrophoretic mobility shift assays (EMSA) provide evidence that the specificity of product binding is dependent upon the base opposite the AP site. The binding of Mig-Mth to DNA containing the non-cleavable substrate analogue difluorotoluene (F) was also analyzed to determine the effect of the opposite base on Mig-Mth binding specificity for substrate-like duplex DNA. The results of these experiments support the idea that opposite strand interactions play roles in determining substrate specificity. Endonuclease IV, which cleaves AP sites in the next step of the BER pathway, was used to analyze the effect of product removal on the overall rate of thymine hydrolysis by Mig-Mth. Our results support the hypothesis that endonuclease IV increases the apparent activity of Mig-Mth significantly under steady-state conditions by preventing reassociation of enzyme to product. PMID:12509271

  15. Thymine DNA Glycosylase Is a Positive Regulator of Wnt Signaling in Colorectal Cancer*

    PubMed Central

    Xu, Xuehe; Yu, Tianxin; Shi, Jiandang; Chen, Xi; Zhang, Wen; Lin, Ting; Liu, Zhihong; Wang, Yadong; Zeng, Zheng; Wang, Chi; Li, Mingsong; Liu, Chunming

    2014-01-01

    Wnt signaling plays an important role in colorectal cancer (CRC). Although the mechanisms of β-catenin degradation have been well studied, the mechanism by which β-catenin activates transcription is still not fully understood. While screening a panel of DNA demethylases, we found that thymine DNA glycosylase (TDG) up-regulated Wnt signaling. TDG interacts with the transcription factor TCF4 and coactivator CREB-binding protein/p300 in the Wnt pathway. Knocking down TDG by shRNAs inhibited the proliferation of CRC cells in vitro and in vivo. In CRC patients, TDG levels were significantly higher in tumor tissues than in the adjacent normal tissues. These results suggest that TDG warrants consideration as a potential biomarker for CRC and as a target for CRC treatment. PMID:24532795

  16. Increased postischemic brain injury in mice deficient in uracil-DNA glycosylase

    PubMed Central

    Endres, Matthias; Biniszkiewicz, Detlev; Sobol, Robert W.; Harms, Christoph; Ahmadi, Michael; Lipski, Andreas; Katchanov, Juri; Mergenthaler, Philipp; Dirnagl, Ulrich; Wilson, Samuel H.; Meisel, Andreas; Jaenisch, Rudolf

    2004-01-01

    Uracil-DNA glycosylase (UNG) is involved in base excision repair of aberrant uracil residues in nuclear and mitochondrial DNA. Ung knockout mice generated by gene targeting are viable, fertile, and phenotypically normal and have regular mutation rates. However, when exposed to a nitric oxide donor, Ung–/– fibroblasts show an increase in the uracil/cytosine ratio in the genome and augmented cell death. After combined oxygen-glucose deprivation, Ung–/– primary cortical neurons have increased vulnerability to cell death, which is associated with early mitochondrial dysfunction. In vivo, UNG expression and activity are low in brains of naive WT mice but increase significantly after reversible middle cerebral artery occlusion and reperfusion. Moreover, major increases in infarct size are observed in Ung–/– mice compared with littermate control mice. In conclusion, our results provide compelling evidence that UNG is of major importance for tissue repair after brain ischemia. PMID:15199406

  17. Neil3 and NEIL1 DNA Glycosylases Remove Oxidative Damages from Quadruplex DNA and Exhibit Preferences for Lesions in the Telomeric Sequence Context*

    PubMed Central

    Zhou, Jia; Liu, Minmin; Fleming, Aaron M.; Burrows, Cynthia J.; Wallace, Susan S.

    2013-01-01

    The telomeric DNA of vertebrates consists of d(TTAGGG)n tandem repeats, which can form quadruplex DNA structures in vitro and likely in vivo. Despite the fact that the G-rich telomeric DNA is susceptible to oxidation, few biochemical studies of base excision repair in telomeric DNA and quadruplex structures have been done. Here, we show that telomeric DNA containing thymine glycol (Tg), 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh), or spiroiminodihydantoin (Sp) can form quadruplex DNA structures in vitro. We have tested the base excision activities of five mammalian DNA glycosylases (NEIL1, NEIL2, mNeil3, NTH1, and OGG1) on these lesion-containing quadruplex substrates and found that only mNeil3 had excision activity on Tg in quadruplex DNA and that the glycosylase exhibited a strong preference for Tg in the telomeric sequence context. Although Sp and Gh in quadruplex DNA were good substrates for mNeil3 and NEIL1, none of the glycosylases had activity on quadruplex DNA containing 8-oxoG. In addition, NEIL1 but not mNeil3 showed enhanced glycosylase activity on Gh in the telomeric sequence context. These data suggest that one role for Neil3 and NEIL1 is to repair DNA base damages in telomeres in vivo and that Neil3 and Neil1 may function in quadruplex-mediated cellular events, such as gene regulation via removal of damaged bases from quadruplex DNA. PMID:23926102

  18. Inhibition of uracil-DNA glycosylase increases SCEs in BrdU-treated and visible light-irradiated cells

    SciTech Connect

    Maldonado, A.; Hernandez, P.; Gutierrez, C.

    1985-11-01

    The authors have approached the study of the ability of different types of lesions produced by DNA-damaging agents to develop sister-chromatid exchanges (SCEs) by analyzing SCE levels observed in Allium cepa L cells with BrdU-substituted DNA and exposed to visible light (VL), an irradiation which produces uracil residues in DNA after debromination of bromouracil and enhances SCE levels but only above a certain dose. They have partially purified an uracil-DNA glycosylase activity from A. cepa L root meristem cells, which removes uracil from DNA, the first step in the excision repair of this lesion. This enzyme was inhibited in vitro by 6-amino-uracil and uracil but not by thymine. When cells exposed to VL, at a dose that did not produce per se an SCE increase, were immediately post-treated with these inhibitors of uracil-DNA glycosylase, a significant increase in SCE levels was obtained. Moreover, SCE levels in irradiated cells dropped to control level when a short holding time elapsed between exposure to VL and the beginning of post-treatment with the inhibitor. Thus, our results showed that inhibitors of uracil-DNA glycosylase enhanced SCE levels in cells with unifilarly BrdU-substituted DNA exposed to visible light; and indicated the existence of a very rapid repair of SCE-inducing lesions produced by visible light irradiation of cells with unifilarly BrdU-containing DNA.

  19. Structural Features of the Interaction between Human 8-Oxoguanine DNA Glycosylase hOGG1 and DNA

    PubMed Central

    Koval, V. V.; Knorre, D. G.; Fedorova, O. S.

    2014-01-01

    The purpose of the present review is to summarize the data related with the structural features of interaction between the human repair enzyme 8-oxoguanine DNA glycosylase (hOGG1) and DNA. The review covers the questions concerning the role of individual amino acids of hOGG1 in the specific recognition of the oxidized DNA bases, formation of the enzyme–substrate complex, and excision of the lesion bases from DNA. Attention is also focused upon conformational changes in the enzyme active site and disruption of enzyme activity as a result of amino acid mutations. The mechanism of damaged bases release from DNA induced by hOGG1 is discussed in the context of structural dynamics. PMID:25349714

  20. Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases

    PubMed Central

    Nelson, Shane R.; Dunn, Andrew R.; Kathe, Scott D.; Warshaw, David M.; Wallace, Susan S.

    2014-01-01

    DNA glycosylases are enzymes that perform the initial steps of base excision repair, the principal repair mechanism that identifies and removes endogenous damages that occur in an organism’s DNA. We characterized the motion of single molecules of three bacterial glycosylases that recognize oxidized bases, Fpg, Nei, and Nth, as they scan for damages on tightropes of λ DNA. We find that all three enzymes use a key “wedge residue” to scan for damage because mutation of this residue to an alanine results in faster diffusion. Moreover, all three enzymes bind longer and diffuse more slowly on DNA that contains the damages they recognize and remove. Using a sliding window approach to measure diffusion constants and a simple chemomechanical simulation, we demonstrate that these enzymes diffuse along DNA, pausing momentarily to interrogate random bases, and when a damaged base is recognized, they stop to evert and excise it. PMID:24799677

  1. Pre-steady-state kinetics shows differences in processing of various DNA lesions by Escherichia coli formamidopyrimidine-DNA glycosylase

    PubMed Central

    Koval, Vladimir V.; Kuznetsov, Nikita A.; Zharkov, Dmitry O.; Ishchenko, Alexander A.; Douglas, Kenneth T.; Nevinsky, Georgy A.; Fedorova, Olga S.

    2004-01-01

    Formamidopyrimidine-DNA-glycosylase (Fpg pro tein, MutM) catalyses excision of 8-oxoguanine (8-oxoG) and other oxidatively damaged purines from DNA in a glycosylase/apurinic/apyrimidinic-lyase reaction. We report pre-steady-state kinetic analysis of Fpg action on oligonucleotide duplexes containing 8-oxo-2′-deoxyguanosine, natural abasic site or tetrahydrofuran (an uncleavable abasic site analogue). Monitoring Fpg intrinsic tryptophan fluorescence in stopped-flow experiments reveals multiple conformational transitions in the protein molecule during the catalytic cycle. At least four and five conformational transitions occur in Fpg during the interaction with abasic and 8-oxoG-containing substrates, respectively, within 2 ms to 10 s time range. These transitions reflect the stages of enzyme binding to DNA and lesion recognition with the mutual adjustment of DNA and enzyme structures to achieve catalytically competent conformation. Unlike these well-defined binding steps, catalytic stages are not associated with discernible fluorescence events. Only a single conformational change is detected for the cleavable substrates at times exceeding 10 s. The data obtained provide evidence that several fast sequential conformational changes occur in Fpg after binding to its substrate, converting the protein into a catalytically active conformation. PMID:14769949

  2. 8-Oxoguanine DNA Glycosylase (OGG1) Deficiency Increases Susceptibility to Obesity and Metabolic Dysfunction

    PubMed Central

    Sampath, Harini; Vartanian, Vladimir; Rollins, M. Rick; Sakumi, Kunihiko; Nakabeppu, Yusaku; Lloyd, R. Stephen

    2012-01-01

    Oxidative damage to DNA is mainly repaired via base excision repair, a pathway that is catalyzed by DNA glycosylases such as 8-oxoguanine DNA glycosylase (OGG1). While OGG1 has been implicated in maintaining genomic integrity and preventing tumorigenesis, we report a novel role for OGG1 in altering cellular and whole body energy homeostasis. OGG1-deficient (Ogg1−/−) mice have increased adiposity and hepatic steatosis following exposure to a high-fat diet (HFD), compared to wild-type (WT) animals. Ogg1−/− animals also have higher plasma insulin levels and impaired glucose tolerance upon HFD feeding, relative to WT counterparts. Analysis of energy expenditure revealed that HFD-fed Ogg1−/− mice have a higher resting VCO2 and consequently, an increased respiratory quotient during the resting phase, indicating a preference for carbohydrate metabolism over fat oxidation in these mice. Additionally, microarray and quantitative PCR analyses revealed that key genes of fatty acid oxidation, including carnitine palmitoyl transferase-1, and the integral transcriptional co-activator Pgc-1α were significantly downregulated in Ogg1−/− livers. Multiple genes involved in TCA cycle metabolism were also significantly reduced in livers of Ogg1−/− mice. Furthermore, hepatic glycogen stores were diminished, and fasting plasma ketones were significantly reduced in Ogg1−/− mice. Collectively, these data indicate that OGG1 deficiency alters cellular substrate metabolism, favoring a fat sparing phenotype, that results in increased susceptibility to obesity and related pathologies in Ogg1−/− mice. PMID:23284747

  3. Association of the Rad9-Rad1-Hus1 checkpoint clamp with MYH DNA glycosylase and DNA.

    PubMed

    Hwang, Bor-Jang; Jin, Jin; Gunther, Randall; Madabushi, Amrita; Shi, Guoli; Wilson, Gerald M; Lu, A-Lien

    2015-07-01

    Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9-1-1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9-1-1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134-155) is a key determinant of MYH binding. Both the N-(residues 1-146) and C-terminal (residues 147-280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1(K136A) mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad9(1-266) (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 9(1-266)-1-1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 9(1-266)-1-1 to 5'-recessed DNA substrates is an intrinsic property of this complex and is dependent on complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9-1-1 subunits to MYH-directed BER based on subunit asymmetry in protein-protein interactions and DNA binding events. PMID:26021743

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

    PubMed

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

    2015-11-12

    Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. Here we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge-dipole and CH-π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision. PMID:26524531

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  6. A dynamic checkpoint in oxidative lesion discrimination by formamidopyrimidine–DNA glycosylase

    PubMed Central

    Li, Haoquan; Endutkin, Anton V.; Bergonzo, Christina; Campbell, Arthur J.; de los Santos, Carlos; Grollman, Arthur; Zharkov, Dmitry O.; Simmerling, Carlos

    2016-01-01

    In contrast to proteins recognizing small-molecule ligands, DNA-dependent enzymes cannot rely solely on interactions in the substrate-binding centre to achieve their exquisite specificity. It is widely believed that substrate recognition by such enzymes involves a series of conformational changes in the enzyme–DNA complex with sequential gates favoring cognate DNA and rejecting nonsubstrates. However, direct evidence for such mechanism is limited to a few systems. We report that discrimination between the oxidative DNA lesion, 8-oxoguanine (oxoG) and its normal counterpart, guanine, by the repair enzyme, formamidopyrimidine-DNA glycosylase (Fpg), likely involves multiple gates. Fpg uses an aromatic wedge to open the Watson–Crick base pair and everts the lesion into its active site. We used molecular dynamics simulations to explore the eversion free energy landscapes of oxoG and G by Fpg, focusing on structural and energetic details of oxoG recognition. The resulting energy profiles, supported by biochemical analysis of site-directed mutants disturbing the interactions along the proposed path, show that Fpg selectively facilitates eversion of oxoG by stabilizing several intermediate states, helping the rapidly sliding enzyme avoid full extrusion of every encountered base for interrogation. Lesion recognition through multiple gating intermediates may be a common theme in DNA repair enzymes. PMID:26553802

  7. A dynamic checkpoint in oxidative lesion discrimination by formamidopyrimidine-DNA glycosylase.

    PubMed

    Li, Haoquan; Endutkin, Anton V; Bergonzo, Christina; Campbell, Arthur J; de los Santos, Carlos; Grollman, Arthur; Zharkov, Dmitry O; Simmerling, Carlos

    2016-01-29

    In contrast to proteins recognizing small-molecule ligands, DNA-dependent enzymes cannot rely solely on interactions in the substrate-binding centre to achieve their exquisite specificity. It is widely believed that substrate recognition by such enzymes involves a series of conformational changes in the enzyme-DNA complex with sequential gates favoring cognate DNA and rejecting nonsubstrates. However, direct evidence for such mechanism is limited to a few systems. We report that discrimination between the oxidative DNA lesion, 8-oxoguanine (oxoG) and its normal counterpart, guanine, by the repair enzyme, formamidopyrimidine-DNA glycosylase (Fpg), likely involves multiple gates. Fpg uses an aromatic wedge to open the Watson-Crick base pair and everts the lesion into its active site. We used molecular dynamics simulations to explore the eversion free energy landscapes of oxoG and G by Fpg, focusing on structural and energetic details of oxoG recognition. The resulting energy profiles, supported by biochemical analysis of site-directed mutants disturbing the interactions along the proposed path, show that Fpg selectively facilitates eversion of oxoG by stabilizing several intermediate states, helping the rapidly sliding enzyme avoid full extrusion of every encountered base for interrogation. Lesion recognition through multiple gating intermediates may be a common theme in DNA repair enzymes. PMID:26553802

  8. An amplified electrochemical strategy using DNA-QDs dendrimer superstructure for the detection of thymine DNA glycosylase activity.

    PubMed

    Liu, Hongying; Lou, Youbing; Zhou, Fei; Zhu, Hao; Abdel-Halim, E S; Zhu, Jun-Jie

    2015-09-15

    A triple-signal amplification strategy was proposed for highly sensitive and selective detection of thymine DNA glycosylase (TDG) by coupling a dendrimer-like DNA label with the electrochemical method and quantum dots (QDs) tagging. The DNA-QDs dendrimer-like superstructure was designed by DNA hybridization and covalent assembling. Benefiting from outstanding performance of the amplification strategy, this assay showed high sensitivity, extraordinary stability, and easy operation. The limit of detection could reach 0.00003 U µL(-1) with a splendid specificity. The TDG content in different concentration of HeLa cell was also determined. This assay opens a new horizon for both qualitative and quantitative detection of TDG, holding great promise for potential application in cancer cell research and clinical diagnostics. PMID:25913445

  9. Characterization of GM-CSF-inhibitory factor and Uracil DNA glycosylase encoding genes from camel pseudocowpoxvirus.

    PubMed

    Nagarajan, G; Swami, Shelesh Kumar; Dahiya, Shyam Singh; Narnaware, S D; Mehta, S C; Singh, P K; Singh, Raghvendar; Tuteja, F C; Patil, N V

    2015-06-01

    The present study describes the PCR amplification of GM-CSF-inhibitory factor (GIF) and Uracil DNA glycosylase (UDG) encoding genes of pseudocowpoxvirus (PCPV) from the Indian Dromedaries (Camelus dromedarius) infected with contagious ecthyma using the primers based on the corresponding gene sequences of human PCPV and reindeer PCPV, respectively. The length of GIF gene of PCPV obtained from camel is 795 bp and due to the addition of one cytosine residue at position 374 and one adenine residue at position 516, the open reading frame (ORF) got altered, resulting in the production of truncated polypeptide. The ORF of UDG encoding gene of camel PCPV is 696 bp encoding a polypeptide of 26.0 kDa. Comparison of amino acid sequence homologies of GIF and UDG of camel PCPV revealed that the camel PCPV is closer to ORFV and PCPV (reference stains of both human and reindeer), respectively. PMID:25816930

  10. Analysis of substrate specificity of Schizosaccharomyces pombe Mag1 alkylpurine DNA glycosylase

    SciTech Connect

    Adhikary, Suraj; Eichman, Brandt F.

    2014-10-02

    DNA glycosylases specialized for the repair of alkylation damage must identify, with fine specificity, a diverse array of subtle modifications within DNA. The current mechanism involves damage sensing through interrogation of the DNA duplex, followed by more specific recognition of the target base inside the active site pocket. To better understand the physical basis for alkylpurine detection, we determined the crystal structure of Schizosaccharomyces pombe Mag1 (spMag1) in complex with DNA and performed a mutational analysis of spMag1 and the close homologue from Saccharomyces cerevisiae (scMag). Despite strong homology, spMag1 and scMag differ in substrate specificity and cellular alkylation sensitivity, although the enzymological basis for their functional differences is unknown. We show that Mag preference for 1,N{sup 6}-ethenoadenine ({var_epsilon}A) is influenced by a minor groove-interrogating residue more than the composition of the nucleobase-binding pocket. Exchanging this residue between Mag proteins swapped their {var_epsilon}A activities, providing evidence that residues outside the extrahelical base-binding pocket have a role in identification of a particular modification in addition to sensing damage.

  11. Role of uracil-DNA glycosylase in mutation avoidance by Streptococcus pneumoniae

    SciTech Connect

    Chen, Jau-Der; Lacks, S.A. )

    1991-01-01

    Uracil-DNA glycosylase activity was found in Streptococcus pneumoniae, and the enzyme was partially purified. An ung mutant lacking the activity was obtained by positive selection of cells transformed with a plasmid containing uracil in its DNA. The effects of the ung mutation on mutagenic processes in S. pneumoniae were examined. The sequence of several malM mutations revertible by nitrous acid showed them to correspond to A {center dot}T{r arrow}G {center dot} C transitions. This confirmed a prior deduction that nitrous acid action on transforming DNA gave only G {center dot} C{r arrow}A {center dot} T mutations. Examination of malM mutant reversion frequencies in ung strains indicated that G {center dot} C{r arrow}A {center dot} T mutation rates generally were 10-fold higher than in wild-type strains, presumably owing to lack of repair of deaminated cytosine residues in DNA. No effect of ung on mutation avoidance by the Hex mismatch repair system was observed, which means that uracil incorporation and removal from nascent DNA cannot be solely responsible for producing strand breaks that target nascent DNA for correction after replication. One malM mutation corresponding to an A {center dot} T{r arrow}G {center dot} C transition showed a 10-fold-higher spontaneous reversion frequency than other such transitions in a wild-type background. This hot spot was located in a directly repeated DNA sequence; it is proposed that transient slippage to the wild-type repeat during replication accounts for the higher reversion frequency.

  12. A Germline Polymorphism of Thymine DNA Glycosylase Induces Genomic Instability and Cellular Transformation

    PubMed Central

    Sjolund, Ashley; Nemec, Antonia A.; Paquet, Nicolas; Prakash, Aishwarya; Sung, Patrick; Doublié, Sylvie; Sweasy, Joann B.

    2014-01-01

    Thymine DNA glycosylase (TDG) functions in base excision repair, a DNA repair pathway that acts in a lesion-specific manner to correct individual damaged or altered bases. TDG preferentially catalyzes the removal of thymine and uracil paired with guanine, and is also active on 5-fluorouracil (5-FU) paired with adenine or guanine. The rs4135113 single nucleotide polymorphism (SNP) of TDG is found in 10% of the global population. This coding SNP results in the alteration of Gly199 to Ser. Gly199 is part of a loop responsible for stabilizing the flipped abasic nucleotide in the active site pocket. Biochemical analyses indicate that G199S exhibits tighter binding to both its substrate and abasic product. The persistent accumulation of abasic sites in cells expressing G199S leads to the induction of double-strand breaks (DSBs). Cells expressing the G199S variant also activate a DNA damage response. When expressed in cells, G199S induces genomic instability and cellular transformation. Together, these results suggest that individuals harboring the G199S variant may have increased risk for developing cancer. PMID:25375110

  13. Ginsenoside Rd Attenuates DNA Damage by Increasing Expression of DNA Glycosylase Endonuclease VIII-like Proteins after Focal Cerebral Ischemia

    PubMed Central

    Yang, Long-Xiu; Zhang, Xiao; Zhao, Gang

    2016-01-01

    Background: Ginsenoside Rd (GSRd), one of the main active ingredients in traditional Chinese herbal Panax ginseng, has been found to have therapeutic effects on ischemic stroke. However, the molecular mechanisms of GSRd's neuroprotective function remain unclear. Ischemic stroke-induced oxidative stress results in DNA damage, which triggers cell death and contributes to poor prognosis. Oxidative DNA damage is primarily processed by the base excision repair (BER) pathway. Three of the five major DNA glycosylases that initiate the BER pathway in the event of DNA damage from oxidation are the endonuclease VIII-like (NEIL) proteins. This study aimed to investigate the effect of GSRd on the expression of DNA glycosylases NEILs in a rat model of focal cerebral ischemia. Methods: NEIL expression patterns were evaluated by quantitative real-time polymerase chain reaction in both normal and middle cerebral artery occlusion (MCAO) rat models. Survival rate and Zea-Longa neurological scores were used to assess the effect of GSRd administration on MCAO rats. Mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damages were evaluated by the way of real-time analysis of mutation frequency. NEIL expressions were measured in both messenger RNA (mRNA) and protein levels by quantitative polymerase chain reaction and Western blotting analysis. Apoptosis level was quantitated by the expression of cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling assay. Results: We found that GSRd administration reduced mtDNA and nDNA damages, which contributed to an improvement in survival rate and neurological function; significantly up-regulated NEIL1 and NEIL3 expressions in both mRNA and protein levels of MCAO rats; and reduced cell apoptosis and the expression of cleaved caspase-3 in rats at 7 days after MCAO. Conclusions: Our results indicated that the neuroprotective function of GSRd for acute ischemic stroke might be partially explained by the up

  14. Purification and characterization of a novel UV lesion-specific DNA glycosylase/AP lyase from Bacillus sphaericus.

    PubMed

    Vasquez, D A; Nyaga, S G; Lloyd, R S

    2000-05-31

    The purification and characterization of a pyrimidine dimer-specific glycosylase/AP lyase from Bacillus sphaericus (Bsp-pdg) are reported. Bsp-pdg is highly specific for DNA containing the cis-syn cyclobutane pyrimidine dimer, displaying no detectable activity on oligonucleotides with trans-syn I, trans-syn II, (6-4), or Dewar photoproducts. Like other glycosylase/AP lyases that sequentially cleave the N--glycosyl bond of the 5' pyrimidine of a cyclobutane pyrimidine dimer, and the phosphodiester backbone, this enzyme appears to utilize a primary amine as the attacking nucleophile. The formation of a covalent enzyme-DNA imino intermediate is evidenced by the ability to trap this protein-DNA complex by reduction with sodium borohydride. Also consistent with its AP lyase activity, Bsp-pdg was shown to incise an AP site-containing oligonucleotide, yielding beta- and delta-elimination products. N-terminal amino acid sequence analysis of this 26 kDa protein revealed little amino acid homology to any previously reported protein. This is the first report of a glycosylase/AP lyase enzyme from Bacillus sphaericus that is specific for cis-syn pyrimidine dimers. PMID:10844244

  15. Electrostatic Properties of Complexes along a DNA Glycosylase Damage Search Pathway

    PubMed Central

    2015-01-01

    Human uracil DNA glycosylase (hUNG) follows an extended reaction coordinate for locating rare uracil bases in genomic DNA. This process begins with diffusion-controlled engagement of undamaged DNA, followed by a damage search step in which the enzyme remains loosely associated with the DNA chain (translocation), and finally, a recognition step that allows the enzyme to efficiently bind and excise uracil when it is encountered. At each step along this coordinate, the enzyme must form DNA interactions that are highly specialized for either rapid damage searching or catalysis. Here we make extensive measurements of hUNG activity as a function of salt concentration to dissect the thermodynamic, kinetic, and electrostatic properties of key enzyme states along this reaction coordinate. We find that the interaction of hUNG with undamaged DNA is electrostatically driven at a physiological concentration of potassium ions (ΔGelect = −3.5 ± 0.5 kcal mol–1), with only a small nonelectrostatic contribution (ΔGnon = −2.0 ± 0.2 kcal mol–1). In contrast, the interaction with damaged DNA is dominated by the nonelectrostatic free energy term (ΔGnon = −7.2 ± 0.1 kcal mol–1), yet retains the nonspecific electrostatic contribution (ΔGelect = −2.3 ± 0.2 kcal mol–1). Stopped-flow kinetic experiments established that the salt sensitivity of damaged DNA binding originates from a reduction of kon, while koff is weakly dependent on salt. Similar findings were obtained from the salt dependences of the steady-state kinetic parameters, where the diffusion-controlled kcat/Km showed a salt dependence similar to kon, while kcat (limited by product release) was weakly dependent on salt. Finally, the salt dependence of translocation between two uracil sites separated by 20 bp in the same DNA chain was indistinguishable from that of kon. This result suggests that the transition-state for translocation over this spacing resembles that for DNA association from bulk solution and

  16. Electrostatic properties of complexes along a DNA glycosylase damage search pathway.

    PubMed

    Cravens, Shannen L; Hobson, Matthew; Stivers, James T

    2014-12-01

    Human uracil DNA glycosylase (hUNG) follows an extended reaction coordinate for locating rare uracil bases in genomic DNA. This process begins with diffusion-controlled engagement of undamaged DNA, followed by a damage search step in which the enzyme remains loosely associated with the DNA chain (translocation), and finally, a recognition step that allows the enzyme to efficiently bind and excise uracil when it is encountered. At each step along this coordinate, the enzyme must form DNA interactions that are highly specialized for either rapid damage searching or catalysis. Here we make extensive measurements of hUNG activity as a function of salt concentration to dissect the thermodynamic, kinetic, and electrostatic properties of key enzyme states along this reaction coordinate. We find that the interaction of hUNG with undamaged DNA is electrostatically driven at a physiological concentration of potassium ions (ΔGelect = -3.5 ± 0.5 kcal mol(-1)), with only a small nonelectrostatic contribution (ΔGnon = -2.0 ± 0.2 kcal mol(-1)). In contrast, the interaction with damaged DNA is dominated by the nonelectrostatic free energy term (ΔGnon = -7.2 ± 0.1 kcal mol(-1)), yet retains the nonspecific electrostatic contribution (ΔGelect = -2.3 ± 0.2 kcal mol(-1)). Stopped-flow kinetic experiments established that the salt sensitivity of damaged DNA binding originates from a reduction of kon, while koff is weakly dependent on salt. Similar findings were obtained from the salt dependences of the steady-state kinetic parameters, where the diffusion-controlled kcat/Km showed a salt dependence similar to kon, while kcat (limited by product release) was weakly dependent on salt. Finally, the salt dependence of translocation between two uracil sites separated by 20 bp in the same DNA chain was indistinguishable from that of kon. This result suggests that the transition-state for translocation over this spacing resembles that for DNA association from bulk solution and that h

  17. Structural Characterization of Viral Ortholog of Human DNA Glycosylase NEIL1 Bound to Thymine Glycol or 5-Hydroxyuracil-containing DNA*

    PubMed Central

    Imamura, Kayo; Averill, April; Wallace, Susan S.; Doublié, Sylvie

    2012-01-01

    Thymine glycol (Tg) and 5-hydroxyuracil (5-OHU) are common oxidized products of pyrimidines, which are recognized and cleaved by two DNA glycosylases of the base excision repair pathway, endonuclease III (Nth) and endonuclease VIII (Nei). Although there are several structures of Nei enzymes unliganded or bound to an abasic (apurinic or apyrimidinic) site, until now there was no structure of an Nei bound to a DNA lesion. Mimivirus Nei1 (MvNei1) is an ortholog of human NEIL1, which was previously crystallized bound to DNA containing an apurinic site (Imamura, K., Wallace, S. S., and Doublié, S. (2009) J. Biol. Chem. 284, 26174–26183). Here, we present two crystal structures of MvNei1 bound to two oxidized pyrimidines, Tg and 5-OHU. Both lesions are flipped out from the DNA helix. Tg is in the anti conformation, whereas 5-OHU adopts both anti and syn conformations in the glycosylase active site. Only two protein side chains (Glu-6 and Tyr-253) are within hydrogen-bonding contact with either damaged base, and mutating these residues did not markedly affect the glycosylase activity. This finding suggests that lesion recognition by Nei occurs before the damaged base flips into the glycosylase active site. PMID:22170059

  18. ATM regulates 3-Methylpurine-DNA glycosylase and promotes therapeutic resistance to alkylating agents

    PubMed Central

    Agnihotri, Sameer; Burrell, Kelly; Buczkowicz, Pawel; Remke, Marc; Golbourn, Brian; Chornenkyy, Yevgen; Gajadhar, Aaron; Fernandez, Nestor A.; Clarke, Ian D.; Barszczyk, Mark S.; Pajovic, Sanja; Ternamian, Christian; Head, Renee; Sabha, Nesrin; Sobol, Robert W.; Taylor, Michael D; Rutka, James T.; Jones, Chris; Dirks, Peter B.; Zadeh, Gelareh; Hawkins, Cynthia

    2014-01-01

    Alkylating agents are a frontline therapy for the treatment of several aggressive cancers including pediatric glioblastoma, a lethal tumor in children. Unfortunately, many tumors are resistant to this therapy. We sought to identify ways of sensitizing tumor cells to alkylating agents while leaving normal cells unharmed; increasing therapeutic response while minimizing toxicity. Using a siRNA screen targeting over 240 DNA damage response genes, we identified novel sensitizers to alkylating agents. In particular the base excision repair (BER) pathway, including 3-methylpurine-DNA glycosylase (MPG), as well as ataxia telangiectasia mutated (ATM) were identified in our screen. Interestingly, we identified MPG as a direct novel substrate of ATM. ATM-mediated phosphorylation of MPG was required for enhanced MPG function. Importantly, combined inhibition or loss of MPG and ATM resulted in increased alkylating agent-induced cytotoxicity in vitro and prolonged survival in vivo. The discovery of the ATM-MPG axis will lead to improved treatment of alkylating agent-resistant tumors. PMID:25100205

  19. Kinetics of substrate recognition and cleavage by human 8-oxoguanine-DNA glycosylase

    PubMed Central

    Kuznetsov, Nikita A.; Koval, Vladimir V.; Zharkov, Dmitry O.; Nevinsky, Georgy A.; Douglas, Kenneth T.; Fedorova, Olga S.

    2005-01-01

    Human 8-oxoguanine-DNA glycosylase (hOgg1) excises 8-oxo-7,8-dihydroguanine (8-oxoG) from damaged DNA. We report a pre-steady-state kinetic analysis of hOgg1 mechanism using stopped-flow and enzyme fluorescence monitoring. The kinetic scheme for hOgg1 processing an 8-oxoG:C-containing substrate was found to include at least three fast equilibrium steps followed by two slow, irreversible steps and another equilibrium step. The second irreversible step was rate-limiting overall. By comparing data from Ogg1 intrinsic fluorescence traces and from accumulation of products of different types, the irreversible steps were attributed to two main chemical steps of the Ogg1-catalyzed reaction: cleavage of the N-glycosidic bond of the damaged nucleotide and β-elimination of its 3′-phosphate. The fast equilibrium steps were attributed to enzyme conformational changes during the recognition of 8-oxoG, and the final equilibrium, to binding of the reaction product by the enzyme. hOgg1 interacted with a substrate containing an aldehydic AP site very slowly, but the addition of 8-bromoguanine (8-BrG) greatly accelerated the reaction, which was best described by two initial equilibrium steps followed by one irreversible chemical step and a final product release equilibrium step. The irreversible step may correspond to β-elimination since it is the very step facilitated by 8-BrG. PMID:16024742

  20. Protein p56 from the Bacillus subtilis phage ϕ29 inhibits DNA-binding ability of uracil-DNA glycosylase

    PubMed Central

    Serrano-Heras, Gemma; Ruiz-Masó, José A.; del Solar, Gloria; Espinosa, Manuel; Bravo, Alicia; Salas, Margarita

    2007-01-01

    Protein p56 (56 amino acids) from the Bacillus subtilis phage ϕ29 inactivates the host uracil-DNA glycosylase (UDG), an enzyme involved in the base excision repair pathway. At present, p56 is the only known example of a UDG inhibitor encoded by a non-uracil containing viral DNA. Using analytical ultracentrifugation methods, we found that protein p56 formed dimers at physiological concentrations. In addition, circular dichroism spectroscopic analyses revealed that protein p56 had a high content of β-strands (around 40%). To understand the mechanism underlying UDG inhibition by p56, we carried out in vitro experiments using the Escherichia coli UDG enzyme. The highly acidic protein p56 was able to compete with DNA for binding to UDG. Moreover, the interaction between p56 and UDG blocked DNA binding by UDG. We also demonstrated that Ugi, a protein that interacts with the DNA-binding domain of UDG, was able to replace protein p56 previously bound to the UDG enzyme. These results suggest that protein p56 could be a novel naturally occurring DNA mimicry. PMID:17698500

  1. Reaction intermediates in the catalytic mechanism of Escherichia coli MutY DNA glycosylase.

    PubMed

    Manuel, Raymond C; Hitomi, Kenichi; Arvai, Andrew S; House, Paul G; Kurtz, Andrew J; Dodson, M L; McCullough, Amanda K; Tainer, John A; Lloyd, R Stephen

    2004-11-01

    The Escherichia coli adenine DNA glycosylase, MutY, plays an important role in the maintenance of genomic stability by catalyzing the removal of adenine opposite 8-oxo-7,8-dihydroguanine or guanine in duplex DNA. Although the x-ray crystal structure of the catalytic domain of MutY revealed a mechanism for catalysis of the glycosyl bond, it appeared that several opportunistically positioned lysine side chains could participate in a secondary beta-elimination reaction. In this investigation, it is established via site-directed mutagenesis and the determination of a 1.35-A structure of MutY in complex with adenine that the abasic site (apurinic/apyrimidinic) lyase activity is alternatively regulated by two lysines, Lys142 and Lys20. Analyses of the crystallographic structure also suggest a role for Glu161 in the apurinic/apyrimidinic lyase chemistry. The beta-elimination reaction is structurally and chemically uncoupled from the initial glycosyl bond scission, indicating that this reaction occurs as a consequence of active site plasticity and slow dissociation of the product complex. MutY with either the K142A or K20A mutation still catalyzes beta and beta-delta elimination reactions, and both mutants can be trapped as covalent enzyme-DNA intermediates by chemical reduction. The trapping was observed to occur both pre- and post-phosphodiester bond scission, establishing that both of these intermediates have significant half-lives. Thus, the final spectrum of DNA products generated reflects the outcome of a delicate balance of closely related equilibrium constants. PMID:15326180

  2. Asbestos-induced pulmonary fibrosis is augmented in 8-oxoguanine DNA glycosylase knockout mice.

    PubMed

    Cheresh, Paul; Morales-Nebreda, Luisa; Kim, Seok-Jo; Yeldandi, Anjana; Williams, David B; Cheng, Yuan; Mutlu, Gökhan M; Budinger, G R Scott; Ridge, Karen; Schumacker, Paul T; Bohr, Vilhelm A; Kamp, David W

    2015-01-01

    Asbestos causes asbestosis and malignancies by mechanisms that are not fully established. Alveolar epithelial cell (AEC) injury and repair are crucial determinants of the fibrogenic potential of noxious agents such as asbestos. We previously showed that mitochondrial reactive oxygen species mediate asbestos-induced AEC intrinsic apoptosis and that mitochondrial human 8-oxoguanine-DNA glycosylase 1 (OGG1), a DNA repair enzyme, prevents oxidant-induced AEC apoptosis. We reasoned that OGG1 deficiency augments asbestos-induced pulmonary fibrosis. Compared with intratracheal instillation of PBS (50 μl) or titanium dioxide (100 μg/50 μl), crocidolite or Libby amphibole asbestos (100 μg/50 μl) each augmented pulmonary fibrosis in wild-type C57BL/6J (WT) mice after 3 weeks as assessed by histology, fibrosis score, lung collagen via Sircol, and type 1 collagen expression; these effects persisted at 2 months. Compared with WT mice, Ogg1 homozygous knockout (Ogg1(-/-)) mice exhibit increased pulmonary fibrosis after crocidolite exposure and apoptosis in cells at the bronchoalveolar duct junctions as assessed via cleaved caspase-3 immunostaining. AEC involvement was verified by colocalization studies using surfactant protein C. Asbestos increased endoplasmic reticulum stress in the lungs of WT and Ogg1(-/-) mice. Compared with WT, alveolar type 2 cells isolated from Ogg1(-/-) mice have increased mtDNA damage, reduced mitochondrial aconitase expression, and increased P53 and cleaved caspase-9 expression, and these changes were enhanced 3 weeks after crocidolite exposure. These findings suggest an important role for AEC mtDNA integrity maintained by OGG1 in the pathogenesis of pulmonary fibrosis that may represent a novel therapeutic target. PMID:24918270

  3. Oxidized dNTPs and the OGG1 and MUTYH DNA glycosylases combine to induce CAG/CTG repeat instability

    PubMed Central

    Cilli, Piera; Ventura, Ilenia; Minoprio, Anna; Meccia, Ettore; Martire, Alberto; Wilson, Samuel H.; Bignami, Margherita; Mazzei, Filomena

    2016-01-01

    DNA trinucleotide repeat (TNR) expansion underlies several neurodegenerative disorders including Huntington's disease (HD). Accumulation of oxidized DNA bases and their inefficient processing by base excision repair (BER) are among the factors suggested to contribute to TNR expansion. In this study, we have examined whether oxidation of the purine dNTPs in the dNTP pool provides a source of DNA damage that promotes TNR expansion. We demonstrate that during BER of 8-oxoguanine (8-oxodG) in TNR sequences, DNA polymerase β (POL β) can incorporate 8-oxodGMP with the formation of 8-oxodG:C and 8-oxodG:A mispairs. Their processing by the OGG1 and MUTYH DNA glycosylases generates closely spaced incisions on opposite DNA strands that are permissive for TNR expansion. Evidence in HD model R6/2 mice indicates that these DNA glycosylases are present in brain areas affected by neurodegeneration. Consistent with prevailing oxidative stress, the same brain areas contained increased DNA 8-oxodG levels and expression of the p53-inducible ribonucleotide reductase. Our in vitro and in vivo data support a model where an oxidized dNTPs pool together with aberrant BER processing contribute to TNR expansion in non-replicating cells. PMID:26980281

  4. Oxidized dNTPs and the OGG1 and MUTYH DNA glycosylases combine to induce CAG/CTG repeat instability.

    PubMed

    Cilli, Piera; Ventura, Ilenia; Minoprio, Anna; Meccia, Ettore; Martire, Alberto; Wilson, Samuel H; Bignami, Margherita; Mazzei, Filomena

    2016-06-20

    DNA trinucleotide repeat (TNR) expansion underlies several neurodegenerative disorders including Huntington's disease (HD). Accumulation of oxidized DNA bases and their inefficient processing by base excision repair (BER) are among the factors suggested to contribute to TNR expansion. In this study, we have examined whether oxidation of the purine dNTPs in the dNTP pool provides a source of DNA damage that promotes TNR expansion. We demonstrate that during BER of 8-oxoguanine (8-oxodG) in TNR sequences, DNA polymerase β (POL β) can incorporate 8-oxodGMP with the formation of 8-oxodG:C and 8-oxodG:A mispairs. Their processing by the OGG1 and MUTYH DNA glycosylases generates closely spaced incisions on opposite DNA strands that are permissive for TNR expansion. Evidence in HD model R6/2 mice indicates that these DNA glycosylases are present in brain areas affected by neurodegeneration. Consistent with prevailing oxidative stress, the same brain areas contained increased DNA 8-oxodG levels and expression of the p53-inducible ribonucleotide reductase. Our in vitro and in vivo data support a model where an oxidized dNTPs pool together with aberrant BER processing contribute to TNR expansion in non-replicating cells. PMID:26980281

  5. Folate Deficiency Induces Neurodegeneration and Brain Dysfunction in Mice Lacking Uracil DNA Glycosylase

    PubMed Central

    Kronenberg, Golo; Harms, Christoph; Sobol, Robert W.; Cardozo-Pelaez, Fernando; Linhart, Heinz; Winter, Benjamin; Balkaya, Mustafa; Gertz, Karen; Gay, Shanna B.; Cox, David; Eckart, Sarah; Ahmadi, Michael; Juckel, Georg; Kempermann, Gerd; Hellweg, Rainer; Sohr, Reinhard; Hörtnagl, Heide; Wilson, Samuel H.; Jaenisch, Rudolf

    2008-01-01

    Folate deficiency and resultant increased homocysteine levels have been linked experimentally and epidemiologically with neurodegenerative conditions like stroke and dementia. Moreover, folate deficiency has been implicated in the pathogenesis of psychiatric disorders, most notably depression. We hypothesized that the pathogenic mechanisms include uracil misincorporation and, therefore, analyzed the effects of folate deficiency in mice lacking uracil DNA glycosylase (Ung−/−) versus wild-type controls. Folate depletion increased nuclear mutation rates in Ung−/− embryonic fibroblasts, and conferred death of cultured Ung−/− hippocampal neurons. Feeding animals a folate-deficient diet (FD) for 3 months induced degeneration of CA3 pyramidal neurons in Ung−/− but not Ung+/+ mice along with decreased hippocampal expression of brain-derived neurotrophic factor protein and decreased brain levels of antioxidant glutathione. Furthermore, FD induced cognitive deficits and mood alterations such as anxious and despair-like behaviors that were aggravated in Ung−/− mice. Independent of Ung genotype, FD increased plasma homocysteine levels, altered brain monoamine metabolism, and inhibited adult hippocampal neurogenesis. These results indicate that impaired uracil repair is involved in neurodegeneration and neuropsychiatric dysfunction induced by experimental folate deficiency. PMID:18614692

  6. The DNA glycosylases OGG1 and NEIL3 influence differentiation potential, proliferation, and senescence-associated signs in neural stem cells

    SciTech Connect

    Reis, Amilcar; Hermanson, Ola

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer DNA glycosylases OGG1 and NEIL3 are required for neural stem cell state. Black-Right-Pointing-Pointer No effect on cell viability by OGG1 or NEIL3 knockdown in neural stem cells. Black-Right-Pointing-Pointer OGG1 or NEIL3 RNA knockdown result in decreased proliferation and differentiation. Black-Right-Pointing-Pointer Increased HP1{gamma} immunoreactivity after NEIL3 knockdown suggests premature senescence. -- Abstract: Embryonic neural stem cells (NSCs) exhibit self-renewal and multipotency as intrinsic characteristics that are key parameters for proper brain development. When cells are challenged by oxidative stress agents the resulting DNA lesions are repaired by DNA glycosylases through the base excision repair (BER) pathway as a means to maintain the fidelity of the genome, and thus, proper cellular characteristics. The functional roles for DNA glycosylases in NSCs have however remained largely unexplored. Here we demonstrate that RNA knockdown of the DNA glycosylases OGG1 and NEIL3 decreased NSC differentiation ability and resulted in decreased expression of both neuronal and astrocytic genes after mitogen withdrawal, as well as the stem cell marker Musashi-1. Furthermore, while cell survival remained unaffected, NEIL3 deficient cells displayed decreased cell proliferation rates along with an increase in HP1{gamma} immunoreactivity, a sign of premature senescence. Our results suggest that DNA glycosylases play multiple roles in governing essential neural stem cell characteristics.

  7. Uracil DNA Glycosylase BKRF3 Contributes to Epstein-Barr Virus DNA Replication through Physical Interactions with Proteins in Viral DNA Replication Complex

    PubMed Central

    Su, Mei-Tzu; Liu, I-Hua; Wu, Chia-Wei; Chang, Shu-Ming; Tsai, Ching-Hwa; Yang, Pei-Wen; Chuang, Yu-Chia; Lee, Chung-Pei

    2014-01-01

    ABSTRACT Epstein-Barr virus (EBV) BKRF3 shares sequence homology with members of the uracil-N-glycosylase (UNG) protein family and has DNA glycosylase activity. Here, we explored how BKRF3 participates in the DNA replication complex and contributes to viral DNA replication. Exogenously expressed Flag-BKRF3 was distributed mostly in the cytoplasm, whereas BKRF3 was translocated into the nucleus and colocalized with the EBV DNA polymerase BALF5 in the replication compartment during EBV lytic replication. The expression level of BKRF3 increased gradually during viral replication, coupled with a decrease of cellular UNG2, suggesting BKRF3 enzyme activity compensates for UNG2 and ensures the fidelity of viral DNA replication. In immunoprecipitation-Western blotting, BKRF3 was coimmunoprecipitated with BALF5, the polymerase processivity factor BMRF1, and the immediate-early transactivator Rta. Coexpression of BMRF1 appeared to facilitate the nuclear targeting of BKRF3 in immunofluorescence staining. Residues 164 to 255 of BKRF3 were required for interaction with Rta and BALF5, whereas residues 81 to 166 of BKRF3 were critical for BMRF1 interaction in glutathione S-transferase (GST) pulldown experiments. Viral DNA replication was defective in cells harboring BKRF3 knockout EBV bacmids. In complementation assays, the catalytic mutant BKRF3(Q90L,D91N) restored viral DNA replication, whereas the leucine loop mutant BKRF3(H213L) only partially rescued viral DNA replication, coupled with a reduced ability to interact with the viral DNA polymerase and Rta. Our data suggest that BKRF3 plays a critical role in viral DNA synthesis predominantly through its interactions with viral proteins in the DNA replication compartment, while its enzymatic activity may be supplementary for uracil DNA glycosylase (UDG) function during virus replication. IMPORTANCE Catalytic activities of both cellular UDG UNG2 and viral UDGs contribute to herpesviral DNA replication. To ensure that the enzyme

  8. DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations

    PubMed Central

    Fortes, F.P.; Kuasne, H.; Marchi, F.A.; Miranda, P.M.; Rogatto, S.R.; Achatz, M.I.

    2015-01-01

    Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG) pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17) in 30 patients with germline TP53mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR) with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results. PMID:25945745

  9. DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations.

    PubMed

    Fortes, F P; Kuasne, H; Marchi, F A; Miranda, P M; Rogatto, S R; Achatz, M I

    2015-07-01

    Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG) pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17) in 30 patients with germline TP53 mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR) with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results. PMID:25945745

  10. Electrostatic interactions play an essential role in DNA repair and cold-adaptation of uracil DNA glycosylase.

    PubMed

    Olufsen, Magne; Smalås, Arne O; Brandsdal, Bjørn O

    2008-03-01

    Life has adapted to most environments on earth, including low and high temperature niches. The increased catalytic efficiency and thermoliability observed for enzymes from organisms living in constantly cold regions when compared to their mesophilic and thermophilic cousins are poorly understood at the molecular level. Uracil DNA glycosylase (UNG) from cod (cUNG) catalyzes removal of uracil from DNA with an increased k(cat) and reduced K(m) relative to its warm-active human (hUNG) counterpart. Specific issues related to DNA repair and substrate binding/recognition (K(m)) are here investigated by continuum electrostatics calculations, MD simulations and free energy calculations. Continuum electrostatic calculations reveal that cUNG has surface potentials that are more complementary to the DNA potential at and around the catalytic site when compared to hUNG, indicating improved substrate binding. Comparative MD simulations combined with free energy calculations using the molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) method show that large opposing energies are involved when forming the enzyme-substrate complexes. Furthermore, the binding free energies obtained reveal that the Michaelis-Menten complex is more stable for cUNG, primarily due to enhanced electrostatic properties, suggesting that energetic fine-tuning of electrostatics can be utilized for enzymatic temperature adaptation. Energy decomposition pinpoints the residual determinants responsible for this adaptation. PMID:18196298

  11. Uracil DNa-glycosylase from HeLa cells: general properties, substrate specificity and effect of uracil analogs.

    PubMed

    Krokan, H; Wittwer, C U

    1981-06-11

    Uracil-DNA glycosylase was partially purified from HeLa cells. Various substrates containing [3H]dUMP residues were prepared by nick-translation of calf thymus DNA. The standard substrate was double-stranded DNA with [3H]dUMP located internally in the chain. Compared to the release of uracil from this substrate, a 3-fold increase in the rate was seen with single-stranded DNA, and a 20-fold reduction in the rate was observed when the [3H]dUMP-residue was located at the 3'end. The rate of [3H]uracil release decreased progressively when one, two or three of the dNMP residues were replaced by the corresponding rNMP; in the extreme case when the substrate contained [3H]dUMP in addition to rCMP, rGMP, and rAMP, the rate of [3H]uracil release was less than 3% of that of the control. The enzyme was inhibited to the same extent by uracil and the uracil analogs 6-aminouracil and 5-azauracil, but very weakly, or not at all, by 5 other analogs. Our results suggest strongly that uracil-DNA glycosylase has a high degree of selectivity for uracil in dUMP residues located internally in DNA chains and that the recognition of the correct substrate also depends on the residues flanking dUMP being deoxyribonucleotides. PMID:7279657

  12. The versatile thymine DNA-glycosylase: a comparative characterization of the human, Drosophila and fission yeast orthologs.

    PubMed

    Hardeland, Ulrike; Bentele, Marc; Jiricny, Josef; Schär, Primo

    2003-05-01

    Human thymine-DNA glycosylase (TDG) is well known to excise thymine and uracil from G.T and G.U mismatches, respectively, and was therefore proposed to play a central role in the cellular defense against genetic mutation through spontaneous deamination of 5-methylcytosine and cytosine. In this study, we characterized two newly discovered orthologs of TDG, the Drosophila melanogaster Thd1p and the Schizosaccharomyces pombe Thp1p proteins, with an objective to address the function of this subfamily of uracil-DNA glycosylases from an evolutionary perspective. A systematic biochemical comparison of both enzymes with human TDG revealed a number of biologically significant facts. (i) All eukaryotic TDG orthologs have broad and species-specific substrate spectra that include a variety of damaged pyrimidine and purine bases; (ii) the common most efficiently processed substrates of all are uracil and 3,N4- ethenocytosine opposite guanine and 5-fluorouracil in any double-stranded DNA context; (iii) 5-methylcytosine and thymine derivatives are processed with an appreciable efficiency only by the human and the Drosophila enzymes; (iv) none of the proteins is able to hydrolyze a non-damaged 5'-methylcytosine opposite G; and (v) the double strand and mismatch dependency of the enzymes varies with the substrate and is not a stringent feature of this subfamily of DNA glycosylases. These findings advance our current view on the role of TDG proteins and document that they have evolved with high structural flexibility to counter a broad range of DNA base damage in accordance with the specific needs of individual species. PMID:12711670

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

    PubMed Central

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

    2016-01-01

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

  14. Expansion Mechanisms and Evolutionary History on Genes Encoding DNA Glycosylases and Their Involvement in Stress and Hormone Signaling

    PubMed Central

    Jiang, Shu-Ye; Ramachandran, Srinivasan

    2016-01-01

    DNA glycosylases catalyze the release of methylated bases. They play vital roles in the base excision repair pathway and might also function in DNA demethylation. At least three families of DNA glycosylases have been identified, which included 3′-methyladenine DNA glycosylase (MDG) I, MDG II, and HhH-GPD (Helix–hairpin–Helix and Glycine/Proline/aspartate (D)). However, little is known on their genome-wide identification, expansion, and evolutionary history as well as their expression profiling and biological functions. In this study, we have genome-widely identified and evolutionarily characterized these family members. Generally, a genome encodes only one MDG II gene in most of organisms. No MDG I or MDG II gene was detected in green algae. However, HhH-GPD genes were detectable in all available organisms. The ancestor species contain small size of MDG I and HhH-GPD families. These two families were mainly expanded through the whole-genome duplication and segmental duplication. They were evolutionarily conserved and were generally under purifying selection. However, we have detected recent positive selection among the Oryza genus, which might play roles in species divergence. Further investigation showed that expression divergence played important roles in gene survival after expansion. All of these family genes were expressed in most of developmental stages and tissues in rice plants. High ratios of family genes were downregulated by drought and fungus pathogen as well as abscisic acid (ABA) and jasmonic acid (JA) treatments, suggesting a negative regulation in response to drought stress and pathogen infection through ABA- and/or JA-dependent hormone signaling pathway. PMID:27026054

  15. Structure of Escherichia coli AlkA in Complex with Undamaged DNA

    SciTech Connect

    Bowman, Brian R.; Lee, Seongmin; Wang, Shuyu; Verdine, Gregory L

    2010-11-22

    Because DNA damage is so rare, DNA glycosylases interact for the most part with undamaged DNA. Whereas the structural basis for recognition of DNA lesions by glycosylases has been studied extensively, less is known about the nature of the interaction between these proteins and undamaged DNA. Here we report the crystal structures of the DNA glycosylase AlkA in complex with undamaged DNA. The structures revealed a recognition mode in which the DNA is nearly straight, with no amino acid side chains inserted into the duplex, and the target base pair is fully intrahelical. A comparison of the present structures with that of AlkA recognizing an extrahelical lesion revealed conformational changes in both the DNA and protein as the glycosylase transitions from the interrogation of undamaged DNA to catalysis of nucleobase excision. Modeling studies with the cytotoxic lesion 3-methyladenine and accompanying biochemical experiments suggested that AlkA actively interrogates the minor groove of the DNA while probing for the presence of lesions.

  16. Sequence-dependent Structural Variation in DNA Undergoing Intrahelical Inspection by the DNA glycosylase MutM

    SciTech Connect

    Sung, Rou-Jia; Zhang, Michael; Qi, Yan; Verdine, Gregory L.

    2012-08-31

    MutM, a bacterial DNA-glycosylase, plays a critical role in maintaining genome integrity by catalyzing glycosidic bond cleavage of 8-oxoguanine (oxoG) lesions to initiate base excision DNA repair. The task faced by MutM of locating rare oxoG residues embedded in an overwhelming excess of undamaged bases is especially challenging given the close structural similarity between oxoG and its normal progenitor, guanine (G). MutM actively interrogates the DNA to detect the presence of an intrahelical, fully base-paired oxoG, whereupon the enzyme promotes extrusion of the target nucleobase from the DNA duplex and insertion into the extrahelical active site. Recent structural studies have begun to provide the first glimpse into the protein-DNA interactions that enable MutM to distinguish an intrahelical oxoG from G; however, these initial studies left open the important question of how MutM can recognize oxoG residues embedded in 16 different neighboring sequence contexts (considering only the 5'- and 3'-neighboring base pairs). In this study we set out to understand the manner and extent to which intrahelical lesion recognition varies as a function of the 5'-neighbor. Here we report a comprehensive, systematic structural analysis of the effect of the 5'-neighboring base pair on recognition of an intrahelical oxoG lesion. These structures reveal that MutM imposes the same extrusion-prone ('extrudogenic') backbone conformation on the oxoG lesion irrespective of its 5'-neighbor while leaving the rest of the DNA relatively free to adjust to the particular demands of individual sequences.

  17. A human nuclear uracil DNA glycosylase is the 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase

    SciTech Connect

    Meyer-Siegler, K.; Mauro, D.J.; Seal, G.; Wurzer, J.; DeRiel, J.K.; Sirover, M.A. )

    1991-10-01

    The authors have isolated and characterized a plasmid (pChug 20.1) that contains the cDNA of a nuclear uracil DNA glycosylase (UDG) gene isolated from normal human placenta. This cDNA directed the synthesis of a fusion protein that exhibited UDG activity. The enzymatic activity was specific for a uracil-containing polynucleotide substrate and was inhibited by a glycosylase antibody or a {beta}-galactosidase antibody. Sequence analysis demonstrated an open reading frame that encoded a protein of 335 amino acids of calculated M{sub r} 36,050 and pI 8.7, corresponding to the M{sub r} 37,000 and pI 8.1 of purified human placental UDG. Surprisingly, a search of the GenBank data base revealed that the cDNA of UDG was completely homologous with the 378-kDa subunit of human glyceraldehyde-3-phosphate dehydrogenase. Human erythrocyte glyceraldehyde-3-phosphate dehydrogenase was obtained commercially in its tetrameric form. A 37-kDa subunit was isolated form it and shown to possess UDG activity equivalent to that seen for the purified human placental UDG. The multiple functions of this 37-kDa protein as here and previously reported indicate that it possesses a series of activities, depending on its oligomeric state. Accordingly, mutation(s) in the gene of this multifunctional protein may conceivably result in the diverse cellular phenotypes of Bloom syndrome.

  18. The C-terminal Lysine of Ogg2 DNA Glycosylases is a Major Molecular Determinant for Guanine/8-Oxoguanine Distinction

    SciTech Connect

    Faucher, Frédérick; Wallace, Susan S.; Doublié, Sylvie

    2010-08-12

    7,8-Dihydro-8-oxoguanine (8-oxoG) is a major oxidative lesion found in DNA. The 8-oxoguanine DNA glycosylases (Ogg) responsible for the removal of 8-oxoG are divided into three families Ogg1, Ogg2 and AGOG. The Ogg2 members are devoid of the recognition loop used by Ogg1 to discriminate between 8-oxoG and guanine and it was unclear until recently how Ogg2 enzymes recognize the oxidized base. We present here the first crystallographic structure of an Ogg2 member, Methanocaldococcus janischii Ogg, in complex with a DNA duplex containing the 8-oxoG lesion. This structure highlights the crucial role of the C-terminal lysine, strictly conserved in Ogg2, in the recognition of 8-oxoG. The structure also reveals that Ogg2 undergoes a conformational change upon DNA binding similar to that observed in Ogg1 glycosylases. Furthermore, this work provides a structural rationale for the lack of opposite base specificity in this family of enzymes.

  19. 8-oxoguanine DNA glycosylase 1-deficiency modifies allergic airway inflammation by regulating STAT6 and IL-4 in cells and in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: 8-oxoguanine-DNA glycosylase (OGG-1) is an enzyme involved in DNA repair. OGG-1 has a potential role in regulating inflammation but its function in modulating allergic diseases remains undefined. Objectives: To investigate the role of OGG-1 in mediating allergic inflammation, we used OGG...

  20. Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration

    PubMed Central

    Weil, Amy F.; Ghosh, Devlina; Zhou, Yan; Seiple, Lauren; McMahon, Moira A.; Spivak, Adam M.; Siliciano, Robert F.; Stivers, James T.

    2013-01-01

    HIV-1 reverse transcriptase discriminates poorly between dUTP and dTTP, and accordingly, viral DNA products become heavily uracilated when viruses infect host cells that contain high ratios of dUTP:dTTP. Uracilation of invading retroviral DNA is thought to be an innate immunity barrier to retroviral infection, but the mechanistic features of this immune pathway and the cellular fate of uracilated retroviral DNA products is not known. Here we developed a model system in which the cellular dUTP:dTTP ratio can be pharmacologically increased to favor dUTP incorporation, allowing dissection of this innate immunity pathway. When the virus-infected cells contained elevated dUTP levels, reverse transcription was found to proceed unperturbed, but integration and viral protein expression were largely blocked. Furthermore, successfully integrated proviruses lacked detectable uracil, suggesting that only nonuracilated viral DNA products were integration competent. Integration of the uracilated proviruses was restored using an isogenic cell line that had no detectable human uracil DNA glycosylase (hUNG2) activity, establishing that hUNG2 is a host restriction factor in cells that contain high dUTP. Biochemical studies in primary cells established that this immune pathway is not operative in CD4+ T cells, because these cells have high dUTPase activity (low dUTP), and only modest levels of hUNG activity. Although monocyte-derived macrophages have high dUTP levels, these cells have low hUNG activity, which may diminish the effectiveness of this restriction pathway. These findings establish the essential elements of this pathway and reconcile diverse observations in the literature. PMID:23341616

  1. Uracil DNA glycosylase (UNG) loss enhances DNA double strand break formation in human cancer cells exposed to pemetrexed

    PubMed Central

    Weeks, L D; Zentner, G E; Scacheri, P C; Gerson, S L

    2014-01-01

    Misincorporation of genomic uracil and formation of DNA double strand breaks (DSBs) are known consequences of exposure to TS inhibitors such as pemetrexed. Uracil DNA glycosylase (UNG) catalyzes the excision of uracil from DNA and initiates DNA base excision repair (BER). To better define the relationship between UNG activity and pemetrexed anticancer activity, we have investigated DNA damage, DSB formation, DSB repair capacity, and replication fork stability in UNG+/+ and UNG−/− cells. We report that despite identical growth rates and DSB repair capacities, UNG−/− cells accumulated significantly greater uracil and DSBs compared with UNG+/+ cells when exposed to pemetrexed. ChIP-seq analysis of γ-H2AX enrichment confirmed fewer DSBs in UNG+/+ cells. Furthermore, DSBs in UNG+/+ and UNG−/− cells occur at distinct genomic loci, supporting differential mechanisms of DSB formation in UNG-competent and UNG-deficient cells. UNG−/− cells also showed increased evidence of replication fork instability (PCNA dispersal) when exposed to pemetrexed. Thymidine co-treatment rescues S-phase arrest in both UNG+/+ and UNG−/− cells treated with IC50-level pemetrexed. However, following pemetrexed exposure, UNG−/− but not UNG+/+ cells are refractory to thymidine rescue, suggesting that deficient uracil excision rather than dTTP depletion is the barrier to cell cycle progression in UNG−/− cells. Based on these findings we propose that pemetrexed-induced uracil misincorporation is genotoxic, contributing to replication fork instability, DSB formation and ultimately cell death. PMID:24503537

  2. Label-free fluorescence turn-on detection of uracil DNA glycosylase activity based on G-quadruplex formation.

    PubMed

    Ma, Changbei; Wu, Kefeng; Liu, Haisheng; Xia, Kun; Wang, Kemin; Wang, Jun

    2016-11-01

    We have developed a new methodology for fluorescence turn-on detection of uracil DNA glycosylase (UDG) activity based on G-quadruplex formation using a thioflavin T probe. In the presence of UDG, it catalyzed the hydrolysis of the uracil bases in the duplex DNA, resulting in the dissociation of the duplex DNA owing to their low melting temperature. Then, the probe DNA can be recognized quickly by the ThT dye and resulting in an increase in fluorescence. This approach is highly selective and sensitive with a detection limit of 0.01U/mL. It is simple and cost effective without requirement of labeling with a fluorophore-quencher pair. This new method could be used to evaluate the inhibition effect of 5-fluorouracil on UDG activity, and become a useful tool in biomedical research. PMID:27591637

  3. N-methylpurine DNA glycosylase overexpression increases alkylation sensitivity by rapidly removing non-toxic 7-methylguanine adducts

    PubMed Central

    Rinne, M. L.; He, Y.; Pachkowski, B. F.; Nakamura, J.; Kelley, M. R.

    2005-01-01

    Previous studies indicate that overexpression of N-methylpurine DNA glycosylase (MPG) dramatically sensitizes cells to alkylating agent-induced cytotoxicity. We recently demonstrated that this sensitivity is preceded by an increased production of AP sites and strand breaks, confirming that overexpression of MPG disrupts normal base excision repair and causes cell death through overproduction of toxic repair intermediates. Here we establish through site-directed mutagenesis that MPG-induced sensitivity to alkylation is dependent on enzyme glycosylase activity. However, in contrast to the sensitivity seen to heterogeneous alkylating agents, MPG overexpression generates no cellular sensitivity to MeOSO2(CH2)2-lexitropsin, an alkylator which exclusively induces 3-meA lesions. Indeed, MPG overexpression has been shown to increase the toxicity of alkylating agents that produce 7-meG adducts, and here we demonstrate that MPG-overexpressing cells have dramatically increased removal of 7-meG from their DNA. These data suggest that the mechanism of MPG-induced cytotoxicity involves the conversion of non-toxic 7-meG lesions into highly toxic repair intermediates. This study establishes a mechanism by which a benign DNA modification can be made toxic through the overexpression of an otherwise well-tolerated gene product, and the application of this principle could lead to improved chemotherapeutic strategies that reduce the peripheral toxicity of alkylating agents. PMID:15905475

  4. Functional Evaluation of Nine Missense-Type Variants of the Human DNA Glycosylase Enzyme MUTYH in the Japanese Population.

    PubMed

    Shinmura, Kazuya; Kato, Hisami; Goto, Masanori; Yamada, Hidetaka; Tao, Hong; Nakamura, Satoki; Sugimura, Haruhiko

    2016-04-01

    Biallelic germline mutations of MUTYH, the gene encoding DNA glycosylase, cause MUTYH-associated polyposis (MAP), characterized by multiple colorectal adenomas and carcinoma(s). However, a considerable number of MUTYH variants are still functionally uncharacterized. Herein, we report the results of functional evaluation of nine missense-type MUTYH variant proteins in the Japanese population. The DNA glycosylase activity and ability to suppress mutations caused by 8-hydroxyguanine, an oxidized form of guanine, were examined for the nine variants of type 2 MUTYH, a nuclear form of the enzyme, by DNA cleavage activity assay and supF forward mutation assay, respectively. Both activities were severely defective in the p.N210S MUTYH type 2 variant corresponding to p.N238S in the reference MUTYH form and partially defective in p.R219G variant corresponding to p.R247G, but nearly fully retained in seven other variants examined. Our results suggest that p.N238S and p.R247G are likely to be pathogenic alleles for MAP. PMID:26694661

  5. Base-Excision-Repair-Induced Construction of a Single Quantum-Dot-Based Sensor for Sensitive Detection of DNA Glycosylase Activity.

    PubMed

    Wang, Li-Juan; Ma, Fei; Tang, Bo; Zhang, Chun-Yang

    2016-08-01

    DNA glycosylase is an initiating enzyme of cellular base excision repair pathway which is responsible for the repair of various DNA lesions and the maintenance of genomic stability, and the dysregulation of DNA glycosylase activity is associated with a variety of human pathology. Accurate detection of DNA glycosylase activity is critical to both clinical diagnosis and therapeutics, but conventional methods for the DNA glycosylase assay are usually time-consuming with poor sensitivity. Here, we demonstrate the base-excision-repair-induced construction of a single quantum dot (QD)-based sensor for highly sensitive measurement of DNA glycosylase activity. We use human 8-oxoguanine-DNA glycosylase 1 (hOGG1), which is responsible for specifically repairing the damaged 8-hydroxyguanine (8-oxoG, one of the most abundant and widely studied DNA damage products), as a model DNA glycosylase. In the presence of biotin-labeled DNA substrate, the hOGG1 may catalyze the removal of 8-oxo G from 8-oxoG·C base pairs to generate an apurinic/apyrimidinic (AP) site. With the assistance of apurinic/apyrimidinic endonuclease (APE1), the cleavage of the AP site results in the generation of a single-nucleotide gap. Subsequently, DNA polymerase β incorporates a Cy5-labeled dGTP into the DNA substrate to fill the gap. With the addition of streptavidin-coated QDs, a QD-DNA-Cy5 nanostructure is formed via specific biotin-streptavidin binding, inducing the occurrence of fluorescence resonance energy transfer (FRET) from the QD to Cy5. The resulting Cy5 signal can be simply monitored by total internal reflection fluorescence (TIRF) imaging. The proposed method enables highly sensitive measurement of hOGG1 activity with a detection limit of 1.8 × 10(-6) U/μL. Moreover, it can be used to measure the enzyme kinetic parameters and detect the hOGG1 activity in crude cell extracts, offering a powerful tool for biomedical research and clinical diagnosis. PMID:27401302

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

    SciTech Connect

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

    2006-02-28

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

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

    PubMed Central

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

    2016-01-01

    Azinomycin B is a hybrid polyketide/nonribosomal peptide natural product and possesses antitumor activity by interacting covalently with duplex DNA and inducing interstrand crosslinks. In the biosynthetic study of azinomycin B, a gene (orf1) adjacent to the azinomycin B gene cluster was found to be essential for the survival of the producer, Streptomyces sahachiroi ATCC33158. Sequence analyses revealed that Orf1 belongs to the HTH_42 superfamily of conserved bacterial proteins which are widely distributed in pathogenic and antibiotic-producing bacteria with unknown functions. The protein exhibits a protective effect against azinomycin B when heterologously expressed in azinomycin-sensitive strains. EMSA assays showed its sequence nonspecific binding to DNA and structure-specific binding to azinomycin B-adducted sites, and ChIP assays revealed extensive association of Orf1 with chromatin in vivo. Interestingly, Orf1 not only protects target sites by protein–DNA interaction but is also capable of repairing azinomycin B-mediated DNA cross-linking. It possesses the DNA glycosylase-like activity and specifically repairs DNA damage induced by azinomycin B through removal of both adducted nitrogenous bases in the cross-link. This bifunctional protein massively binds to genomic DNA to reduce drug attack risk as a novel DNA binding protein and triggers the base excision repair system as a novel DNA glycosylase. PMID:26400161

  8. Partial uracil-DNA-glycosylase treatment for screening of ancient DNA.

    PubMed

    Rohland, Nadin; Harney, Eadaoin; Mallick, Swapan; Nordenfelt, Susanne; Reich, David

    2015-01-19

    The challenge of sequencing ancient DNA has led to the development of specialized laboratory protocols that have focused on reducing contamination and maximizing the number of molecules that are extracted from ancient remains. Despite the fact that success in ancient DNA studies is typically obtained by screening many samples to identify a promising subset, ancient DNA protocols have not, in general, focused on reducing the time required to screen samples. We present an adaptation of a popular ancient library preparation method that makes screening more efficient. First, the DNA extract is treated using a protocol that causes characteristic ancient DNA damage to be restricted to the terminal nucleotides, while nearly eliminating it in the interior of the DNA molecules, allowing a single library to be used both to test for ancient DNA authenticity and to carry out population genetic analysis. Second, the DNA molecules are ligated to a unique pair of barcodes, which eliminates undetected cross-contamination from this step onwards. Third, the barcoded library molecules include incomplete adapters of short length that can increase the specificity of hybridization-based genomic target enrichment. The adapters are completed just before sequencing, so the same DNA library can be used in multiple experiments, and the sequences distinguished. We demonstrate this protocol on 60 ancient human samples. PMID:25487342

  9. Phosphorylation Sites Identified in the NEIL1 DNA Glycosylase Are Potential Targets for the JNK1 Kinase

    PubMed Central

    Prakash, Aishwarya; Cao, Vy Bao; Doublié, Sylvie

    2016-01-01

    The NEIL1 DNA glycosylase is one of eleven mammalian DNA glycosylases that partake in the first step of the base excision repair (BER) pathway. NEIL1 recognizes and cleaves mainly oxidized pyrimidines from DNA. The past decade has witnessed the identification of an increasing number of post-translational modifications (PTMs) in BER enzymes including phosphorylation, acetylation, and sumoylation, which modulate enzyme function. In this work, we performed the first comprehensive analysis of phosphorylation sites in human NEIL1 expressed in human cells. Mass spectrometry (MS) analysis revealed phosphorylation at three serine residues: S207, S306, and a third novel site, S61. We expressed, purified, and characterized phosphomimetic (glutamate) and phosphoablating (alanine) mutants of the three phosphorylation sites in NEIL1 revealed by the MS analysis. All mutant enzymes were active and bound tightly to DNA, indicating that phosphorylation does not affect DNA binding and enzyme activity at these three serine sites. We also characterized phosphomimetic mutants of two other sites of phosphorylation, Y263 and S269, reported previously, and observed that mutation of Y263 to E yielded a completely inactive enzyme. Furthermore, based on sequence motifs and kinase prediction algorithms, we identified the c-Jun N-terminal kinase 1 (JNK1) as the kinase involved in the phosphorylation of NEIL1. JNK1, a member of the mitogen activated protein kinase (MAPK) family, was detected in NEIL1 immunoprecipitates, interacted with NEIL1 in vitro, and was able to phosphorylate the enzyme at residues S207, S306, and S61. PMID:27518429

  10. Down-regulation of 8-oxoguanine DNA glycosylase 1 expression in the airway epithelium ameliorates allergic lung inflammation.

    PubMed

    Bacsi, Attila; Aguilera-Aguirre, Leopoldo; Szczesny, Bartosz; Radak, Zsolt; Hazra, Tapas K; Sur, Sanjiv; Ba, Xueqing; Boldogh, Istvan

    2013-01-01

    Allergic airway inflammation is characterized by increased expression of pro-inflammatory mediators, inflammatory cell infiltration, mucus hypersecretion, and airway hyperresponsiveness, in parallel with oxidative DNA base and strand damage, whose etiological role is not understood. Our goal was to establish the role of 8-oxoguanine (8-oxoG), a common oxidatively damaged base, and its repair by 8-oxoguanine DNA glycosylase 1 (Ogg1) in allergic airway inflammatory processes. Airway inflammation was induced by intranasally administered ragweed (Ambrosia artemisiifolia) pollen grain extract (RWPE) in sensitized BALB/c mice. We utilized siRNA technology to deplete Ogg1 from airway epithelium; 8-oxoG and DNA strand break levels were quantified by Comet assays. Inflammatory cell infiltration and epithelial methaplasia were determined histologically, mucus and cytokines levels biochemically and enhanced pause was used as the main index of airway hyperresponsiveness. Decreased Ogg1 expression and thereby 8-oxoG repair in the airway epithelium conveyed a lower inflammatory response after RWPE challenge of sensitized mice, as determined by expression of Th2 cytokines, eosinophilia, epithelial methaplasia, and airway hyperresponsiveness. In contrast, 8-oxoG repair in Ogg1-proficient airway epithelium was coupled to an increase in DNA single-strand break (SSB) levels and exacerbation of allergen challenge-dependent inflammation. Decreased expression of the Nei-like glycosylases Neil1 and Neil2 that preferentially excise ring-opened purines and 5-hydroxyuracil, respectively, did not alter the above parameters of allergic immune responses to RWPE. These results show that DNA SSBs formed during Ogg1-mediated repair of 8-oxoG augment antigen-driven allergic immune responses. A transient modulation of OGG1 expression/activity in airway epithelial cells could have clinical benefits. PMID:23127499

  11. Synthetic Routes to N-9 Alkylated 8-Oxoguanines; Weak Inhibitors of the Human DNA Glycosylase OGG1.

    PubMed

    Mahajan, Tushar R; Ytre-Arne, Mari Eknes; Strøm-Andersen, Pernille; Dalhus, Bjørn; Gundersen, Lise-Lotte

    2015-01-01

    The human 8-oxoguanine DNA glycosylase OGG1 is involved in base excision repair (BER), one of several DNA repair mechanisms that may counteract the effects of chemo- and radiation therapy for the treatment of cancer. We envisage that potent inhibitors of OGG1 may be found among the 9-alkyl-8-oxoguanines. Thus we explored synthetic routes to 8-oxoguanines and examined these as OGG1 inhibitors. The best reaction sequence started from 6-chloroguanine and involved N-9 alkylation, C-8 bromination, and finally simultaneous hydrolysis of both halides. Bromination before N-alkylation should only be considered when the N-substituent is not compatible with bromination conditions. The 8-oxoguanines were found to be weak inhibitors of OGG1. 6-Chloro-8-oxopurines, byproducts in the hydrolysis of 2,6-halopurines, turned out to be slightly better inhibitors than the corresponding 8-oxoguanines. PMID:26364627

  12. Solution-state NMR Investigation of DNA Binding Interactions in Escherichia coli Formamidopyrimidine-DNA Glycosylase (Fpg): A Dynamic Description of the DNA/Protein Interface

    SciTech Connect

    Buchko, Garry W.; McAteer, Kathleen; Wallace, Susan S.; Kennedy, Michael A.

    2005-03-02

    Formamidopyrimidine-DNA glycosylase (Fpg) is a base excision repair protein that removes oxidative DNA lesions. Recent crystal structures of Fpg bound to DNA revealed residues involved in damage recognition and enzyme catalysis, but failed to shed light on the dynamic nature of the processes. To examine the structural and dynamic changes that occur in solution when Fpg binds DNA, NMR spectroscopy was used to study Escherichia coli Fpg free and bound to a double-stranded DNA oligomer (13-PD) containing propanediol, a non-hydrolyzable abasic-site analogue. Only 209 out of a possible 252 (83%) free-precession HSQC cross peaks were observed and 180 of these were assignable, indicating that ~30% of the residues undergo intermediate timescale motion that makes them intractable in backbone assignment experiments. DNA titration experiments revealed line broadening and chemical shift perturbations for backbone amides nearby and distant from the DNA binding surface, but failed to quench the intermediate time-scale motion observed for free Fpg. CPMG-HSQC experiments revealed millisecond to microsecond motion for the backbone amides of D91 and H92 that was quenched upon binding 13-PD. Collectively, these observations reveal that, in solution, Fpg contains highly flexible regions. The dynamic nature of Fpg, especially at the DNA binding surface, may be key to its processive search mechanism.

  13. Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase.

    PubMed

    Lavrukhin, O V; Lloyd, R S

    2000-12-12

    Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates. PMID:11106507

  14. Slow base excision by human alkyladenine DNA glycosylase limits the rate of formation of AP sites and AP endonuclease 1 does not stimulate base excision.

    PubMed

    Maher, Robyn L; Vallur, Aarthy C; Feller, Joyce A; Bloom, Linda B

    2007-01-01

    The base excision repair pathway removes damaged DNA bases and resynthesizes DNA to replace the damage. Human alkyladenine DNA glycosylase (AAG) is one of several damage-specific DNA glycosylases that recognizes and excises damaged DNA bases. AAG removes primarily damaged adenine residues. Human AP endonuclease 1 (APE1) recognizes AP sites produced by DNA glycosylases and incises the phophodiester bond 5' to the damaged site. The repair process is completed by a DNA polymerase and DNA ligase. If not tightly coordinated, base excision repair could generate intermediates that are more deleterious to the cell than the initial DNA damage. The kinetics of AAG-catalyzed excision of two damaged bases, hypoxanthine and 1,N6-ethenoadenine, were measured in the presence and absence of APE1 to investigate the mechanism by which the base excision activity of AAG is coordinated with the AP incision activity of APE1. 1,N6-ethenoadenine is excised significantly slower than hypoxanthine and the rate of excision is not affected by APE1. The excision of hypoxanthine is inhibited to a small degree by accumulated product, and APE1 stimulates multiple turnovers by alleviating product inhibition. These results show that APE1 does not significantly affect the kinetics of base excision by AAG. It is likely that slow excision by AAG limits the rate of AP site formation in vivo such that AP sites are not created faster than can be processed by APE1. PMID:17018265

  15. Using structural-based protein engineering to modulate the differential inhibition effects of SAUGI on human and HSV uracil DNA glycosylase.

    PubMed

    Wang, Hao-Ching; Ho, Chun-Han; Chou, Chia-Cheng; Ko, Tzu-Ping; Huang, Ming-Fen; Hsu, Kai-Cheng; Wang, Andrew H-J

    2016-05-19

    Uracil-DNA glycosylases (UDGs) are highly conserved proteins that can be found in a wide range of organisms, and are involved in the DNA repair and host defense systems. UDG activity is controlled by various cellular factors, including the uracil-DNA glycosylase inhibitors, which are DNA mimic proteins that prevent the DNA binding sites of UDGs from interacting with their DNA substrate. To date, only three uracil-DNA glycosylase inhibitors, phage UGI, p56, and Staphylococcus aureus SAUGI, have been determined. We show here that SAUGI has differential inhibitory effects on UDGs from human, bacteria, Herpes simplex virus (HSV; human herpesvirus 1) and Epstein-Barr virus (EBV; human herpesvirus 4). Newly determined crystal structures of SAUGI/human UDG and a SAUGI/HSVUDG complex were used to explain the differential binding activities of SAUGI on these two UDGs. Structural-based protein engineering was further used to modulate the inhibitory ability of SAUGI on human UDG and HSVUDG. The results of this work extend our understanding of DNA mimics as well as potentially opening the way for novel therapeutic applications for this kind of protein. PMID:26980279

  16. Using structural-based protein engineering to modulate the differential inhibition effects of SAUGI on human and HSV uracil DNA glycosylase

    PubMed Central

    Wang, Hao-Ching; Ho, Chun-Han; Chou, Chia-Cheng; Ko, Tzu-Ping; Huang, Ming-Fen; Hsu, Kai-Cheng; Wang, Andrew H.-J.

    2016-01-01

    Uracil-DNA glycosylases (UDGs) are highly conserved proteins that can be found in a wide range of organisms, and are involved in the DNA repair and host defense systems. UDG activity is controlled by various cellular factors, including the uracil-DNA glycosylase inhibitors, which are DNA mimic proteins that prevent the DNA binding sites of UDGs from interacting with their DNA substrate. To date, only three uracil-DNA glycosylase inhibitors, phage UGI, p56, and Staphylococcus aureus SAUGI, have been determined. We show here that SAUGI has differential inhibitory effects on UDGs from human, bacteria, Herpes simplex virus (HSV; human herpesvirus 1) and Epstein-Barr virus (EBV; human herpesvirus 4). Newly determined crystal structures of SAUGI/human UDG and a SAUGI/HSVUDG complex were used to explain the differential binding activities of SAUGI on these two UDGs. Structural-based protein engineering was further used to modulate the inhibitory ability of SAUGI on human UDG and HSVUDG. The results of this work extend our understanding of DNA mimics as well as potentially opening the way for novel therapeutic applications for this kind of protein. PMID:26980279

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

    SciTech Connect

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

    2010-11-18

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

  18. Opinion: uracil DNA glycosylase (UNG) plays distinct and non-canonical roles in somatic hypermutation and class switch recombination

    PubMed Central

    Yousif, Ashraf S.; Stanlie, Andre; Begum, Nasim A.

    2014-01-01

    Activation-induced cytidine deaminase (AID) is essential to class switch recombination (CSR) and somatic hypermutation (SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair complex, is required for CSR. The role of UNG in CSR and SHM is extremely controversial. AID deficiency in mice abolishes both CSR and SHM, while UNG-deficient mice have drastically reduced CSR but augmented SHM raising a possibility of differential functions of UNG in CSR and SHM. Interestingly, UNG has been associated with a CSR-specific repair adapter protein Brd4, which interacts with acetyl histone 4, γH2AX and 53BP1 to promote non-homologous end joining during CSR. A non-canonical scaffold function of UNG, but not the catalytic activity, can be attributed to the recruitment of essential repair proteins associated with the error-free repair during SHM, and the end joining during CSR. PMID:24994819

  19. Opinion: uracil DNA glycosylase (UNG) plays distinct and non-canonical roles in somatic hypermutation and class switch recombination.

    PubMed

    Yousif, Ashraf S; Stanlie, Andre; Begum, Nasim A; Honjo, Tasuku

    2014-10-01

    Activation-induced cytidine deaminase (AID) is essential to class switch recombination (CSR) and somatic hypermutation (SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair complex, is required for CSR. The role of UNG in CSR and SHM is extremely controversial. AID deficiency in mice abolishes both CSR and SHM, while UNG-deficient mice have drastically reduced CSR but augmented SHM raising a possibility of differential functions of UNG in CSR and SHM. Interestingly, UNG has been associated with a CSR-specific repair adapter protein Brd4, which interacts with acetyl histone 4, γH2AX and 53BP1 to promote non-homologous end joining during CSR. A non-canonical scaffold function of UNG, but not the catalytic activity, can be attributed to the recruitment of essential repair proteins associated with the error-free repair during SHM, and the end joining during CSR. PMID:24994819

  20. Excision of 5-halogenated Uracils by Human Thymine DNA Glycosylase: Robust Activity for DNA Contexts other than CpG*

    PubMed Central

    Morgan, Michael T.; Bennett, Matthew T.; Drohat, Alexander C.

    2010-01-01

    Thymine DNA glycosylase (TDG) excises thymine from G·T mispairs, and removes a variety of damaged bases (X), with a preference for lesions in a CpG·X context. We recently reported that human TDG rapidly excises 5-halogenated uracils, exhibiting much greater activity for CpG·FU, CpG·ClU, and CpG·BrU than for CpG·T. Here, we examine the effects of altering the CpG context on the excision activity for U, T, FU, ClU, and BrU. We show that the maximal activity (kmax) for G·X substrates depends significantly on the 5′ base pair. For example, kmax decreases by 6-, 11-, and 82-fold for TpG·ClU, GpG·ClU, and ApG·ClU, respectively, as compared to CpG·ClU. For the other G·X substrates, the 5′-neighbor effects have a similar trend but vary in magnitude. The activity for G·FU, G·ClU, and G·BrU, with any 5′-flanking pair, meets and in most cases significantly exceeds the CpG·T activity. Strikingly, hTDG activity is reduced 102.3- to 104.3-fold for A·X relative to G·X pairs, and reduced further for A·X pairs with a 5′ pair other than C·G. The effect of altering the 5′ pair and/or the opposing base (G·X versus A·X) is greater for substrates that are larger (BrdU, dT) or have a more stable N-glycosidic bond (such as dT). The largest CpG context effects are observed for the excision of thymine. The potential role played by hTDG in the cytotoxic effects of ClU and BrU incorporation into DNA, which can occur under inflammatory conditions, and in the cytotoxicity of FU, a widely used anticancer agent, are discussed. PMID:17602166

  1. Deficiency of the oxidative damage-specific DNA glycosylase NEIL1 leads to reduced germinal center B cell expansion

    PubMed Central

    Mori, Hiromi; Ouchida, Rika; Hijikata, Atsushi; Kitamura, Hiroshi; Ohara, Osamu; Li, Yingqian; Gao, Xiang; Yasui, Akira; Lloyd, R. Stephen; Wang, Ji-Yang

    2016-01-01

    Mammalian cells possess multiple DNA glycosylases, including OGG1, NTH1, NEIL1, NEIL2 and NEIL3, for the repair of oxidative DNA damage. Among these, NEIL1 and NEIL2 are able to excise oxidized bases on single stranded or bubble-structured DNA and has been implicated in repair of oxidative damage associated with DNA replication or transcription. We found that Neil1 was highly constitutively expressed in the germinal center (GC) B cells, a rapidly dividing cell population that is undergoing immunoglobulin (Ig) gene hypermutation and isotype switching. While Neil1−/− mice exhibited normal B and T cell development and maturation, these mice contained a significantly lower frequency of GC B cells than did WT mice after immunization with a T-dependent antigen. Consistent with the reduced expansion of GC B cells, Neil1−/− mice had a decreased frequency of Ig gene hypermutation and produced less antibody against a T-dependent antigen during both primary and secondary immune responses. These results suggest that repair of endogenous oxidative DNA damage by NEIL1 is important for the rapid expansion of GC B cells and efficient induction of humoral immune responses. PMID:19782007

  2. Structural basis for removal of adenine mispaired with 8-oxoguanine by MutY adenine DNA glycosylase.

    PubMed

    Fromme, J Christopher; Banerjee, Anirban; Huang, Susan J; Verdine, Gregory L

    2004-02-12

    The genomes of aerobic organisms suffer chronic oxidation of guanine to the genotoxic product 8-oxoguanine (oxoG). Replicative DNA polymerases misread oxoG residues and insert adenine instead of cytosine opposite the oxidized base. Both bases in the resulting A*oxoG mispair are mutagenic lesions, and both must undergo base-specific replacement to restore the original C*G pair. Doing so represents a formidable challenge to the DNA repair machinery, because adenine makes up roughly 25% of the bases in most genomes. The evolutionarily conserved enzyme adenine DNA glycosylase (called MutY in bacteria and hMYH in humans) initiates repair of A*oxoG to C*G by removing the inappropriately paired adenine base from the DNA backbone. A central issue concerning MutY function is the mechanism by which A*oxoG mispairs are targeted among the vast excess of A*T pairs. Here we report the use of disulphide crosslinking to obtain high-resolution crystal structures of MutY-DNA lesion-recognition complexes. These structures reveal the basis for recognizing both lesions in the A*oxoG pair and for catalysing removal of the adenine base. PMID:14961129

  3. Differential regulation of S-region hypermutation and class-switch recombination by noncanonical functions of uracil DNA glycosylase

    PubMed Central

    Yousif, Ashraf S.; Stanlie, Andre; Mondal, Samiran; Honjo, Tasuku; Begum, Nasim A.

    2014-01-01

    Activation-induced cytidine deaminase (AID) is essential to class-switch recombination (CSR) and somatic hypermutation (SHM) in both V region SHM and S region SHM (s-SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair (BER) complex, is required for CSR. Strikingly, however, UNG deficiency causes augmentation of SHM, suggesting involvement of distinct functions of UNG in SHM and CSR. Here, we show that noncanonical scaffold functions of UNG regulate s-SHM negatively and CSR positively. The s-SHM suppressive function of UNG is attributed to the recruitment of faithful BER components at the cleaved DNA locus, with competition against error-prone polymerases. By contrast, the CSR-promoting function of UNG enhances AID-dependent S-S synapse formation by recruiting p53-binding protein 1 and DNA-dependent protein kinase, catalytic subunit. Several loss-of-catalysis mutants of UNG discriminated CSR-promoting activity from s-SHM suppressive activity. Taken together, the noncanonical function of UNG regulates the steps after AID-induced DNA cleavage: error-prone repair suppression in s-SHM and end-joining promotion in CSR. PMID:24591630

  4. Differential regulation of S-region hypermutation and class-switch recombination by noncanonical functions of uracil DNA glycosylase.

    PubMed

    Yousif, Ashraf S; Stanlie, Andre; Mondal, Samiran; Honjo, Tasuku; Begum, Nasim A

    2014-03-18

    Activation-induced cytidine deaminase (AID) is essential to class-switch recombination (CSR) and somatic hypermutation (SHM) in both V region SHM and S region SHM (s-SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair (BER) complex, is required for CSR. Strikingly, however, UNG deficiency causes augmentation of SHM, suggesting involvement of distinct functions of UNG in SHM and CSR. Here, we show that noncanonical scaffold functions of UNG regulate s-SHM negatively and CSR positively. The s-SHM suppressive function of UNG is attributed to the recruitment of faithful BER components at the cleaved DNA locus, with competition against error-prone polymerases. By contrast, the CSR-promoting function of UNG enhances AID-dependent S-S synapse formation by recruiting p53-binding protein 1 and DNA-dependent protein kinase, catalytic subunit. Several loss-of-catalysis mutants of UNG discriminated CSR-promoting activity from s-SHM suppressive activity. Taken together, the noncanonical function of UNG regulates the steps after AID-induced DNA cleavage: error-prone repair suppression in s-SHM and end-joining promotion in CSR. PMID:24591630

  5. Base excision repair enzymes protect abasic sites in duplex DNA from interstrand cross-links.

    PubMed

    Admiraal, Suzanne J; O'Brien, Patrick J

    2015-03-10

    Hydrolysis of the N-glycosyl bond between a nucleobase and deoxyribose leaves an abasic site within duplex DNA. The abasic site can react with exocyclic amines of nucleobases on the complementary strand to form interstrand DNA-DNA cross-links (ICLs). We find that several enzymes from the base excision repair (BER) pathway protect an abasic site on one strand of a DNA duplex from cross-linking with an amine on the opposing strand. Human alkyladenine DNA glycosylase (AAG) and Escherichia coli 3-methyladenine DNA glycosylase II (AlkA) accomplish this by binding tightly to the abasic site and sequestering it. AAG protects an abasic site opposite T, the product of its canonical glycosylase reaction, by a factor of ∼10-fold, as estimated from its inhibition of the reaction of an exogenous amine with the damaged DNA. Human apurinic/apyrimidinic site endonuclease 1 and E. coli endonuclease III both decrease the amount of ICL at equilibrium by generating a single-strand DNA nick at the abasic position as it is liberated from the cross-link. The reversibility of the reaction between amines and abasic sites allows BER enzymes to counter the potentially disruptive effects of this type of cross-link on DNA transactions. PMID:25679877

  6. Potential role of 8-oxoguanine DNA glycosylase 1 as a STAT1 coactivator in endotoxin-induced inflammatory response.

    PubMed

    Kim, Hong Sook; Kim, Byung-Hak; Jung, Joo Eun; Lee, Chang Seok; Lee, Hyun Gyu; Lee, Jung Weon; Lee, Kun Ho; You, Ho Jin; Chung, Myung-Hee; Ye, Sang-Kyu

    2016-04-01

    Human 8-oxoguanine DNA glycosylase 1 (OGG1) is the major DNA repair enzyme that plays a key role in excision of oxidative damaged DNA bases such as 8-oxoguainine (8-oxoG). Recent studies suggest another function of OGG1, namely that it may be involved in the endotoxin- or oxidative stress-induced inflammatory response. In this study, we investigated the role of OGG1 in the inflammatory response. OGG1 expression is increased in the organs of endotoxin-induced or myelin oligodendrocyte glycoprotein (MOG)-immunized mice and immune cells, resulting in induction of the expression of pro-inflammatory mediators at the transcriptional levels. Biochemical studies showed that signal transducer and activator of transcription 1 (STAT1) plays a key role in endotoxin-induced OGG1 expression and inflammatory response. STAT1 regulates the transcriptional activity of OGG1 through recruiting and binding to the gamma-interferon activation site (GAS) motif of the OGG1 promoter region, and chromatin remodeling by acetylation and dimethylation of lysine-14 and -4 residues of histone H3. In addition, OGG1 acts as a STAT1 coactivator and has transcriptional activity in the presence of endotoxin. The data presented here identifies a novel mechanism, and may provide new therapeutic strategies for the treatment of endotoxin-mediated inflammatory diseases. PMID:26496208

  7. Structure/Function Analysis of DNA-glycosylases That Repair Oxidized Purines and Pyrimidines and the Influence of Surrounding DNA Sequence on Their Interactions

    SciTech Connect

    Wallace, Susan S.

    2005-08-22

    The overall goal of this project was to elucidate the structure/function relationships between oxidized DNA bases and the DNA repair enzymes that recognize and remove them. The NMR solution structure of formamidopyrimidine DNA glycosylase (Fpg) that recognizes oxidized DNA purines was to be determined. Furthermore, the solution structures of DNA molecules containing specific lesions recognized by Fpg was to be determined in sequence contexts that either facilitate or hinder this recognition. These objectives were in keeping with the long-term goals of the Principal Investigator's laboratory, that is, to understand the basic mechanisms that underpin base excision repair processing of oxidative DNA lesions and to elucidate the interactions of unrepaired lesions with DNA polymerases. The results of these two DNA transactions can ultimately determine the fate of the cell. These objectives were also in keeping with the goals of our collaborator, Dr. Michael Kennedy, who is studying the repair and recognition of damaged DNA. Overall the goals of this project were congruent with those of the Department of Energy's Health Effects and Life Sciences Research Program, especially to the Structural Biology, the Human Genome and the Health Effects Programs. The mission of the latter Program includes understanding the biological effects and consequences of DNA damages produced by toxic agents in the many DOE waste sites so that cleanup can be accomplished in a safe, effective and timely manner.

  8. Absence of the Uracil DNA Glycosylase of Murine Gammaherpesvirus 68 Impairs Replication and Delays the Establishment of Latency In Vivo

    PubMed Central

    Minkah, Nana; Macaluso, Marc; Oldenburg, Darby G.; Paden, Clinton R.; White, Douglas W.; McBride, Kevin M.

    2015-01-01

    ABSTRACT Uracil DNA glycosylases (UNG) are highly conserved proteins that preserve DNA fidelity by catalyzing the removal of mutagenic uracils. All herpesviruses encode a viral UNG (vUNG), and yet the role of the vUNG in a pathogenic course of gammaherpesvirus infection is not known. First, we demonstrated that the vUNG of murine gammaherpesvirus 68 (MHV68) retains the enzymatic function of host UNG in an in vitro class switch recombination assay. Next, we generated a recombinant MHV68 with a stop codon in ORF46/UNG (ΔUNG) that led to loss of UNG activity in infected cells and a replication defect in primary fibroblasts. Acute replication of MHV68ΔUNG in the lungs of infected mice was reduced 100-fold and was accompanied by a substantial delay in the establishment of splenic latency. Latency was largely, yet not fully, restored by an increase in virus inoculum or by altering the route of infection. MHV68 reactivation from latent splenocytes was not altered in the absence of the vUNG. A survey of host UNG activity in cells and tissues targeted by MHV68 indicated that the lung tissue has a lower level of enzymatic UNG activity than the spleen. Taken together, these results indicate that the vUNG plays a critical role in the replication of MHV68 in tissues with limited host UNG activity and this vUNG-dependent expansion, in turn, influences the kinetics of latency establishment in distal reservoirs. IMPORTANCE Herpesviruses establish chronic lifelong infections using a strategy of replicative expansion, dissemination to latent reservoirs, and subsequent reactivation for transmission and spread. We examined the role of the viral uracil DNA glycosylase, a protein conserved among all herpesviruses, in replication and latency of murine gammaherpesvirus 68. We report that the viral UNG of this murine pathogen retains catalytic activity and influences replication in culture. The viral UNG was impaired for productive replication in the lung. This defect in expansion at the

  9. Triphlorethol-A from Ecklonia cava up-regulates the oxidant sensitive 8-oxoguanine DNA glycosylase 1.

    PubMed

    Kim, Ki Cheon; Lee, In Kyung; Kang, Kyoung Ah; Piao, Mei Jing; Ryu, Min Ju; Kim, Jeong Mi; Lee, Nam Ho; Hyun, Jin Won

    2014-11-01

    This study investigated the protective mechanisms of triphlorethol-A, isolated from Ecklonia cava, against oxidative stress-induced DNA base damage, especially 8-oxoguanine (8-oxoG), in Chinese hamster lung fibroblast V79-4 cells. 8-Oxoguanine DNA glycosylase-1 (OGG1) plays an important role in the removal of 8-oxoG during the cellular response to DNA base damage. Triphlorethol-A significantly decreased the levels of 8-oxoG induced by H2O2, and this correlated with increases in OGG1 mRNA and OGG1 protein levels. Furthermore, siOGG1-transfected cell attenuated the protective effect of triphlorethol-A against H2O2 treatment. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor for OGG1, and Nrf2 combines with small Maf proteins in the nucleus to bind to antioxidant response elements (ARE) in the upstream promoter region of the OGG1 gene. Triphlorethol-A restored the expression of nuclear Nrf2, small Maf protein, and the Nrf2-Maf complex, all of which were reduced by oxidative stress. Furthermore, triphlorethol-A increased Nrf2 binding to ARE sequences and the resulting OGG1 promoter activity, both of which were also reduced by oxidative stress. The levels of the phosphorylated forms of Akt kinase, downstream of phosphatidylinositol 3-kinase (PI3K), and Erk, which are regulators of OGG1, were sharply decreased by oxidative stress, but these decreases were prevented by triphlorethol-A. Specific PI3K, Akt, and Erk inhibitors abolished the cytoprotective effects of triphlorethol-A, suggesting that OGG1 induction by triphlorethol-A involves the PI3K/Akt and Erk pathways. Taken together, these data indicate that by activating the DNA repair system, triphlorethol-A exerts protective effects against DNA base damage induced by oxidative stress. PMID:25353254

  10. Targeted deletion of the genes encoding NTH1 and NEIL1 DNA N-glycosylases reveals the existence of novel carcinogenic oxidative damage to DNA☆

    PubMed Central

    Chan, Michael K.; Ocampo-Hafalla, Maria T.; Vartanian, Vladimir; Jaruga, Pawel; Kirkali, Güldal; Koenig, Karen L.; Brown, Stuart; Lloyd, R. Stephen; Dizdaroglu, Miral; Teebor, George W.

    2016-01-01

    We have generated a strain of mice lacking two DNA N-glycosylases of base excision repair (BER), NTH1 and NEIL1, homologs of bacterial Nth (endonuclease three) and Nei (endonuclease eight). Although these enzymes remove several oxidized bases from DNA, they do not remove the well-known carcinogenic oxidation product of guanine: 7,8-dihydro-8-oxoguanine (8-OH-Gua), which is removed by another DNA N-glycosylase, OGG1. The Nth1−/−Neil1−/− mice developed pulmonary and hepatocellular tumors in much higher incidence than either of the single knockouts, Nth1−/− and Neil1−/−. The pulmonary tumors contained, exclusively, activating GGT→GAT transitions in codon 12 of K-ras of their DNA. Such transitions contrast sharply with the activating GGT→GTT transversions in codon 12 of K-ras of the pathologically similar pulmonary tumors, which arose in mice lacking OGG1 and a second DNA N-glycosylase, MUTY. To characterize the biochemical phenotype of the knockout mice, the content of oxidative DNA base damage was analyzed from three tissues isolated from control, single and double knockout mice. The content of 8-OH-Gua was indistinguishable among all genotypes. In contrast, the content of 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) derived from adenine and guanine, respectively, were increased in some but not all tissues of Neil1−/− and Neil1−/−Nth1−/− mice. The high incidence of tumors in our Nth1−/−Neil1−/− mice together with the nature of the activating mutation in the K-ras gene of their pulmonary tumors, reveal for the first time, the existence of mutagenic and carcinogenic oxidative damage to DNA which is not 8-OH-Gua. PMID:19346169

  11. Thermodynamics of the multi-stage DNA lesion recognition and repair by formamidopyrimidine-DNA glycosylase using pyrrolocytosine fluorescence—stopped-flow pre-steady-state kinetics

    PubMed Central

    Kuznetsov, Nikita A.; Vorobjev, Yuri N.; Krasnoperov, Lev N.; Fedorova, Olga S.

    2012-01-01

    Formamidopyrimidine-DNA glycosylase, Fpg protein from Escherichia coli, initiates base excision repair in DNA by removing a wide variety of oxidized lesions. In this study, we perform thermodynamic analysis of the multi-stage interaction of Fpg with specific DNA-substrates containing 7,8-dihydro-8-oxoguanosine (oxoG), or tetrahydrofuran (THF, an uncleavable abasic site analog) and non-specific (G) DNA-ligand based on stopped-flow kinetic data. Pyrrolocytosine, highly fluorescent analog of the natural nucleobase cytosine, is used to record multi-stage DNA lesion recognition and repair kinetics over a temperature range (10–30°C). The kinetic data were used to obtain the standard Gibbs energy, enthalpy and entropy of the specific stages using van’t Hoff approach. The data suggest that not only enthalpy-driven exothermic oxoG recognition, but also the desolvation-accompanied entropy-driven enzyme-substrate complex adjustment into the catalytically active state play equally important roles in the overall process. PMID:22584623

  12. Partial uracil–DNA–glycosylase treatment for screening of ancient DNA

    PubMed Central

    Rohland, Nadin; Harney, Eadaoin; Mallick, Swapan; Nordenfelt, Susanne; Reich, David

    2015-01-01

    The challenge of sequencing ancient DNA has led to the development of specialized laboratory protocols that have focused on reducing contamination and maximizing the number of molecules that are extracted from ancient remains. Despite the fact that success in ancient DNA studies is typically obtained by screening many samples to identify a promising subset, ancient DNA protocols have not, in general, focused on reducing the time required to screen samples. We present an adaptation of a popular ancient library preparation method that makes screening more efficient. First, the DNA extract is treated using a protocol that causes characteristic ancient DNA damage to be restricted to the terminal nucleotides, while nearly eliminating it in the interior of the DNA molecules, allowing a single library to be used both to test for ancient DNA authenticity and to carry out population genetic analysis. Second, the DNA molecules are ligated to a unique pair of barcodes, which eliminates undetected cross-contamination from this step onwards. Third, the barcoded library molecules include incomplete adapters of short length that can increase the specificity of hybridization-based genomic target enrichment. The adapters are completed just before sequencing, so the same DNA library can be used in multiple experiments, and the sequences distinguished. We demonstrate this protocol on 60 ancient human samples. PMID:25487342

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

    PubMed

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

    2014-04-01

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

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

    PubMed Central

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

    2014-01-01

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

  15. 3-methyladenine, an autophagic inhibitor, attenuates therapeutic effects of sirolimus on scopolamine-induced cognitive dysfunction in a rat model

    PubMed Central

    Zhu, Bin; Yang, Chun; Ding, Liang-Cai; Liu, Ning

    2014-01-01

    Previous studies have demonstrated that sirolimus has therapeutic effects for Alzheimer’s disease which characterized by cognitive dysfunction. However, its underlying mechanisms have not been fully elucidated. In the present study, we aimed to investigate the mechanisms of therapeutic effects of sirolimus for cognitive dysfunction rat model which induced by chronic administration of scopolamine. Forty Wistar rats were randomly divided into 4 groups (n=10 each): saline group and scopolamine group, sirolimus plus scopolamine group and 3-methyladenine pretreatment group. Morris water maze test was applied to measure the cognitive function of rat. After behavioral test, rats were sacrificed and prefrontal cortex and hippocampus were harvested for measuring amyloid-β (Aβ), Beclin-1 and mammalian target of rapamycin (mTOR). Compared with saline group, scopolamine administered significantly decreased the cognitive performance of rats during the Morris water maze test and changed Aβ, Beclin-1 and mTOR levels in rat prefrontal cortex and hippocampus (P<0.05); In addition, rats in sirolimus plus scopolamine group significantly reversed scopolamine-induced effects (P<0.05). Most importantly, 3-methyladenine abrogated the effects of sirolimus on scopolamine-induced cognitive dysfunction (P<0.05). In conclusion, the mechanism of sirolimus exerting therapeutic effects for scopolamine-induced cognitive dysfunction is likely related to the activation of autophagy. PMID:25419365

  16. Evaluation of the Role of the Vaccinia Virus Uracil DNA Glycosylase and A20 Proteins as Intrinsic Components of the DNA Polymerase Holoenzyme*

    PubMed Central

    Boyle, Kathleen A.; Stanitsa, Eleni S.; Greseth, Matthew D.; Lindgren, Jill K.; Traktman, Paula

    2011-01-01

    The vaccinia virus DNA polymerase is inherently distributive but acquires processivity by associating with a heterodimeric processivity factor comprised of the viral A20 and D4 proteins. D4 is also an enzymatically active uracil DNA glycosylase (UDG). The presence of an active repair protein as an essential component of the polymerase holoenzyme is a unique feature of the replication machinery. We have shown previously that the A20-UDG complex has a stoichiometry of ∼1:1, and our data suggest that A20 serves as a bridge between polymerase and UDG. Here we show that conserved hydrophobic residues in the N′ terminus of A20 are important for its binding to UDG. Our data argue against the assembly of D4 into higher order multimers, suggesting that the processivity factor does not form a toroidal ring around the DNA. Instead, we hypothesize that the intrinsic, processive DNA scanning activity of UDG tethers the holoenzyme to the DNA template. The inclusion of UDG as an essential holoenzyme component suggests that replication and base excision repair may be coupled. Here we show that the DNA polymerase can utilize dUTP as a substrate in vitro. Moreover, uracil moieties incorporated into the nascent strand during holoenzyme-mediated DNA synthesis can be excised by the viral UDG present within this holoenzyme, leaving abasic sites. Finally, we show that the polymerase stalls upon encountering an abasic site in the template strand, indicating that, like many replicative polymerases, the poxviral holoenzyme cannot perform translesion synthesis across an abasic site. PMID:21572084

  17. Standard role for a conserved aspartate or more direct involvement in deglycosylation? An ONIOM and MD investigation of adenine-DNA glycosylase.

    PubMed

    Kellie, Jennifer L; Wilson, Katie A; Wetmore, Stacey D

    2013-12-01

    8-Oxoguanine (OG) is one of the most frequently occurring forms of DNA damage and is particularly deleterious since it forms a stable Hoogsteen base pair with adenine (A). The repair of an OG:A mispair is initiated by adenine-DNA glycosylase (MutY), which hydrolyzes the sugar-nucleobase bond of the adenine residue before the lesion is processed by other proteins. MutY has been proposed to use a two-part chemical step involving protonation of the adenine nucleobase, followed by SN1 hydrolysis of the glycosidic bond. However, differences between a recent (fluorine recognition complex, denoted as the FLRC) crystal structure and the structure on which most mechanistic conclusions have been based to date (namely, the lesion recognition complex or LRC) raise questions regarding the mechanism used by MutY and the discrete role of various active-site residues. The present work uses both molecular dynamics (MD) and quantum mechanical (ONIOM) models to compare the active-site conformational dynamics in the two crystal structures, which suggests that only the understudied FLRC leads to a catalytically competent reactant. Indeed, all previous computational studies on MutY have been initiated from the LRC structure. Subsequently, for the first time, various mechanisms are examined with detailed ONIOM(M06-2X:PM6) reaction potential energy surfaces (PES) based on the FLRC structure, which significantly extends the mechanistic picture. Specifically, our work reveals that the reaction proceeds through a different route than the commonly accepted mechanism and the catalytic function of various active-site residues (Geobacillus stearothermophilus numbering). Specifically, contrary to proposals based on the LRC, E43 is determined to solely be involved in the initial adenine protonation step and not the deglycosylation reaction as the general base. Additionally, a novel catalytic role is proposed for Y126, whereby this residue plays a significant role in stabilizing the highly charged

  18. Naturally occurring polyphenol, morin hydrate, inhibits enzymatic activity of N-methylpurine DNA glycosylase, a DNA repair enzyme with various roles in human disease

    PubMed Central

    Dixon, Monica; Woodrick, Jordan; Gupta, Suhani; Karmahapatra, Soumendra Krishna; Devito, Stephen; Vasudevan, Sona; Dakshanamurthy, Sivanesan; Adhikari, Sanjay; Yenugonda, Venkata M.; Roy, Rabindra

    2015-01-01

    Interest in the mechanisms of DNA repair pathways, including the base excision repair (BER) pathway specifically, has heightened since these pathways have been shown to modulate important aspects of human disease. Modulation of the expression or activity of a particular BER enzyme, N-methylpurine DNA glycosylase (MPG), has been demonstrated to play a role in carcinogenesis and resistance to chemotherapy as well as neurodegenerative diseases, which has intensified the focus on studying MPG-related mechanisms of repair. A specific small molecule inhibitor for MPG activity would be a valuable biochemical tool for understanding these repair mechanisms. By screening several small molecule chemical libraries, we identified a natural polyphenolic compound, morin hydrate, which inhibits MPG activity specifically (IC50 = 2.6 µM). Detailed mechanism analysis showed that morin hydrate inhibited substrate DNA binding of MPG, and eventually the enzymatic activity of MPG. Computational docking studies with an x-ray derived MPG structure as well as comparison studies with other structurally-related flavanoids offer a rationale for the inhibitory activity of morin hydrate observed. The results of this study suggest that the morin hydrate could be an effective tool for studying MPG function and it is possible that morin hydrate and its derivatives could be utilized in future studies focused on the role of MPG in human disease. PMID:25650313

  19. "Light-up" Sensing of human 8-oxoguanine DNA glycosylase activity by target-induced autocatalytic DNAzyme-generated rolling circle amplification.

    PubMed

    Kong, Xiang-Juan; Wu, Shuang; Cen, Yao; Yu, Ru-Qin; Chu, Xia

    2016-05-15

    Human 8-oxoguanine DNA glycosylase (hOGG1) plays a crucial role in maintaining the genomic integrity of living organisms for its capability of repairing DNA oxidative damage. The expression level of hOGG1 is closely associated with many diseases including various kinds of cancers. In this study, a novel "light-up" sensor based on target-induced formation of 5' phosphorylated probe and autocatalytic DNAzyme-generated rolling circle amplification has been developed for highly sensitive human 8-oxoguanine DNA glycosylase (hOGG1) activity assay. The approach reaches detection limit as low as 0.001U/mL for hOGG1 via scarcely increased background signal and dual signal amplification strategy. To the best of our knowledge, it is one of the most sensitive methods for the detection of base excision repair enzyme. Moreover, the approach shows excellent specificity over other nonspecific enzymes would interfere with the assay and holds great promise for application in real sample analysis. Hence, the proposed method provides a highly sensitive, selective, and desirable hOGG1 sensing platform. PMID:26765532

  20. Association between oxidative DNA damage and the expression of 8-oxoguanine DNA glycosylase 1 in lung epithelial cells of neonatal rats exposed to hyperoxia

    PubMed Central

    JIN, LINLIN; YANG, HAIPING; FU, JIANHUA; XUE, XINDONG; YAO, LI; QIAO, LIN

    2015-01-01

    Previous studies have demonstrated that oxidative stress-induced lung injury is involved in the occurrence and developmental process of bronchopulmonary dysplasia (BPD). The present study assessed whether oxidative DNA damage occurs in the early stages of hyperoxia-induced BPD in neonatal rats and evaluated the expression and localization of the DNA repair gene, 8-oxoguanine DNA glycosylase 1 (OGG1), upon exposure to hyperoxia. Neonatal rats and primary cultured neonatal rat alveolar epithelial type II (AECII) cells were exposed to hyperoxia (90% O2) or normoxia (21% O2) and the expression levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in the lung tissues and AECII cells were determined using a competitive enzyme-linked immunosorbent assay. DNA strand breaks in the AECII cells were detected using a comet assay. The expression and localization of the OGG1 protein in the lung tissues and AECII cells were determined by immunofluorescence confocal microscopy and western blotting. The mRNA expression levels of OGG1 in the lung tissues and AECII cells were determined by reverse transcription polymerase chain reaction. The expression of 8-OHdG was elevated in the hyperoxia-exposed neonatal rat lung tissue and the AECII cells compared with the normoxic controls. The occurrence of DNA strand breaks in the AECII cells increased with increasing duration of hyperoxia exposure. The protein expression of OGG1 was significantly increased in the hyperoxia-exposed lung tissues and AECII cells, with OGG1 preferentially localized to the cytoplasm. No concomitant increase in the mRNA expression of OGG1 was detected. These results revealed that oxidative DNA damage occurred in lung epithelial cells during early-stage BPD, as confirmed by in vitro and in vivo hyperoxia exposure experiments, and the increased expression of OGG1 was associated with this process. PMID:25672835

  1. Mitochondrial-targeted DNA repair enzyme 8-oxoguanine DNA glycosylase 1 protects against ventilator-induced lung injury in intact mice

    PubMed Central

    Hashizume, Masahiro; Mouner, Marc; Chouteau, Joshua M.; Gorodnya, Olena M.; Ruchko, Mykhaylo V.; Potter, Barry J.; Wilson, Glenn L.; Gillespie, Mark N.

    2013-01-01

    This study tested the hypothesis that oxidative mitochondrial-targeted DNA (mtDNA) damage triggered ventilator-induced lung injury (VILI). Control mice and mice infused with a fusion protein targeting the DNA repair enzyme, 8-oxoguanine-DNA glycosylase 1 (OGG1) to mitochondria were mechanically ventilated with a range of peak inflation pressures (PIP) for specified durations. In minimal VILI (1 h at 40 cmH2O PIP), lung total extravascular albumin space increased 2.8-fold even though neither lung wet/dry (W/D) weight ratios nor bronchoalveolar lavage (BAL) macrophage inflammatory protein (MIP)-2 or IL-6 failed to differ from nonventilated or low PIP controls. This increase in albumin space was attenuated by OGG1. Moderately severe VILI (2 h at 40 cmH2O PIP) produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio and marked increases in BAL MIP-2 and IL-6, accompanied by oxidative mitochondrial DNA damage, as well as decreases in the total tissue glutathione (GSH) and GSH/GSSH ratio compared with nonventilated lungs. All of these injury indices were attenuated in OGG1-treated mice. At the highest level of VILI (2 h at 50 cmH2O PIP), OGG1 failed to protect against massive lung edema and BAL cytokines or against depletion of the tissue GSH pool. Interestingly, whereas untreated mice died before completing the 2-h protocol, OGG1-treated mice lived for the duration of observation. Thus mitochondrially targeted OGG1 prevented VILI over a range of ventilation times and pressures and enhanced survival in the most severely injured group. These findings support the concept that oxidative mtDNA damage caused by high PIP triggers induction of acute lung inflammation and injury. PMID:23241530

  2. A highly conserved family of domains related to the DNA-glycosylase fold helps predict multiple novel pathways for RNA modifications

    PubMed Central

    Burroughs, A Maxwell; Aravind, L

    2014-01-01

    A protein family including mammalian NEMF, Drosophila caliban, yeast Tae2, and bacterial FpbA-like proteins was first defined over a decade ago and found to be universally distributed across the three domains/superkingdoms of life. Since its initial characterization, this family of proteins has been tantalizingly linked to a wide range of biochemical functions. Tapping the enormous wealth of genome information that has accumulated since the initial characterization of these proteins, we perform a detailed computational analysis of the family, identifying multiple conserved domains. Domains identified include an enzymatic domain related to the formamidopyrimidine (Fpg), MutM, and Nei/EndoVIII family of DNA glycosylases, a novel, predicted RNA-binding domain, and a domain potentially mediating protein–protein interactions. Through this characterization, we predict that the DNA glycosylase-like domain catalytically operates on double-stranded RNA, as part of a hitherto unknown base modification mechanism that probably targets rRNAs. At least in archaea, and possibly eukaryotes, this pathway might additionally include the AMMECR1 family of proteins. The predicted RNA-binding domain associated with this family is also observed in distinct architectural contexts in other proteins across phylogenetically diverse prokaryotes. Here it is predicted to play a key role in a new pathway for tRNA 4-thiouridylation along with TusA-like sulfur transfer proteins. PMID:24646681

  3. Selective inhibition by methoxyamine of the apurinic/apyrimidinic endonuclease activity associated with pyrimidine dimer-DNA glycosylases from Micrococcus luteus and bacteriophage T4

    SciTech Connect

    Liuzzi, M.; Weinfeld, M.; Paterson, M.C.

    1987-06-16

    The UV endonucleases from Micrococcus luteus and bacteriophage T4 possess two catalytic activities specific for the site of cyclobutane pyrimidine dimers in UV-irradiated DNA: a DNA glycosylase that cleaves the 5'-glycosyl bond of the dimerized pyrimidines and an apurinic/apyrimidinic (AP) endonuclease that thereupon incises the phosphodiester bond 3' to the resulting apyrimidinic site. The authors have explored the potential use of methoxyamine, a chemical that reacts at neutral pH with AP sites in DNA, as a selective inhibitor of the AP endonuclease activities residing in the M. luteus and T4 enzymes. The presence of 50 mM methoxyamine during incubation of UV-treated, (/sup 3/H)thymine-labeled poly(dA) x poly(dT) with either enzyme preparation was found to protect completely the irradiated copolymer from endonucleolytic attack at dimer sites, as assayed by yield of acid-soluble radioactivity. In contrast, the dimer-DNA glycosylase activity of each enzyme remained fully functional, as monitored retrospectively by release of free thymine after either photochemical-(5 kJ/m/sup 2/, 254 nm) or photoenzymic- (Escherichia coli photolyase plus visible light) induced reversal of pyrimidine dimers in the UV-damaged substrate. The data demonstrate that the inhibition of the strand-incision reaction arises because of chemical modification of the AP sites and is not due to inactivation of the enzyme by methoxyamine. The results, combined with earlier findings for 5'-acting AP endonucleases, strongly suggest that methoxyamine is a highly specific inhibitor of virtually all AP endonucleases, irrespective of their modes of action, and may therefore prove useful in a wide variety of DNA repair studies.

  4. Influence of local duplex stability and N6-methyladenine on uracil recognition by mismatch-specific uracil-DNA glycosylase (Mug).

    PubMed

    Valinluck, Victoria; Liu, Pingfang; Burdzy, Artur; Ryu, Junichi; Sowers, Lawrence C

    2002-12-01

    To maintain genomic integrity, DNA repair enzymes continually remove damaged bases and lesions resulting from endogenous and exogenous processes. These repair enzymes must distinguish damaged bases from normal bases to prevent the inadvertent removal of normal bases, which would promote genomic instability. The mechanisms by which this high level of specificity is accomplished are as yet unresolved. One member of the uracil-DNA glycosylase family of repair enzymes, Escherichia coli mismatch-specific uracil-DNA glycosylase (Mug), is reported to distinguish U:G mispairs from U:A base pairs based upon specific contacts with the mispaired guanine after flipping the target uracil out of the duplex. However, recent studies suggest other mechanisms for base selection, including local duplex stability. In this study, we used the modified base N6-methyladenine to probe the effect of local helix perturbation on Mug recognition of uracil. N6-Methyladenine is found in E. coli as part of both the mismatch repair and restriction-modification systems. In its cis isomer, N6-methyladenine destabilizes hydrogen bonding by interfering with pseudo-Watson-Crick base pairing. It is observed that the selection of uracil by Mug is sequence dependent and that uracil residues in sequences of reduced thermostability are preferentially removed. The replacement of adenine by N6-methyladenine increases the frequency of removal of the uracil residue paired opposite the modified adenine. These results are in accord with suggestions that local helix stability is an important determinant of base recognition by some DNA repair enzymes and provide a potential strategy for identifying the sequence location of modified bases in DNA. PMID:12482242

  5. The levels of 7,8-dihydrodeoxyguanosine (8-oxoG) and 8-oxoguanine DNA glycosylase 1 (OGG1) - A potential diagnostic biomarkers of Alzheimer's disease.

    PubMed

    Sliwinska, Agnieszka; Kwiatkowski, Dominik; Czarny, Piotr; Toma, Monika; Wigner, Paulina; Drzewoski, Jozef; Fabianowska-Majewska, Krystyna; Szemraj, Janusz; Maes, Michael; Galecki, Piotr; Sliwinski, Tomasz

    2016-09-15

    Evidence indicates that oxidative stress contributes to neuronal cell death in Alzheimer's disease (AD). Increased oxidative DNA damage l, as measured with 8-oxoguanine (8-oxoG), and reduced capacity of proteins responsible for removing of DNA damage, including 8-oxoguanine DNA glycosylase 1 (OGG1), were detected in brains of AD patients. In the present study we assessed peripheral blood biomarkers of oxidative DNA damage, i.e. 8- oxoG and OGG1, in AD diagnosis, by comparing their levels between the patients and the controls. Our study was performed on DNA and serum isolated from peripheral blood taken from 100 AD patients and 110 controls. For 8-oxoG ELISA was employed. The OGG1 level was determined using ELISA and Western blot technique. Levels of 8-oxoG were significantly higher in DNA of AD patients. Both ELISA and Western blot showed decreased levels of OGG1 in serum of AD patients. Our results show that oxidative DNA damage biomarkers detected in peripheral tissue could reflect the changes occurring in the brain of patients with AD. These results also suggest that peripheral blood samples may be useful to measure oxidative stress biomarkers in AD. PMID:27538622

  6. Label-free and selective photoelectrochemical detection of chemical DNA methylation damage using DNA repair enzymes.

    PubMed

    Wu, Yiping; Zhang, Bintian; Guo, Liang-Hong

    2013-07-16

    Exogenous chemicals may produce DNA methylation that is potentially toxic to living systems. Methylated DNA bases are difficult to detect with biosensors because the methyl group is small and chemically inert. In this report, a label-free photoelectrochemical sensor was developed for the selective detection of chemically methylated bases in DNA films. The sensor employed two DNA repair enzymes, human alkyladenine DNA glycosylase and human apurinic/apyrimidinic endonuclease, to convert DNA methylation sites in DNA films on indium tin oxide electrodes into strand breaks. A DNA intercalator, Ru(bpy)2(dppz)(2+) (bpy=2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) was then used as the photoelectrochemical signal indicator to detect the DNA strand breaks. Its photocurrent signal was found to correlate inversely with the amount of 3-methyladenines (metAde) produced with a methylating agent, methylmethane sulfonate (MMS). The sensor detected the methylated bases produced with as low as 1 mM MMS, at which concentration the amount of metAde on the sensor surface was estimated to be 0.5 pg, or 1 metAde in 1.6 × 10(5) normal bases. Other DNA base modification products, such as 5-methylcytosine and DNA adducts with ethyl and styrene groups did not attenuate the photocurrent, demonstrating good selectivity of the sensor. This strategy can be utilized to develop sensors for the detection of other modified DNA bases with specific DNA repair enzymes. PMID:23777269

  7. Aberrant repair initiated by mismatch-specific thymine-DNA glycosylases provides a mechanism for the mutational bias observed in CpG islands

    PubMed Central

    Talhaoui, Ibtissam; Couve, Sophie; Gros, Laurent; Ishchenko, Alexander A.; Matkarimov, Bakhyt; Saparbaev, Murat K.

    2014-01-01

    The human thymine-DNA glycosylase (TDG) initiates the base excision repair (BER) pathway to remove spontaneous and induced DNA base damage. It was first biochemically characterized for its ability to remove T mispaired with G in CpG context. TDG is involved in the epigenetic regulation of gene expressions by protecting CpG-rich promoters from de novo DNA methylation. Here we demonstrate that TDG initiates aberrant repair by excising T when it is paired with a damaged adenine residue in DNA duplex. TDG targets the non-damaged DNA strand and efficiently excises T opposite of hypoxanthine (Hx), 1,N6-ethenoadenine, 7,8-dihydro-8-oxoadenine and abasic site in TpG/CpX context, where X is a modified residue. In vitro reconstitution of BER with duplex DNA containing Hx•T pair and TDG results in incorporation of cytosine across Hx. Furthermore, analysis of the mutation spectra inferred from single nucleotide polymorphisms in human population revealed a highly biased mutation pattern within CpG islands (CGIs), with enhanced mutation rate at CpA and TpG sites. These findings demonstrate that under experimental conditions used TDG catalyzes sequence context-dependent aberrant removal of thymine, which results in TpG, CpA→CpG mutations, thus providing a plausible mechanism for the putative evolutionary origin of the CGIs in mammalian genomes. PMID:24692658

  8. Structural and biophysical analysis of interactions between cod and human uracil-DNA N-glycosylase (UNG) and UNG inhibitor (Ugi)

    SciTech Connect

    Assefa, Netsanet Gizaw; Niiranen, Laila; Johnson, Kenneth A.; Leiros, Hanna-Kirsti Schrøder; Smalås, Arne Oskar; Willassen, Nils Peder; Moe, Elin

    2014-08-01

    A structural and biophysical study of the interactions between cod and human uracil-DNA N-glycosylase (UNG) and their inhibitor Ugi is presented. The stronger interaction between cod UNG and Ugi can be explained by a greater positive electrostatic surface potential. Uracil-DNA N-glycosylase from Atlantic cod (cUNG) shows cold-adapted features such as high catalytic efficiency, a low temperature optimum for activity and reduced thermal stability compared with its mesophilic homologue human UNG (hUNG). In order to understand the role of the enzyme–substrate interaction related to the cold-adapted properties, the structure of cUNG in complex with a bacteriophage encoded natural UNG inhibitor (Ugi) has been determined. The interaction has also been analyzed by isothermal titration calorimetry (ITC). The crystal structure of cUNG–Ugi was determined to a resolution of 1.9 Å with eight complexes in the asymmetric unit related through noncrystallographic symmetry. A comparison of the cUNG–Ugi complex with previously determined structures of UNG–Ugi shows that they are very similar, and confirmed the nucleotide-mimicking properties of Ugi. Biophysically, the interaction between cUNG and Ugi is very strong and shows a binding constant (K{sub b}) which is one order of magnitude larger than that for hUNG–Ugi. The binding of both cUNG and hUNG to Ugi was shown to be favoured by both enthalpic and entropic forces; however, the binding of cUNG to Ugi is mainly dominated by enthalpy, while the entropic term is dominant for hUNG. The observed differences in the binding properties may be explained by an overall greater positive electrostatic surface potential in the protein–Ugi interface of cUNG and the slightly more hydrophobic surface of hUNG.

  9. Entrapment and Structure of an Extrahelical Guanine Attempting to Enter the Active Site of a Bacterial DNA Glycosylase, MutM

    SciTech Connect

    Qi, Yan; Spong, Marie C.; Nam, Kwangho; Karplus, Martin; Verdine, Gregory L.

    2010-09-21

    MutM, a bacterial DNA glycosylase, protects genome integrity by catalyzing glycosidic bond cleavage of 8-oxoguanine (oxoG) lesions, thereby initiating base excision DNA repair. The process of searching for and locating oxoG lesions is especially challenging, because of the close structural resemblance of oxoG to its million-fold more abundant progenitor, G. Extrusion of the target nucleobase from the DNA double helix to an extrahelical position is an essential step in lesion recognition and catalysis by MutM. Although the interactions between the extruded oxoG and the active site of MutM have been well characterized, little is known in structural detail regarding the interrogation of extruded normal DNA bases by MutM. Here we report the capture and structural elucidation of a complex in which MutM is attempting to present an undamaged G to its active site. The structure of this MutM-extrahelical G complex provides insights into the mechanism MutM employs to discriminate against extrahelical normal DNA bases and into the base extrusion process in general.

  10. Exercise-Induced Neuroprotection of Hippocampus in APP/PS1 Transgenic Mice via Upregulation of Mitochondrial 8-Oxoguanine DNA Glycosylase

    PubMed Central

    Kang, Weimin; Jiang, Ning; Wang, Xun; Zhang, Yong; Ji, Li Li

    2014-01-01

    Improving mitochondrial function has been proposed as a reasonable therapeutic strategy to reduce amyloid-β (Aβ) load and to modify the progression of Alzheimer's disease (AD). However, the relationship between mitochondrial adaptation and brain neuroprotection caused by physical exercise in AD is poorly understood. This study was undertaken to investigate the effects of long-term treadmill exercise on mitochondrial 8-oxoguanine DNA glycosylase-1 (OGG1) level, mtDNA oxidative damage, and mitochondrial function in the hippocampus of APP/PS1 transgenic mouse model of AD. In the present study, twenty weeks of treadmill training significantly improved the cognitive function and reduced the expression of Aβ-42 in APP/PS1 transgenic (Tg) mice. Training also ameliorated mitochondrial respiratory function by increasing the complexes I, and IV and ATP synthase activities, whereas it attenuated ROS generation and mtDNA oxidative damage in Tg mice. Furthermore, the impaired mitochondrial antioxidant enzymes and mitochondrial OGG1 activities seen in Tg mice were restored with training. Acetylation level of mitochondrial OGG1 and MnSOD was markedly suppressed in Tg mice after exercise training, in parallel with increased level of SIRT3. These findings suggest that exercise training could increase mtDNA repair capacity in the mouse hippocampus, which in turn would result in protection against AD-related mitochondrial dysfunction and phenotypic deterioration. PMID:25538817

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

    PubMed

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

    2016-04-22

    Thymine-DNA glycosylase (TDG) plays critical roles in DNA base excision repair and DNA demethylation. It has been proposed, based on structural studies and in vitro biochemistry, that sumoylation is required for efficient TDG enzymatic turnover following base excision. However, whether sumoylation is required for TDG activity in vivo has not previously been tested. We have developed an in vivo assay for TDG activity that takes advantage of its recently discovered role in DNA demethylation and selective recognition and repair of 5-carboxylcytosine. Using this assay, we investigated the role of sumoylation in regulating TDG activity through the use of TDG mutants defective for sumoylation and Small Ubiquitin-like Modifier (SUMO) binding and by altering TDG sumoylation through SUMO and SUMO protease overexpression experiments. Our findings indicate that sumoylation and SUMO binding are not essential for TDG-mediated excision and repair of 5-carboxylcytosine bases. Moreover, in vitro assays revealed that apurinic/apyrimidinic nuclease 1 provides nearly maximum stimulation of TDG processing of G·caC substrates. Thus, under our assay conditions, apurinic/apyrimidinic nuclease 1-mediated stimulation or other mechanisms sufficiently alleviate TDG product inhibition and promote its enzymatic turnover in vivo. PMID:26917720

  12. Age-dependent changes in 8-oxoguanine-DNA-glycosylase activity is modulated by adaptive responses to physical exercise in human skeletal muscle

    PubMed Central

    Radak, Zsolt; Bori, Zoltan; Koltai, Erika; Fatouros, Ioannis G.; Jamurtas, Athanasios Z.; Douroudos, Ioannis I.; Terzis, Gerasimos; Nikolaidis, Michalis G.; Chatzinikolaou, Athanasios; Sovatzidis, Apostolos; Kumagai, Shuzo; Naito, Hisahi; Boldogh, Istvan

    2012-01-01

    8-Oxo-7,8 dihydroguanine (8-oxoG) accumulates in the genome over time and is believed to contribute to the development of aging characteristics of skeletal muscle and various aging-related diseases. Here, we show a significantly increased level of intrahelical 8-oxoG and 8-oxoguanine DNA glycosylase (OGG1) expression in aged human skeletal muscle compared to that of young individuals. In response to exercise, the 8-oxoG level was found to be lastingly elevated in sedentary young and old subjects, but returned rapidly to pre-exercise levels in the DNA of physically active individuals independent of age. 8-OxoG levels in DNA were inversely correlated with the abundance of acetylated OGG1 (Ac-OGG1), but not with total OGG1, apurinic/apyrimidinic endonuclease (AP)-1 or Ac-APE1. The actual Ac-OGG1 level was linked to exercise-induced oxidative stress, as shown by changes in lipid peroxide levels and expression of Cu,Zn-SOD, Mn-SOD and SIRT3, as well as the balance between acetyl transferase p300/CBP and the deacetylase SIRT1, but not SIRT6 expression. Together these data suggest that that acetylated form of OGG1, and not OGGl itself, correlates inversely with the 8-oxoG level in the DNA of human skeletal muscle, and the Ac-OGG1 level is dependent on adaptive cellular responses to physical activity, but is age independent. PMID:21569841

  13. Exercise-induced neuroprotection of hippocampus in APP/PS1 transgenic mice via upregulation of mitochondrial 8-oxoguanine DNA glycosylase.

    PubMed

    Bo, Hai; Kang, Weimin; Jiang, Ning; Wang, Xun; Zhang, Yong; Ji, Li Li

    2014-01-01

    Improving mitochondrial function has been proposed as a reasonable therapeutic strategy to reduce amyloid-β (Aβ) load and to modify the progression of Alzheimer's disease (AD). However, the relationship between mitochondrial adaptation and brain neuroprotection caused by physical exercise in AD is poorly understood. This study was undertaken to investigate the effects of long-term treadmill exercise on mitochondrial 8-oxoguanine DNA glycosylase-1 (OGG1) level, mtDNA oxidative damage, and mitochondrial function in the hippocampus of APP/PS1 transgenic mouse model of AD. In the present study, twenty weeks of treadmill training significantly improved the cognitive function and reduced the expression of Aβ-42 in APP/PS1 transgenic (Tg) mice. Training also ameliorated mitochondrial respiratory function by increasing the complexes I, and IV and ATP synthase activities, whereas it attenuated ROS generation and mtDNA oxidative damage in Tg mice. Furthermore, the impaired mitochondrial antioxidant enzymes and mitochondrial OGG1 activities seen in Tg mice were restored with training. Acetylation level of mitochondrial OGG1 and MnSOD was markedly suppressed in Tg mice after exercise training, in parallel with increased level of SIRT3. These findings suggest that exercise training could increase mtDNA repair capacity in the mouse hippocampus, which in turn would result in protection against AD-related mitochondrial dysfunction and phenotypic deterioration. PMID:25538817

  14. Hyperosmotic stress memory in Arabidopsis is mediated by distinct epigenetically labile sites in the genome and is restricted in the male germline by DNA glycosylase activity

    PubMed Central

    Wibowo, Anjar; Becker, Claude; Marconi, Gianpiero; Durr, Julius; Price, Jonathan; Hagmann, Jorg; Papareddy, Ranjith; Putra, Hadi; Kageyama, Jorge; Becker, Jorg; Weigel, Detlef; Gutierrez-Marcos, Jose

    2016-01-01

    Inducible epigenetic changes in eukaryotes are believed to enable rapid adaptation to environmental fluctuations. We have found distinct regions of the Arabidopsis genome that are susceptible to DNA (de)methylation in response to hyperosmotic stress. The stress-induced epigenetic changes are associated with conditionally heritable adaptive phenotypic stress responses. However, these stress responses are primarily transmitted to the next generation through the female lineage due to widespread DNA glycosylase activity in the male germline, and extensively reset in the absence of stress. Using the CNI1/ATL31 locus as an example, we demonstrate that epigenetically targeted sequences function as distantly-acting control elements of antisense long non-coding RNAs, which in turn regulate targeted gene expression in response to stress. Collectively, our findings reveal that plants use a highly dynamic maternal ‘short-term stress memory’ with which to respond to adverse external conditions. This transient memory relies on the DNA methylation machinery and associated transcriptional changes to extend the phenotypic plasticity accessible to the immediate offspring. DOI: http://dx.doi.org/10.7554/eLife.13546.001 PMID:27242129

  15. Uracil DNA Glycosylase Is Dispensable for Human Immunodeficiency Virus Type 1 Replication and Does Not Contribute to the Antiviral Effects of the Cytidine Deaminase Apobec3G

    PubMed Central

    Kaiser, Shari M.; Emerman, Michael

    2006-01-01

    It is well established that many host factors are involved in the replication of human immunodeficiency virus (HIV) type 1. One host protein, uracil DNA glycosylase 2 (UNG2), binds to multiple viral proteins and is packaged into HIV type 1 virions. UNG initiates the removal of uracils from DNA, and this has been proposed to be important both for reverse transcription and as a mediator to the antiviral effect of virion-incorporated Apobec3G, a cytidine deaminase that generates numerous uracils in the viral DNA during virus replication. We used a natural human UNG−/− cell line as well as cells that express a potent catalytic active-site inhibitor of UNG to assess the effects of removing UNG activity on HIV infectivity. In both cases, we find UNG2 activity and protein to be completely dispensable for virus replication. Moreover, we find that virion-associated UNG2 does not affect the loss of infectivity caused by Apobec3G. PMID:16378989

  16. Hyperosmotic stress memory in Arabidopsis is mediated by distinct epigenetically labile sites in the genome and is restricted in the male germline by DNA glycosylase activity.

    PubMed

    Wibowo, Anjar; Becker, Claude; Marconi, Gianpiero; Durr, Julius; Price, Jonathan; Hagmann, Jorg; Papareddy, Ranjith; Putra, Hadi; Kageyama, Jorge; Becker, Jorg; Weigel, Detlef; Gutierrez-Marcos, Jose

    2016-01-01

    Inducible epigenetic changes in eukaryotes are believed to enable rapid adaptation to environmental fluctuations. We have found distinct regions of the Arabidopsis genome that are susceptible to DNA (de)methylation in response to hyperosmotic stress. The stress-induced epigenetic changes are associated with conditionally heritable adaptive phenotypic stress responses. However, these stress responses are primarily transmitted to the next generation through the female lineage due to widespread DNA glycosylase activity in the male germline, and extensively reset in the absence of stress. Using the CNI1/ATL31 locus as an example, we demonstrate that epigenetically targeted sequences function as distantly-acting control elements of antisense long non-coding RNAs, which in turn regulate targeted gene expression in response to stress. Collectively, our findings reveal that plants use a highly dynamic maternal 'short-term stress memory' with which to respond to adverse external conditions. This transient memory relies on the DNA methylation machinery and associated transcriptional changes to extend the phenotypic plasticity accessible to the immediate offspring. PMID:27242129

  17. The simian varicella virus uracil DNA glycosylase and dUTPase genes are expressed in vivo, but are non-essential for replication in cell culture

    PubMed Central

    Ward, Toby M.; Williams, Marshall V.; Traina-Dorge, Vicki; Gray, Wayne L.

    2012-01-01

    Neurotropic herpesviruses express viral deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and uracil DNA glycosylase (UDG) enzymes which may reduce uracil misincorporation into viral DNA, particularly in neurons of infected ganglia. The simian varicella virus (SVV) dUTPase (ORF 8) and UDG (ORF 59) share 37.7% and 53.9% amino acid identity, respectively, with varicella-zoster virus (VZV) homologs. Infectious SVV mutants defective in either dUTPase (SVV-dUTPase−) or UDG (SVV-UDG−) activity or both (SVV-dUTPase−/UDG−) were constructed using recA assisted endonuclease cleavage (RARE) and a cosmid recombination system. Loss of viral dUTPase and UDG enzymatic activity was confirmed in CV-1 cells infected with the SVV mutants. The SVV-dUTPase−, SVV-UDG−, and SVV-dUTPase−/UDG− mutants replicated as efficiently as wild-type SVV in cell culture. SVV dUTPase and UDG expression was detected in tissues derived from acutely infected animals, but not in tissues derived from latently infected animals. Further studies will evaluate the pathogenesis of SVV dUTPase and UDG mutants and their potential as varicella vaccines. PMID:19200445

  18. Abnormal Expressions of DNA Glycosylase Genes NEIL1, NEIL2, and NEIL3 Are Associated with Somatic Mutation Loads in Human Cancer

    PubMed Central

    Shinmura, Kazuya; Kato, Hisami; Kawanishi, Yuichi; Igarashi, Hisaki; Goto, Masanori; Tao, Hong; Inoue, Yusuke; Nakamura, Satoki; Misawa, Kiyoshi; Mineta, Hiroyuki; Sugimura, Haruhiko

    2016-01-01

    The effects of abnormalities in the DNA glycosylases NEIL1, NEIL2, and NEIL3 on human cancer have not been fully elucidated. In this paper, we found that the median somatic total mutation loads and the median somatic single nucleotide mutation loads exhibited significant inverse correlations with the median NEIL1 and NEIL2 expression levels and a significant positive correlation with the median NEIL3 expression level using data for 13 cancer types from the Cancer Genome Atlas (TCGA) database. A subset of the cancer types exhibited reduced NEIL1 and NEIL2 expressions and elevated NEIL3 expression, and such abnormal expressions of NEIL1, NEIL2, and NEIL3 were also significantly associated with the mutation loads in cancer. As a mechanism underlying the reduced expression of NEIL1 in cancer, the epigenetic silencing of NEIL1 through promoter hypermethylation was found. Finally, we investigated the reason why an elevated NEIL3 expression level was associated with an increased number of somatic mutations in cancer and found that NEIL3 expression was positively correlated with the expression of APOBEC3B, a potent inducer of mutations, in diverse cancers. These results suggested that the abnormal expressions of NEIL1, NEIL2, and NEIL3 are involved in cancer through their association with the somatic mutation load. PMID:27042257

  19. X4 and R5 HIV-1 have distinct post-entry requirements for uracil DNA glycosylase during infection of primary cells.

    PubMed

    Jones, Kate L; Roche, Michael; Gantier, Michael P; Begum, Nasim A; Honjo, Tasuku; Caradonna, Salvatore; Williams, Bryan R G; Mak, Johnson

    2010-06-11

    It has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinct requirements for host cell uracil DNA glycosylase (UNG2) during the early stage of infection. UNG2 has been previously implicated in HIV infection, but its precise role remains controversial. In this study we show that, although UNG2 is highly expressed in different cell lines, UNG2 levels are low in the natural host cells of HIV. Short interfering RNA knockdown of endogenous UNG2 in primary cells showed that UNG2 is required for R5 but not X4 HIV infection and that this requirement is bypassed when HIV enters the target cell via vesicular stomatitis virus envelope-glycoprotein-mediated endocytosis. We also show that short interfering RNA knockdown of UNG2 in virus-producing primary cells leads to defective R5 HIV virions that are unable to complete viral cDNA synthesis. Quantitative PCR analysis revealed that endogenous UNG2 levels are transiently up-regulated post HIV infection, and this increase in UNG2 mRNA is approximately 10-20 times higher in R5 versus X4 HIV-infected cells. Our data show that both virion-associated UNG2 and HIV infection-induced UNG2 expression are critical for reverse transcription during R5 but not X4 HIV infection. More importantly, we have made the novel observation that R5 and X4 HIV have distinct host cell factor requirements and differential capacities to induce gene expression during the early stages of infection. These differences may result from activation of distinct signaling cascades and/or infection of divergent T-lymphocyte subpopulations. PMID:20371602

  20. X4 and R5 HIV-1 Have Distinct Post-entry Requirements for Uracil DNA Glycosylase during Infection of Primary Cells

    PubMed Central

    Jones, Kate L.; Roche, Michael; Gantier, Michael P.; Begum, Nasim A.; Honjo, Tasuku; Caradonna, Salvatore; Williams, Bryan R. G.; Mak, Johnson

    2010-01-01

    It has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinct requirements for host cell uracil DNA glycosylase (UNG2) during the early stage of infection. UNG2 has been previously implicated in HIV infection, but its precise role remains controversial. In this study we show that, although UNG2 is highly expressed in different cell lines, UNG2 levels are low in the natural host cells of HIV. Short interfering RNA knockdown of endogenous UNG2 in primary cells showed that UNG2 is required for R5 but not X4 HIV infection and that this requirement is bypassed when HIV enters the target cell via vesicular stomatitis virus envelope-glycoprotein-mediated endocytosis. We also show that short interfering RNA knockdown of UNG2 in virus-producing primary cells leads to defective R5 HIV virions that are unable to complete viral cDNA synthesis. Quantitative PCR analysis revealed that endogenous UNG2 levels are transiently up-regulated post HIV infection, and this increase in UNG2 mRNA is ∼10–20 times higher in R5 versus X4 HIV-infected cells. Our data show that both virion-associated UNG2 and HIV infection-induced UNG2 expression are critical for reverse transcription during R5 but not X4 HIV infection. More importantly, we have made the novel observation that R5 and X4 HIV have distinct host cell factor requirements and differential capacities to induce gene expression during the early stages of infection. These differences may result from activation of distinct signaling cascades and/or infection of divergent T-lymphocyte subpopulations. PMID:20371602

  1. 8-Oxoguanine DNA glycosylase-1 augments pro-inflammatory gene expression by facilitating the recruitment of site-specific transcription factors

    PubMed Central

    Ba, Xueqing; Bacsi, Attila; Luo, Jixian; Aguilera-Aguirre, Leopoldo; Zeng, Xianlu; Radak, Zsolt; Brasier, Allan R; Boldogh, Istvan

    2014-01-01

    Among the insidious DNA base lesions, 8-oxo-7,8-dihydroguanine (8-oxoG) is one of the most abundant, a lesion that arises through the attack by reactive oxygen species on guanine, especially when located in cis-regulatory elements. 8-oxoG is repaired by the 8-oxoguanine glycosylase 1 (OGG1)-initiated DNA base excision repair (BER) pathway. Here we investigated whether 8-oxoG repair by OGG1 in promoter regions is compatible with a prompt gene expression and a host innate immune response. For this purpose, we utilized a mouse model of airway inflammation, supplemented with cell cultures, chromatin immunoprecipitation, siRNA knockdown, real-time PCR, Comet and reporter transcription assays. Our data show that exposure of cells to tumor necrosis factor alpha (TNF-α) altered cellular redox, increased the 8-oxoG level in DNA, recruited OGG1 to promoter sequences and transiently inhibited BER of 8-oxoG. Promoter-associated OGG1 then enhanced NF-êB/RelA binding to cis-elements and facilitated recruitment of Specificity Protein 1 (SP1), transcription initiation factor II-D (TFIID), and phospho-RNA polymerase II, resulting in the rapid expression of chemokines/cytokines and inflammatory cell accumulation in mouse airways. siRNA depletion of OGG1 or prevention of guanine oxidation significantly decreased TNF-α-induced inflammatory responses. Together, these results show that non-productive binding of OGG1 to 8-oxoG in promoter sequences could be an epigenetic mechanism to modulate gene expression for a prompt innate immune response. PMID:24489103

  2. Effects of vaccinia virus uracil DNA glycosylase catalytic site and deoxyuridine triphosphatase deletion mutations individually and together on replication in active and quiescent cells and pathogenesis in mice

    PubMed Central

    De Silva, Frank S; Moss, Bernard

    2008-01-01

    Background Low levels of uracil in DNA result from misincorporation of dUMP or cytosine deamination. Vaccinia virus (VACV), the prototype poxvirus, encodes two enzymes that can potentially reduce the amount of uracil in DNA. Deoxyuridine triphosphatase (dUTPase) hydrolyzes dUTP, generating dUMP for biosynthesis of thymidine nucleotides while decreasing the availability of dUTP for misincorporation; uracil DNA glycosylase (UNG) cleaves uracil N-glycosylic bonds in DNA initiating base excision repair. Studies with actively dividing cells showed that the VACV UNG protein is required for DNA replication but the UNG catalytic site is not, whereas the dUTPase gene can be deleted without impairing virus replication. Recombinant VACV with an UNG catalytic site mutation was attenuated in vivo, while a dUTPase deletion mutant was not. However, the importance of the two enzymes for replication in quiescent cells, their possible synergy and roles in virulence have not been fully assessed. Results VACV mutants lacking the gene encoding dUTPase or with catalytic site mutations in UNG and double UNG/dUTPase mutants were constructed. Replication of UNG and UNG/dUTPase mutants were slightly reduced compared to wild type or the dUTPase mutant in actively dividing cells. Viral DNA replication was reduced about one-third under these conditions. After high multiplicity infection of quiescent fibroblasts, yields of wild type and mutant viruses were decreased by 2-logs with relative differences similar to those observed in active fibroblasts. However, under low multiplicity multi-step growth conditions in quiescent fibroblasts, replication of the dUTPase/UNG mutant was delayed and 5-fold lower than that of either single mutant or parental virus. This difference was exacerbated by 1-day serial passages on quiescent fibroblasts, resulting in 2- to 3-logs lower titer of the double mutant compared to the parental and single mutant viruses. Each mutant was more attenuated than a revertant

  3. A unique dual recognition hairpin probe mediated fluorescence amplification method for sensitive detection of uracil-DNA glycosylase and endonuclease IV activities.

    PubMed

    Wu, Yushu; Yan, Ping; Xu, Xiaowen; Jiang, Wei

    2016-03-01

    Uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) play cooperative roles in uracil base-excision repair (UBER) and inactivity of either will interrupt the UBER to cause disease. Detection of UDG and Endo IV activities is crucial to evaluate the UBER process in fundamental research and diagnostic application. Here, a unique dual recognition hairpin probe mediated fluorescence amplification method was developed for sensitively and selectively detecting UDG and Endo IV activities. For detecting UDG activity, the uracil base in the probe was excised by the target enzyme to generate an apurinic/apyrimidinic (AP) site, achieving the UDG recognition. Then, the AP site was cleaved by a tool enzyme Endo IV, releasing a primer to trigger rolling circle amplification (RCA) reaction. Finally, the RCA reaction produced numerous repeated G-quadruplex sequences, which interacted with N-methyl-mesoporphyrin IX to generate an enhanced fluorescence signal. Alternatively, for detecting Endo IV activity, the uracil base in the probe was first converted into an AP site by a tool enzyme UDG. Next, the AP site was cleaved by the target enzyme, achieving the Endo IV recognition. The signal was then generated and amplified in the same way as those in the UDG activity assay. The detection limits were as low as 0.00017 U mL(-1) for UDG and 0.11 U mL(-1) for Endo IV, respectively. Moreover, UDG and Endo IV can be well distinguished from their analogs. This method is beneficial for properly evaluating the UBER process in function studies and disease prognoses. PMID:26899234

  4. Polymorphisms of human 8-oxoguanine DNA glycosylase 1 and 8-hydroxydeoxyguanosine increase susceptibility to arsenic methylation capacity-related urothelial carcinoma.

    PubMed

    Huang, Chao-Yuan; Pu, Yeong-Shiau; Shiue, Horng-Sheng; Chen, Wei-Jen; Lin, Ying-Chin; Hsueh, Yu-Mei

    2016-08-01

    Arsenic causes oxidative stress in cultured animal and human cells, and it is a well-documented human carcinogen. We conducted a hospital-based case-control study including 167 cases of urothelial carcinoma (UC) and 334 age- and gender-matched healthy controls to evaluate the relationships between urinary arsenic profiles, urinary 8-hydroxydeoxyguanosine (8-OHdG) levels, and human 8-oxoguanine DNA glycosylase (hOGG1) genotypes and UC. The urinary arsenic species were analyzed by high-performance liquid chromatography and hydride generator-atomic absorption spectrometry. Genotyping for hOGG1 (Ser326Cys) and hOGG1 (-15C>G) was performed using the Sequenom MassARRAY platform with iPLEX Gold chemistry. Urinary 8-OHdG was measured with high-sensitivity enzyme-linked immunosorbent assay kits. The results indicated that the hOGG1 326 Cys/Cys genotype and the hOGG1 -15C>G G/G genotype were associated with an increased risk of UC (OR [95 % CI] 1.57 [1.04-2.35] and 1.57 [1.04-2.35], respectively). Participants with high urinary total arsenic, regardless of the haplotype of hOGG1 Ser326Cys and the -15C>G polymorphism, had significantly higher urinary 8-OHdG compared to participants with low urinary total arsenic. This is the first study to investigate the joint effects of high urinary total arsenic or inefficient arsenic methylation capacity indices, and the high-risk G-G haplotype of hOGG1 on the risk of UC. The findings are especially meaningful for participants with risk factors such as high urinary total arsenic, inefficient arsenic methylation indices, high urinary 8-OHdG, and the high-risk G-G haplotype of hOGG1 which are all associated with an increased UC risk. PMID:26359225

  5. Structural and biophysical analysis of interactions between cod and human uracil-DNA N-glycosylase (UNG) and UNG inhibitor (Ugi).

    PubMed

    Assefa, Netsanet Gizaw; Niiranen, Laila; Johnson, Kenneth A; Leiros, Hanna-Kirsti Schrøder; Smalås, Arne Oskar; Willassen, Nils Peder; Moe, Elin

    2014-08-01

    Uracil-DNA N-glycosylase from Atlantic cod (cUNG) shows cold-adapted features such as high catalytic efficiency, a low temperature optimum for activity and reduced thermal stability compared with its mesophilic homologue human UNG (hUNG). In order to understand the role of the enzyme-substrate interaction related to the cold-adapted properties, the structure of cUNG in complex with a bacteriophage encoded natural UNG inhibitor (Ugi) has been determined. The interaction has also been analyzed by isothermal titration calorimetry (ITC). The crystal structure of cUNG-Ugi was determined to a resolution of 1.9 Å with eight complexes in the asymmetric unit related through noncrystallographic symmetry. A comparison of the cUNG-Ugi complex with previously determined structures of UNG-Ugi shows that they are very similar, and confirmed the nucleotide-mimicking properties of Ugi. Biophysically, the interaction between cUNG and Ugi is very strong and shows a binding constant (Kb) which is one order of magnitude larger than that for hUNG-Ugi. The binding of both cUNG and hUNG to Ugi was shown to be favoured by both enthalpic and entropic forces; however, the binding of cUNG to Ugi is mainly dominated by enthalpy, while the entropic term is dominant for hUNG. The observed differences in the binding properties may be explained by an overall greater positive electrostatic surface potential in the protein-Ugi interface of cUNG and the slightly more hydrophobic surface of hUNG. PMID:25084329

  6. 8-Oxoguanine DNA glycosylase 1 (ogg1) maintains the function of cardiac progenitor cells during heart formation in zebrafish

    SciTech Connect

    Yan, Lifeng; Zhou, Yong; Yu, Shanhe; Ji, Guixiang; Liu, Wei; Gu, Aihua

    2013-11-15

    Genomic damage may devastate the potential of progenitor cells and consequently impair early organogenesis. We found that ogg1, a key enzyme initiating the base-excision repair, was enriched in the embryonic heart in zebrafish. So far, little is known about DNA repair in cardiogenesis. Here, we addressed the critical role of ogg1 in cardiogenesis for the first time. ogg1 mainly expressed in the anterior lateral plate mesoderm (ALPM), the primary heart tube, and subsequently the embryonic myocardium by in situ hybridisation. Loss of ogg1 resulted in severe cardiac morphogenesis and functional abnormalities, including the short heart length, arrhythmia, decreased cardiomyocytes and nkx2.5{sup +} cardiac progenitor cells. Moreover, the increased apoptosis and repressed proliferation of progenitor cells caused by ogg1 deficiency might contribute to the heart phenotype. The microarray analysis showed that the expression of genes involved in embryonic heart tube morphogenesis and heart structure were significantly changed due to the lack of ogg1. Among those, foxh1 is an important partner of ogg1 in the cardiac development in response to DNA damage. Our work demonstrates the requirement of ogg1 in cardiac progenitors and heart development in zebrafish. These findings may be helpful for understanding the aetiology of congenital cardiac deficits. - Highlights: • A key DNA repair enzyme ogg1 is expressed in the embryonic heart in zebrafish. • We found that ogg1 is essential for normal cardiac morphogenesis in zebrafish. • The production of embryonic cardiomyocytes requires appropriate ogg1 expression. • Ogg1 critically regulated proliferation of cardiac progenitor cells in zebrafish. • foxh1 is a partner of ogg1 in the cardiac development in response to DNA damage.

  7. Intrathecal Injection of 3-Methyladenine Reduces Neuronal Damage and Promotes Functional Recovery via Autophagy Attenuation after Spinal Cord Ischemia/Reperfusion Injury in Rats.

    PubMed

    Wei, Xing; Zhou, Zhentao; Li, Lingyun; Gu, Jun; Wang, Chen; Xu, Fuqi; Dong, Qirong; Zhou, Xiaozhong

    2016-01-01

    The present study aimed to determine the occurrence of autophagy following ischemia/reperfusion (I/R) injury in the rat spinal cord and whether autophagy inhibition contributes to neural tissue damage and locomotor impairment. A spinal cord I/R model was induced via descending thoracic aorta occlusion for 10 min using systemic hypotension (40 mmHg) in adult male Sprague-Dawley rats. Then, 600 nmol 3-methyladenine (3-MA) or vehicle was intrathecally administered. Ultrastructural spinal cord changes were observed via transmission electron microscopy (TEM) and immunofluorescent double-labeling. Western blots were used to determine the protein expression of microtubule-associated protein light chain 3 (LC3) and Beclin 1. Autophagy was activated after spinal cord I/R injury as demonstrated by significantly increased LC3 and Beclin 1 expression at 3-48 h after injury. Furthermore, TEM images indicated the presence of autophagosomes and autolysosomes in the injured spinal cord. 3-MA significantly decreased LC3 and Beclin 1 expression and the number of LC3-positive cells in spinal cord of I/R versus vehicle groups. Moreover, the 3-MA-treated rats exhibited better neurobehavioral scores compared with control rats. These findings suggest activation of autophagy leading to neuronal cell death in the I/R injured spinal cord. These effects were significantly inhibited by intrathecal 3-MA administration. Thus intrathecal 3-MA administration may represent a novel treatment target following spinal cord I/R injury. PMID:27150140

  8. Amplified expression of the tag+ and alkA+ genes in Escherichia coli: identification of gene products and effects on alkylation resistance.

    PubMed Central

    Kaasen, I; Evensen, G; Seeberg, E

    1986-01-01

    We have constructed plasmids which overproduce the tag and alkA gene products of Escherichia coli, i.e., 3-methyladenine DNA glycosylases I and II. The tag and alkA gene products were identified radiochemically in maxi- or minicells as polypeptides of 21 and 30 kilodaltons, respectively, which are consistent with the gel filtration molecular weights of the enzyme activities, thus confirming the identity of the cloned genes. High expression of the tag+-coded glycosylase almost completely suppressed the alkylation sensitivity of alkA mutants, indicating that high levels of 3-methyladenine DNA glycosylase I will eliminate the need for 3-methyladenine DNA glycosylase II in repair of alkylated DNA. Furthermore, overproduction of the alkA+-coded glycosylase greatly sensitizes wild-type cells to alkylation, suggesting that only a limited expression of this enzyme will allow efficient DNA repair. Images PMID:3536857

  9. Hyperglycemia-induced inflammation caused down-regulation of 8-oxoG-DNA glycosylase levels in murine macrophages is mediated by oxidative-nitrosative stress-dependent pathways.

    PubMed

    Kumar, Premranjan; Swain, Mitali Madhusmita; Pal, Arttatrana

    2016-04-01

    High glucose-induced increase in production of reactive oxygen/nitrogen species (ROS/RNS) is recognized as a major cause of the clinical complications associated with diabetes. ROS/RNS apart from being redox agents, cause an unwanted severe physiological load to cells, also act as cellular messengers, and play a key role in activation of circulating macrophages. However, the molecular mechanisms of activation of macrophages by hyperglycemic conditions are currently unclear. In the present study, we report that high glucose (HG) causes a dramatic increase in the production of inflammatory cytokines and chemokines, at least in part through enhanced mRNA transcription. The increase in levels of inflammatory cytokines/chemokines corresponds to increased levels of ROS/RNS, which is accompanied by increased activities of Akt, ERK1/2, tuberin, down regulation of 8-oxoG-DNA glycosylase (OGG1), and increase in 8-hydroxydeoxyguanosine (8-OHdG) accumulation in DNA. Elevated levels of ROS/RNS are triggering alteration in antioxidants level, biomolecules damage, cell cycle dysregulation, and apoptosis in macrophage cells. Pretreatment of antioxidants caused decrease in the levels of ROS/RNS leads to an increase in the levels of antioxidants, decrease in biomolecules damage, alterations in Akt, ERK1/2, tuberin, upregulation of OGG1, and decrease in 8-OHdG accumulations in DNA. Further, antioxidants treatments inhibit the effects of HG on the transcriptional activity of cytokines and chemokines. Our results demonstrate that intracellular signaling pathways mediated by ROS/RNS are linked to each other by elevated glucose in macrophages activation leading to inflammation. These findings provide a mechanistic explanation of how ROS/RNS cooperate to conduct inflammatory intracellular signals in macrophages related complications in hyperglycemic conditions. PMID:26860957

  10. Enhancement of Borrelia burgdorferi PCR by uracil N-glycosylase.

    PubMed Central

    Loewy, Z G; Mecca, J; Diaco, R

    1994-01-01

    Uracil DNA glycosylases are DNA repair enzymes present in virtually every organism. These enzymes function by excising from DNA uracil residues resulting from either misincorporation of dUMP residues by a DNA polymerase or deamination of cytosine. Recently, the enzyme has been exploited in PCRs as a means for controlling carryover contamination from previously amplified DNA. When the enzyme is used in amplifications of Borrelia burgdorferi target sequences, we have observed an enhancement in signal detected by a microwell plate DNA hybridization assay. This increase in signal is dependent upon the length of the target, is titratable with enzyme concentration, and has been observed with amplifications performed with both symmetric and asymmetric PCR profiles. The enhancement is shown to occur at the level of the target genomic DNA. PMID:8126168

  11. Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus.

    PubMed

    Tang, Yi; Chen, Hao; Diao, Youxiang

    2016-01-01

    Tembusu virus (TMUV) is a mosquito-borne flavivirus which threatens both poultry production and public health. In this study we developed a complete open reading frame alignment-based rRT-LAMP method for the universal detection of TUMV. To prevent false-positive results, the reaction was supplemented with uracil DNA glycosylase (UDG) to eliminate carryover contamination. The detection limit of the newly developed UDG-rRT-LAMP for TMUV was as low as 100 copies/reaction of viral RNA and 1 × 10(0.89) - 1 × 10(1.55) tissue culture infectious dose/100 μL of viruses. There were no cross-reactions with other viruses, and the reproducibility of the assay was confirmed by intra- and inter-assay tests with variability ranging from 0.22-3.33%. The new UDG-rRT-LAMP method for TMUV produced the same results as viral isolation combined with RT-PCR as the "gold standard" in 96.88% of cases for 81 clinical samples from subjects with suspected TMUV infection. The addition of UDG can eliminate as much as 1 × 10(-16) g/reaction of contaminants, which can significantly reduce the likelihood of false-positive results during the rRT-LAMP reaction. Our result indicated that our UDG-rRT-LAMP is a rapid, sensitive, specific, and reliable method that can effectively prevent carryover contamination in the detection of TMUV. PMID:27270462

  12. Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus

    PubMed Central

    Tang, Yi; Chen, Hao; Diao, Youxiang

    2016-01-01

    Tembusu virus (TMUV) is a mosquito-borne flavivirus which threatens both poultry production and public health. In this study we developed a complete open reading frame alignment-based rRT-LAMP method for the universal detection of TUMV. To prevent false-positive results, the reaction was supplemented with uracil DNA glycosylase (UDG) to eliminate carryover contamination. The detection limit of the newly developed UDG-rRT-LAMP for TMUV was as low as 100 copies/reaction of viral RNA and 1 × 100.89 − 1 × 101.55 tissue culture infectious dose/100 μL of viruses. There were no cross-reactions with other viruses, and the reproducibility of the assay was confirmed by intra- and inter-assay tests with variability ranging from 0.22–3.33%. The new UDG-rRT-LAMP method for TMUV produced the same results as viral isolation combined with RT-PCR as the “gold standard” in 96.88% of cases for 81 clinical samples from subjects with suspected TMUV infection. The addition of UDG can eliminate as much as 1 × 10−16 g/reaction of contaminants, which can significantly reduce the likelihood of false-positive results during the rRT-LAMP reaction. Our result indicated that our UDG-rRT-LAMP is a rapid, sensitive, specific, and reliable method that can effectively prevent carryover contamination in the detection of TMUV. PMID:27270462

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

    PubMed Central

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

    2010-01-01

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

  14. Expression of human oxoguanine glycosylase 1 or formamidopyrimidine glycosylase in human embryonic kidney 293 cells exacerbates methylmercury toxicity in vitro

    SciTech Connect

    Ondovcik, Stephanie L.; Preston, Thomas J.; McCallum, Gordon P.; Wells, Peter G.

    2013-08-15

    Exposure to methylmercury (MeHg) acutely at high levels, or via chronic low-level dietary exposure from daily fish consumption, can lead to adverse neurological effects in both the adult and developing conceptus. To determine the impact of variable DNA repair capacity, and the role of reactive oxygen species (ROS) and oxidatively damaged DNA in the mechanism of toxicity, transgenic human embryonic kidney (HEK) 293 cells that stably express either human oxoguanine glycosylase 1 (hOgg1) or its bacterial homolog, formamidopyrimidine glycosylase (Fpg), which primarily repair the oxidative lesion 8-oxo-2′-deoxyguanosine (8-oxodG), were used to assess the in vitro effects of MeHg. Western blotting confirmed the expression of hOgg1 or Fpg in both the nuclear and mitochondrial compartments of their respective cell lines. Following acute (1–2 h) incubations with 0–10 μM MeHg, concentration-dependent decreases in clonogenic survival and cell growth accompanied concentration-dependent increases in lactate dehydrogenase (LDH) release, ROS formation, 8-oxodG levels and apurinic/apyrimidinic (AP) sites, consistent with the onset of cytotoxicity. Paradoxically, hOgg1- and Fpg-expressing HEK 293 cells were more sensitive than wild-type cells stably transfected with the empty vector control to MeHg across all cellular and biochemical parameters, exhibiting reduced clonogenic survival and cell growth, and increased LDH release and DNA damage. Accordingly, upregulation of specific components of the base excision repair (BER) pathway may prove deleterious potentially due to the absence of compensatory enhancement of downstream processes to repair toxic intermediary abasic sites. Thus, interindividual variability in DNA repair activity may constitute an important risk factor for environmentally-initiated, oxidatively damaged DNA and its pathological consequences. - Highlights: • hOgg1 and Fpg repair oxidatively damaged DNA. • hOgg1- and Fpg-expressing cells are more

  15. Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase

    PubMed Central

    Hill, Jeff W.; Evans, Michele K.

    2006-01-01

    Human 8-oxoguanine-DNA glycosylase (OGG1) is the major enzyme for repairing 8-oxoguanine (8-oxoG), a mutagenic guanine base lesion produced by reactive oxygen species (ROS). A frequently occurring OGG1 polymorphism in human populations results in the substitution of serine 326 for cysteine (S326C). The 326 C/C genotype is linked to numerous cancers, although the mechanism of carcinogenesis associated with the variant is unclear. We performed detailed enzymatic studies of polymorphic OGG1 and found functional defects in the enzyme. S326C OGG1 excised 8-oxoG from duplex DNA and cleaved abasic sites at rates 2- to 6-fold lower than the wild-type enzyme, depending upon the base opposite the lesion. Binding experiments showed that the polymorphic OGG1 binds DNA damage with significantly less affinity than the wild-type enzyme. Remarkably, gel shift, chemical cross-linking and gel filtration experiments showed that S326C both exists in solution and binds damaged DNA as a dimer. S326C OGG1 enzyme expressed in human cells was also found to have reduced activity and a dimeric conformation. The glycosylase activity of S326C OGG1 was not significantly stimulated by the presence of AP-endonuclease. The altered substrate specificity, lack of stimulation by AP-endonuclease 1 (APE1) and anomalous DNA binding conformation of S326C OGG1 may contribute to its linkage to cancer incidence. PMID:16549874

  16. Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor

    SciTech Connect

    Singh, Prem; Talawar, Ramappa K.; Krishna, P. D. V.; Varshney, Umesh; Vijayan, M.

    2006-12-01

    Uracil N-glycosylase from M. tuberculosis has been crystallized in complex with a proteinaceous inhibitor (Ugi) and X-ray diffraction data have been collected. Uracil N-glycosylase is an enzyme which initiates the pathway of uracil-excision repair of DNA. The enzyme from Mycobacterium tuberculosis was co-expressed with a proteinaceous inhibitor from Bacillus subtilis phage and was crystallized in monoclinic space group C2, with unit-cell parameters a = 201.14, b = 64.27, c = 203.68 Å, β = 109.7°. X-ray data from the crystal have been collected for structure analysis.

  17. Dispensability of the [4Fe-4S] cluster in novel homologues of adenine glycosylase MutY.

    PubMed

    Trasviña-Arenas, Carlos H; Lopez-Castillo, Laura M; Sanchez-Sandoval, Eugenia; Brieba, Luis G

    2016-02-01

    7,8-Dihydro-8-deoxyguanine (8oG) is one of the most common oxidative lesions in DNA. DNA polymerases misincorporate an adenine across from this lesion. Thus, 8oG is a highly mutagenic lesion responsible for G:C→T:A transversions. MutY is an adenine glycosylase, part of the base excision repair pathway that removes adenines, when mispaired with 8oG or guanine. Its catalytic domain includes a [4Fe-4S] cluster motif coordinated by cysteinyl ligands. When this cluster is absent, MutY activity is depleted and several studies concluded that the [4Fe-4S] cluster motif is an indispensable component for DNA binding, substrate recognition and enzymatic activity. In the present study, we identified 46 MutY homologues that lack the canonical cysteinyl ligands, suggesting an absence of the [4Fe-4S] cluster. A phylogenetic analysis groups these novel MutYs into two different clades. One clade is exclusive of the order Lactobacillales and another clade has a mixed composition of anaerobic and microaerophilic bacteria and species from the protozoan genus Entamoeba. Structural modeling and sequence analysis suggests that the loss of the [4Fe-4S] cluster is compensated by a convergent solution in which bulky amino acids substitute the [4Fe-4S] cluster. We functionally characterized MutYs from Lactobacillus brevis and Entamoeba histolytica as representative members from each clade and found that both enzymes are active adenine glycosylases. Furthermore, chimeric glycosylases, in which the [4Fe-4S] cluster of Escherichia coli MutY is replaced by the corresponding amino acids of LbY and EhY, are also active. Our data indicates that the [4Fe-4S] cluster plays a structural role in MutYs and evidences the existence of alternative functional solutions in nature. PMID:26613369

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-29

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

  20. Enforced Presentation of an Extrahelical Guanine to the Lesion Recognition Pocket of Human 8-Oxoguanine Glycosylase, hOGG1*

    PubMed Central

    Crenshaw, Charisse M.; Nam, Kwangho; Oo, Kimberly; Kutchukian, Peter S.; Bowman, Brian R.; Karplus, Martin; Verdine, Gregory L.

    2012-01-01

    A poorly understood aspect of DNA repair proteins is their ability to identify exceedingly rare sites of damage embedded in a large excess of nearly identical undamaged DNA, while catalyzing repair only at the damaged sites. Progress toward understanding this problem has been made by comparing the structures and biochemical behavior of these enzymes when they are presented with either a target lesion or a corresponding undamaged nucleobase. Trapping and analyzing such DNA-protein complexes is particularly difficult in the case of base extrusion DNA repair proteins because of the complexity of the repair reaction, which involves extrusion of the target base from DNA followed by its insertion into the active site where glycosidic bond cleavage is catalyzed. Here we report the structure of a human 8-oxoguanine (oxoG) DNA glycosylase, hOGG1, in which a normal guanine from DNA has been forcibly inserted into the enzyme active site. Although the interactions of the nucleobase with the active site are only subtly different for G versus oxoG, hOGG1 fails to catalyze excision of the normal nucleobase. This study demonstrates that even if hOGG1 mistakenly inserts a normal base into its active site, the enzyme can still reject it on the basis of catalytic incompatibility. PMID:22511791

  1. Enforced Presentation of an Extrahelical Guanine to the Lesion Recognition Pocket of Human 8-Oxoguanine Glycosylase, hOGG1

    SciTech Connect

    Crenshaw, Charisse M.; Nam, Kwangho; Oo, Kimberly; Kutchukian, Peter S.; Bowman, Brian R.; Karplus, Martin; Verdine, Gregory L.

    2012-09-05

    A poorly understood aspect of DNA repair proteins is their ability to identify exceedingly rare sites of damage embedded in a large excess of nearly identical undamaged DNA, while catalyzing repair only at the damaged sites. Progress toward understanding this problem has been made by comparing the structures and biochemical behavior of these enzymes when they are presented with either a target lesion or a corresponding undamaged nucleobase. Trapping and analyzing such DNA-protein complexes is particularly difficult in the case of base extrusion DNA repair proteins because of the complexity of the repair reaction, which involves extrusion of the target base from DNA followed by its insertion into the active site where glycosidic bond cleavage is catalyzed. Here we report the structure of a human 8-oxoguanine (oxoG) DNA glycosylase, hOGG1, in which a normal guanine from DNA has been forcibly inserted into the enzyme active site. Although the interactions of the nucleobase with the active site are only subtly different for G versus oxoG, hOGG1 fails to catalyze excision of the normal nucleobase. This study demonstrates that even if hOGG1 mistakenly inserts a normal base into its active site, the enzyme can still reject it on the basis of catalytic incompatibility.

  2. Effect of 8-oxoguanine glycosylase deficiency on aflatoxin B1 tumourigenicity in mice

    PubMed Central

    Mulder, Jeanne E.; Turner, Patricia V.; Massey, Thomas E.

    2015-01-01

    The mycotoxin aflatoxin B1 (AFB1) may initiate cancer by causing oxidatively damaged DNA, specifically by causing 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) lesions. Base excision repair removes these lesions, with 8-oxoguanine glycosylase (OGG1) being the rate-limiting enzyme. The aim of this study was to determine the effect of ogg1 deficiency on AFB1-induced oxidatively damaged DNA and tumourigenesis. Female wild-type, heterozygous and homozygous ogg1 null mice were given a single dose of 50mg/kg AFB1 or 40 µl dimethyl sulfoxide (DMSO) ip. Neither ogg1 genotype nor AFB1 treatment affected levels of oxidised guanine in lung or liver 2h post-treatment. AFB1-treated ogg1 null mice showed exacerbated weight loss and mortality relative to DMSO-treated ogg1 null mice, but AFB1 treatment did not significantly increase lung or liver tumour incidence compared with controls, regardless of ogg1 genotype. Suspect lung masses from three of the AFB1-treated mice were adenomas, and masses from two of the mice were osteosarcomas. No osteosarcomas were observed in DMSO-treated mice. All liver masses from AFB1-treated mice were adenomas, and one also contained a hepatocellular carcinoma. In DNA from the lung tumours, the K-ras mutation pattern was inconsistent with initiation by AFB1. In conclusion, ogg1 status did not have a significant effect on AFB1-induced oxidatively damaged DNA or tumourigenesis, but deletion of one or both alleles of ogg1 did increase susceptibility to other aspects of AFB1 toxicity. PMID:25583175

  3. Autophagy promotes DNA-protein crosslink clearance.

    PubMed

    Mu, Haibo; Liu, Qianjin; Niu, Hong; Wang, Dongdong; Tang, Jiangjiang; Duan, Jinyou

    2016-02-01

    Toxic DNA-protein crosslinks (DPCs) can result from exposure to radiation or chemotherapeutic agents. DPCs can also accumulate during aging or stress. However, the cellular mechanisms underlying clearance of DPCs remain largely unknown. Here, we have identified an important role of autophagy in the processing of DPCs induced by three representative agents: formaldehyde, a chemical used widely in industry; UV light; and camptothecin, a cytotoxic anticancer drug. Autophagy inhibitors, 3-methyladenine (3-MA) or chloroquine (CQ), promoted the accumulation of DPCs in damaged cells and injured organs. siRNA-mediated silencing of Atg5 or Atg7, two essential components for the formation of the autophagosome, gave similar results. In contrast, the autophagy inducer rapamycin (RAP) attenuated DPCs in vitro and in vivo. Our findings reveal the importance of autophagy in controlling the level of DPCs, and may open up a new avenue for understanding the formation and clearance of this detrimental DNA adduct. PMID:26921017

  4. Radiolysis of DNA-protein complexes

    NASA Astrophysics Data System (ADS)

    Běgusová, Marie; Gillard, Nathalie; Sy, Denise; Castaing, Bertrand; Charlier, Michel; Spotheim-Maurizot, Melanie

    2005-02-01

    We discuss here modifications of DNA and protein radiolysis due to the interaction of these two partners in specific complexes. Experimental patterns of frank strand breaks (FSB) and alkali revealed breaks (ARB) obtained for DNA lac operator bound to the lac repressor and for a DNA containing an abasic site analog bound to the formamidopyrimidine-DNA glycosylase are reported. Experimental data are compared to predicted damage distribution obtained using the theoretical model RADACK.

  5. Nonspecific DNA Binding and Coordination of the First Two Steps of Base Excision Repair

    PubMed Central

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

    2010-01-01

    The base excision repair (BER) pathway repairs a wide variety of damaged nucleobases in DNA. This pathway is initiated by a DNA repair glycosylase, which locates the site of damage and catalyzes the excision of the damaged nucleobase. The resulting abasic site is further processed by apurinic/apyrimidinic site endonuclease 1 (APE1) to create a single strand nick with the 3'-hydroxyl that serves as a primer for DNA repair synthesis. Since an abasic site is highly mutagenic it is critical that the steps of the BER pathway be coordinated. Most human glycosylases bind tightly to their abasic product. APE1 displaces the bound glycosylase, thereby stimulating multiple turnover base excision. It has been proposed that direct protein-protein interactions are involved in the stimulation by APE1, but no common interaction motifs have been identified among the glycosylases that are stimulated by APE1. We characterized the APE1 stimulation of alkyladenine DNA glycosylase (AAG) using a variety of symmetric and asymmetric lesion-containing oligonucleotides. Efficient stimulation on a wide variety of substrates favors a model whereby both AAG and APE1 can simultaneously bind to DNA, but may not interact directly. Rather, nonspecific DNA binding by both AAG and APE1 enables APE1 to replace AAG at the abasic site. AAG is not displaced into solution, but remains bound to an adjacent undamaged site. We propose that nonspecific DNA binding interactions allow transient exposure of the abasic site so that it can be captured by APE1. PMID:20701268

  6. Differential modulation of base excision repair activities during brain ontogeny: implications for repair of transcribed DNA.

    PubMed

    Englander, Ella W; Ma, Huaxian

    2006-01-01

    DNA repair sustains fidelity of genomic replication in proliferating cells and integrity of transcribed sequences in postmitotic tissues. The repair process is critical in the brain, because high oxygen consumption exacerbates the risk for accumulation of oxidative DNA lesions in postmitotic neurons. Most oxidative DNA damage is repaired by the base excision repair (BER) pathway, which is initiated by specialized DNA glycosylases. Because the newly discovered Nei-like mammalian DNA glycosylases (NEIL1/2) proficiently excise oxidized bases from bubble structured DNA, it was suggested that NEILs favor repair of transcribed or replicated DNA. In addition, since NEILs generate 3'-phosphate termini, which are poor targets for AP endonuclease (APE1), it was proposed that APE1-dependent and independent BER sub-pathways exist in mammalian cells. We measured expression and activities of BER enzymes during brain ontogeny, i.e., during a physiologic transition from proliferative to postmitotic differentiated state. While a subset of BER enzymes, exhibited declining expression and excision activities, expression of NEIL1 and NEIL2 glycosylases increased during brain development. Furthermore, the capacity for excision of 5-hydroxyuracil from bubble structured DNA was retained in the mature rat brain suggesting a role for NEIL glycosylases in maintaining the integrity of transcribed DNA in postmitotic brain. PMID:16257035

  7. Catalysts of DNA Strand Cleavage at Apurinic/Apyrimidinic Sites.

    PubMed

    Minko, Irina G; Jacobs, Aaron C; de Leon, Arnie R; Gruppi, Francesca; Donley, Nathan; Harris, Thomas M; Rizzo, Carmelo J; McCullough, Amanda K; Lloyd, R Stephen

    2016-01-01

    Apurinic/apyrimidinic (AP) sites are constantly formed in cellular DNA due to instability of the glycosidic bond, particularly at purines and various oxidized, alkylated, or otherwise damaged nucleobases. AP sites are also generated by DNA glycosylases that initiate DNA base excision repair. These lesions represent a significant block to DNA replication and are extremely mutagenic. Some DNA glycosylases possess AP lyase activities that nick the DNA strand at the deoxyribose moiety via a β- or β,δ-elimination reaction. Various amines can incise AP sites via a similar mechanism, but this non-enzymatic cleavage typically requires high reagent concentrations. Herein, we describe a new class of small molecules that function at low micromolar concentrations as both β- and β,δ-elimination catalysts at AP sites. Structure-activity relationships have established several characteristics that appear to be necessary for the formation of an iminium ion intermediate that self-catalyzes the elimination at the deoxyribose ring. PMID:27363485

  8. Catalysts of DNA Strand Cleavage at Apurinic/Apyrimidinic Sites

    PubMed Central

    Minko, Irina G.; Jacobs, Aaron C.; de Leon, Arnie R.; Gruppi, Francesca; Donley, Nathan; Harris, Thomas M.; Rizzo, Carmelo J.; McCullough, Amanda K.; Lloyd, R. Stephen

    2016-01-01

    Apurinic/apyrimidinic (AP) sites are constantly formed in cellular DNA due to instability of the glycosidic bond, particularly at purines and various oxidized, alkylated, or otherwise damaged nucleobases. AP sites are also generated by DNA glycosylases that initiate DNA base excision repair. These lesions represent a significant block to DNA replication and are extremely mutagenic. Some DNA glycosylases possess AP lyase activities that nick the DNA strand at the deoxyribose moiety via a β- or β,δ-elimination reaction. Various amines can incise AP sites via a similar mechanism, but this non-enzymatic cleavage typically requires high reagent concentrations. Herein, we describe a new class of small molecules that function at low micromolar concentrations as both β- and β,δ-elimination catalysts at AP sites. Structure-activity relationships have established several characteristics that appear to be necessary for the formation of an iminium ion intermediate that self-catalyzes the elimination at the deoxyribose ring. PMID:27363485

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

    PubMed

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

    2016-06-21

    In actively proliferating cells, such as the cells of the developing embryo, DNA repair is crucial for preventing the accumulation of mutations and synchronizing cell division. Sea urchin embryo growth was analyzed and extracts were prepared. The relative activity of DNA polymerase, apurinic/apyrimidinic (AP) endonuclease, uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and other glycosylases was analyzed using specific oligonucleotide substrates of these enzymes; the reaction products were resolved by denaturing 20% polyacrylamide gel electrophoresis. We have characterized the profile of several key base excision repair activities in the developing embryos (2 blastomers to mid-pluteus) of the grey sea urchin, Strongylocentrotus intermedius. The uracil-DNA glycosylase specific activity sharply increased after blastula hatching, whereas the specific activity of 8-oxoguanine-DNA glycosylase steadily decreased over the course of the development. The AP-endonuclease activity gradually increased but dropped at the last sampled stage (mid-pluteus 2). The DNA polymerase activity was high at the first cleavage division and then quickly decreased, showing a transient peak at blastula hatching. It seems that the developing sea urchin embryo encounters different DNA-damaging factors early in development within the protective envelope and later as a free-floating larva, with hatching necessitating adaptation to the shift in genotoxic stress conditions. No correlation was observed between the dynamics of the enzyme activities and published gene expression data from developing congeneric species, S. purpuratus. The results suggest that base excision repair enzymes may be regulated in the sea urchin embryos at the level of covalent modification or protein stability. PMID:27158700

  10. 3CAPS – a structural AP–site analogue as a tool to investigate DNA base excision repair

    PubMed Central

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

    2016-01-01

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

  11. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  12. Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III*

    PubMed Central

    Kuznetsov, Nikita A.; Kladova, Olga A.; Kuznetsova, Alexandra A.; Ishchenko, Alexander A.; Saparbaev, Murat K.; Zharkov, Dmitry O.; Fedorova, Olga S.

    2015-01-01

    Escherichia coli endonuclease III (Endo III or Nth) is a DNA glycosylase with a broad substrate specificity for oxidized or reduced pyrimidine bases. Endo III possesses two types of activities: N-glycosylase (hydrolysis of the N-glycosidic bond) and AP lyase (elimination of the 3′-phosphate of the AP-site). We report a pre-steady-state kinetic analysis of structural rearrangements of the DNA substrates and uncleavable ligands during their interaction with Endo III. Oligonucleotide duplexes containing 5,6-dihydrouracil, a natural abasic site, its tetrahydrofuran analog, and undamaged duplexes carried fluorescent DNA base analogs 2-aminopurine and 1,3-diaza-2-oxophenoxazine as environment-sensitive reporter groups. The results suggest that Endo III induces several fast sequential conformational changes in DNA during binding, lesion recognition, and adjustment to a catalytically competent conformation. A comparison of two fluorophores allowed us to distinguish between the events occurring in the damaged and undamaged DNA strand. Combining our data with the available structures of Endo III, we conclude that this glycosylase uses a multistep mechanism of damage recognition, which likely involves Gln41 and Leu81 as DNA lesion sensors. PMID:25869130

  13. Listeria monocytogenes DNA glycosylase AdiP affects flagellar motility, biofilm formation, virulence, and stress responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The temperature-dependent alteration of flagellar motility gene expression is critical for the foodborne pathogen Listeria monocytogenes to respond to a changing environment. In this study, a genetic determinant, L. monocytogenes f2365_0220 (lmof2365_0220), encoding a putative protein that is struct...

  14. TET-mediated oxidation of methylcytosine causes TDG or NEIL glycosylase dependent gene reactivation

    PubMed Central

    Müller, Udo; Bauer, Christina; Siegl, Michael; Rottach, Andrea; Leonhardt, Heinrich

    2014-01-01

    The discovery of hydroxymethyl-, formyl- and carboxylcytosine, generated through oxidation of methylcytosine by TET dioxygenases, raised the question how these modifications contribute to epigenetic regulation. As they are subjected to complex regulation in vivo, we dissected links to gene expression with in vitro modified reporter constructs. We used an Oct4 promoter-driven reporter gene and demonstrated that in vitro methylation causes gene silencing while subsequent oxidation with purified catalytic domain of TET1 leads to gene reactivation. To identify proteins involved in this pathway we screened for TET interacting factors and identified TDG, PARP1, XRCC1 and LIG3 that are involved in base-excision repair. Knockout and rescue experiments demonstrated that gene reactivation depended on the glycosylase TDG, but not MBD4, while NEIL1, 2 and 3 could partially rescue the loss of TDG. These results clearly show that oxidation of methylcytosine by TET dioxygenases and subsequent removal by TDG or NEIL glycosylases and the BER pathway results in reactivation of epigenetically silenced genes. PMID:24948610

  15. Hippocampal adult neurogenesis is maintained by Neil3-dependent repair of oxidative DNA lesions in neural progenitor cells.

    PubMed

    Regnell, Christine Elisabeth; Hildrestrand, Gunn Annette; Sejersted, Yngve; Medin, Tirill; Moldestad, Olve; Rolseth, Veslemøy; Krokeide, Silje Zandstra; Suganthan, Rajikala; Luna, Luisa; Bjørås, Magnar; Bergersen, Linda H

    2012-09-27

    Accumulation of oxidative DNA damage has been proposed as a potential cause of age-related cognitive decline. The major pathway for removal of oxidative DNA base lesions is base excision repair, which is initiated by DNA glycosylases. In mice, Neil3 is the main DNA glycosylase for repair of hydantoin lesions in single-stranded DNA of neural stem/progenitor cells, promoting neurogenesis. Adult neurogenesis is crucial for maintenance of hippocampus-dependent functions involved in behavior. Herein, behavioral studies reveal learning and memory deficits and reduced anxiety-like behavior in Neil3(-/-) mice. Neural stem/progenitor cells from aged Neil3(-/-) mice show impaired proliferative capacity and reduced DNA repair activity. Furthermore, hippocampal neurons in Neil3(-/-) mice display synaptic irregularities. It appears that Neil3-dependent repair of oxidative DNA damage in neural stem/progenitor cells is required for maintenance of adult neurogenesis to counteract the age-associated deterioration of cognitive performance. PMID:22959434

  16. An AP endonuclease functions in active DNA demethylation and gene imprinting in Arabidopsis [corrected].

    PubMed

    Li, Yan; Córdoba-Cañero, Dolores; Qian, Weiqiang; Zhu, Xiaohong; Tang, Kai; Zhang, Huiming; Ariza, Rafael R; Roldán-Arjona, Teresa; Zhu, Jian-Kang

    2015-01-01

    Active DNA demethylation in plants occurs through base excision repair, beginning with removal of methylated cytosine by the ROS1/DME subfamily of 5-methylcytosine DNA glycosylases. Active DNA demethylation in animals requires the DNA glycosylase TDG or MBD4, which functions after oxidation or deamination of 5-methylcytosine, respectively. However, little is known about the steps following DNA glycosylase action in the active DNA demethylation pathways in plants and animals. We show here that the Arabidopsis APE1L protein has apurinic/apyrimidinic endonuclease activities and functions downstream of ROS1 and DME. APE1L and ROS1 interact in vitro and co-localize in vivo. Whole genome bisulfite sequencing of ape1l mutant plants revealed widespread alterations in DNA methylation. We show that the ape1l/zdp double mutant displays embryonic lethality. Notably, the ape1l+/-zdp-/- mutant shows a maternal-effect lethality phenotype. APE1L and the DNA phosphatase ZDP are required for FWA and MEA gene imprinting in the endosperm and are important for seed development. Thus, APE1L is a new component of the active DNA demethylation pathway and, together with ZDP, regulates gene imprinting in Arabidopsis. PMID:25569774

  17. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  18. Coordination of DNA repair by NEIL1 and PARP-1: a possible link to aging

    PubMed Central

    Noren Hooten, Nicole; Fitzpatrick, Megan; Kompaniez, Kari; Jacob, Kimberly D.; Moore, Brittany R.; Nagle, Julia; Barnes, Janice; Lohani, Althaf; Evans, Michele K.

    2012-01-01

    Oxidative DNA damage accumulates with age and is repaired primarily via the base excision repair (BER) pathway. This process is initiated by DNA glycosylases, which remove damaged bases in a substrate-specific manner. The DNA glycosylases human 8-oxoguanine-DNA glycosylase (OGG1) and NEIL1, a mammalian homolog of Escherichia coli endonuclease VIII, have overlapping yet distinct substrate specificity. Recently, we reported that OGG1 binds to the Poly(ADP-ribose) polymerase 1 (PARP-1), a DNA damage sensor protein that poly(ADP-ribosyl)ates nuclear proteins in response to DNA damage and other cellular signals. Here, we show that NEIL1 and PARP-1 bind both in vitro and in vivo. PARP-1 binds to the C-terminal-100 amino acids of NEIL1 and NEIL1 binds to the BRCT domain of PARP-1. NEIL1 stimulates the poly(ADP-ribosyl)ation activity of PARP-1. Furthermore, NEIL-deficient fibroblasts have impaired poly(ADP-ribosyl)ation of cellular proteins after DNA damage, which can be rescued by NEIL1 expression. Additionally, PARP-1 inhibits NEIL1 incision activity in a concentration-dependent manner. Consistent with the idea of impaired DNA repair during aging, we observed differential binding of PARP-1 to recombinant NEIL1 in older mice compared to younger mice. These data further support the idea that dynamic interplay between different base excision repair proteins is important for efficient BER. PMID:23104860

  19. DNA repair in bacterial cultures and plasmid DNA exposed to infrared laser for treatment of pain

    NASA Astrophysics Data System (ADS)

    Canuto, K. S.; Sergio, L. P. S.; Marciano, R. S.; Guimarães, O. R.; Polignano, G. A. C.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2013-06-01

    Biostimulation of tissues by low intensity lasers has been described on a photobiological basis and clinical protocols are recommended for treatment of various diseases, but their effects on DNA are controversial. The objective of this work was to evaluate effects of low intensity infrared laser exposure on survival and bacterial filamentation in Escherichia coli cultures, and induction of DNA lesions in bacterial plasmids. In E. coli cultures and plasmids exposed to an infrared laser at fluences used to treat pain, bacterial survival and filamentation and DNA lesions in plasmids were evaluated by electrophoretic profile. Data indicate that the infrared laser (i) increases survival of E. coli wild type in 24 h of stationary growth phase, (ii) induces bacterial filamentation, (iii) does not alter topological forms of plasmids and (iv) does not alter the electrophoretic profile of plasmids incubated with exonuclease III or formamidopyrimidine DNA glycosylase. A low intensity infrared laser at the therapeutic fluences used to treat pain can alter survival of E. coli wild type, induce filamentation in bacterial cells, depending on physiologic conditions and DNA repair, and induce DNA lesions other than single or double DNA strand breaks or alkali-labile sites, which are not targeted by exonuclease III or formamidopyrimidine DNA glycosylase.

  20. Finding and Producing Probiotic Glycosylases for the Biocatalysis of Ginsenosides: A Mini Review.

    PubMed

    Ku, Seockmo

    2016-01-01

    Various microorganisms have been widely applied in nutraceutical industries for the processing of phytochemical conversion. Specifically, in the Asian food industry and academia, notable attention is paid to the biocatalytic process of ginsenosides (ginseng saponins) using probiotic bacteria that produce high levels of glycosyl-hydrolases. Multiple groups have conducted experiments in order to determine the best conditions to produce more active and stable enzymes, which can be applicable to produce diverse types of ginsenosides for commercial applications. In this sense, there are various reviews that cover the biofunctional effects of multiple types of ginsenosides and the pathways of ginsenoside deglycosylation. However, little work has been published on the production methods of probiotic enzymes, which is a critical component of ginsenoside processing. This review aims to investigate current preparation methods, results on the discovery of new glycosylases, the application potential of probiotic enzymes and their use for biocatalysis of ginsenosides in the nutraceutical industry. PMID:27196878

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

    PubMed

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

    2016-07-12

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

  2. Sensitivity to methylmercury toxicity is enhanced in oxoguanine glycosylase 1 knockout murine embryonic fibroblasts and is dependent on cellular proliferation capacity

    SciTech Connect

    Ondovcik, Stephanie L.; Tamblyn, Laura; McPherson, John Peter; Wells, Peter G.

    2013-07-01

    Methylmercury (MeHg) is a persistent environmental contaminant with potent neurotoxic action for which the underlying molecular mechanisms remain to be conclusively delineated. Our objectives herein were twofold: first, to corroborate our previous findings of an increased sensitivity of spontaneously-immortalized oxoguanine glycosylase 1-null (Ogg1{sup −/−}) murine embryonic fibroblasts (MEFs) to MeHg through generation of Simian virus 40 (SV40) large T antigen-immortalized wild-type and Ogg1{sup −/−} MEFs; and second, to determine whether MeHg toxicity is proliferation-dependent. As with the spontaneously-immortalized cells used previously, the SV40 large T antigen-immortalized cells exhibited similar tendencies to undergo MeHg-initiated cell cycle arrest, with increased sensitivity in the Ogg1{sup −/−} MEFs as measured by clonogenic survival and DNA damage. Compared to exponentially growing cells, those seeded at a higher density exhibited compromised proliferation, which proved protective against MeHg-mediated cell cycle arrest and induction of DNA double strand breaks (DSBs), measured by phosphorylation of the core histone H2A variant (H2AX) on serine 139 (γH2AX), and by its functional confirmation by micronucleus assessment. This enhanced sensitivity of Ogg1{sup −/−} MEFs to MeHg toxicity using discrete SV40 immortalization corroborates our previous studies, and suggests a novel role for OGG1 in minimizing MeHg-initiated DNA lesions that trigger replication-associated DSBs. Furthermore, proliferative capacity may determine MeHg toxicity in vivo and in utero. Accordingly, variations in cellular proliferative capacity and interindividual variability in repair activity may modulate the risk of toxicological consequences following MeHg exposure. - Highlights: • SV40 large T antigen-immortalized Ogg1{sup −/−} cells are more sensitive to MeHg. • Sensitivity to MeHg is dependent on cellular proliferation capacity. • OGG1 maintains genomic

  3. Selective enzymatic cleavage and labeling for sensitive capillary electrophoresis laser-induced fluorescence analysis of oxidized DNA bases.

    PubMed

    Li, Cuiping; Wang, Hailin

    2015-08-01

    Oxidatively generated DNA damage is considered to be a significant contributing factor to cancer, aging, and age-related human diseases. It is important to detect oxidatively generated DNA damage to understand and clinically diagnosis diseases caused by oxidative damage. In this study, using selective enzymatic cleavage and quantum dot (QD) labeling, we developed a novel capillary electrophoresis-laser induced fluorescence method for the sensitive detection of oxidized DNA bases. First, oxidized DNA bases are recognized and removed by one DNA base excision repair glycosylase, leaving apurinic and apyrimidinic sites (AP sites) at the oxidized positions. The AP sites are further excised by the AP nicking activity of the chosen glycosylase, generating a nucleotide gap with 5'- and 3'- phosphate groups. After dephosphorylation with one alkaline phosphatase, a biotinylated ddNTP is introduced into the nucleotide space within the DNA strand by DNA polymerase I. The biotin-tagged DNA is further labeled with a QD-streptavidin conjugate via non-covalent interactions. The DNA-bound QD is well-separated from excess DNA-unbound QD by highly efficient capillary electrophoresis and is sensitively detected by online coupled laser-induced fluorescence analysis. Using this method, we can assess the trace levels of oxidized DNA bases induced by the Fenton reaction and UV irradiation. Interestingly, the use of the formamidopyrimidine glycosylase (FPG) protein and endonuclease VIII enables the detection of oxidized purine and pyrimidine bases, respectively. Using the synthesized standard DNA, the approach has low limits of detection of 1.1×10(-19)mol in mass and 2.9pM in concentration. PMID:26105778

  4. Methods for Efficient Elimination of Mitochondrial DNA from Cultured Cells

    PubMed Central

    Spadafora, Domenico; Kozhukhar, Nataliya; Chouljenko, Vladimir N.; Kousoulas, Konstantin G.; Alexeyev, Mikhail F.

    2016-01-01

    Here, we document that persistent mitochondria DNA (mtDNA) damage due to mitochondrial overexpression of the Y147A mutant uracil-N-glycosylase as well as mitochondrial overexpression of bacterial Exonuclease III or Herpes Simplex Virus protein UL12.5M185 can induce a complete loss of mtDNA (ρ0 phenotype) without compromising the viability of cells cultured in media supplemented with uridine and pyruvate. Furthermore, we use these observations to develop rapid, sequence-independent methods for the elimination of mtDNA, and demonstrate utility of these methods for generating ρ0 cells of human, mouse and rat origin. We also demonstrate that ρ0 cells generated by each of these three methods can serve as recipients of mtDNA in fusions with enucleated cells. PMID:27136098

  5. Genetic Variability in DNA Repair Proteins in Age-Related Macular Degeneration

    PubMed Central

    Blasiak, Janusz; Synowiec, Ewelina; Salminen, Antero; Kaarniranta, Kai

    2012-01-01

    The pathogenesis of age-related macular degeneration (AMD) is complex and involves interactions between environmental and genetic factors, with oxidative stress playing an important role inducing damage in biomolecules, including DNA. Therefore, genetic variability in the components of DNA repair systems may influence the ability of the cell to cope with oxidative stress and in this way contribute to the pathogenesis of AMD. However, few reports have been published on this subject so far. We demonstrated that the c.977C>G polymorphism (rs1052133) in the hOGG1 gene and the c.972G>C polymorphism (rs3219489) in the MUTYH gene, the products of which play important roles in the repair of oxidatively damaged DNA, might be associated with the risk of AMD. Oxidative stress may promote misincorporation of uracil into DNA, where it is targeted by several DNA glycosylases. We observed that the g.4235T>C (rs2337395) and c.–32A>G (rs3087404) polymorphisms in two genes encoding such glycosylases, UNG and SMUG1, respectively, could be associated with the occurrence of AMD. Polymorphisms in some other DNA repair genes, including XPD (ERCC2), XRCC1 and ERCC6 (CSB) have also been reported to be associated with AMD. These data confirm the importance of the cellular reaction to DNA damage, and this may be influenced by variability in DNA repair genes, in AMD pathogenesis. PMID:23202958

  6. Complexities of the DNA Base Excision Repair Pathway for Repair of Oxidative DNA Damage

    PubMed Central

    Mitra, Sankar; Boldogh, Istvan; Izumi, Tadahide; Hazra, Tapas K.

    2016-01-01

    Oxidative damage represents the most significant insult to organisms because of continuous production of the reactive oxygen species (ROS) in vivo. Oxidative damage in DNA, a critical target of ROS, is repaired primarily via the base excision repair (BER) pathway which appears to be the simplest among the three excision repair pathways. However, it is now evident that although BER can be carried with four or five enzymes in vitro, a large number of proteins, including some required for nucleotide excision repair (NER), are needed for in vivo repair of oxidative damage. Furthermore, BER in transcribed vs. nontranscribed DNA regions requires distinct sets of proteins, as in the case of NER. We propose an additional complexity in repair of replicating vs. nonreplicating DNA. Unlike DNA bulky adducts, the oxidized base lesions could be incorporated in the nascent DNA strand, repair of which may share components of the mismatch repair process. Distinct enzyme specificities are thus warranted for repair of lesions in the parental vs. nascent DNA strand. Repair synthesis may be carried out by DNA polymerase β or replicative polymerases δ and ε. Thus, multiple subpathways are needed for repairing oxidative DNA damage, and the pathway decision may require coordination of the successive steps in repair. Such coordination includes transfer of the product of a DNA glycosylase to AP-endonuclease, the next enzyme in the pathway. Interactions among proteins in the pathway may also reflect such coordination, characterization of which should help elucidate these subpathways and their in vivo regulation. PMID:11746753

  7. Oxidative DNA adducts and DNA-protein cross-links are the major DNA lesions induced by arsenite.

    PubMed

    Bau, Da-Tian; Wang, Tsu-Shing; Chung, Chiao-Hui; Wang, Alexander S S; Wang, Alexander S S; Jan, Kun-Yan

    2002-10-01

    Arsenic is recognized to be a nonmutagenic carcinogen because it induces DNA damage only at very high concentrations. However, many more DNA strand breaks could be detected by digesting the DNA of arsenite-treated cells with endonuclease III, formamidopyrimidine-DNA glycosylase, and proteinase K. By doing so, arsenite could be shown to induce DNA damage in human cells within a pathologically meaningful concentration range. Oxidized guanine products were detected in all arsenite-treated human cells examined. DNA-protein cross-links were also detected in arsenite-treated NB4 and HL60 cells. In human umbilical vein endothelial cells, the induction of oxidized guanine products by arsenite was sensitive to inhibitors of nitric oxide (NO) synthase but not to oxidant modulators, whereas the opposite result was obtained in vascular smooth muscle cells. On the other hand, the arsenite-induced oxidized guanine products and DNA-protein cross-links in NB4 and HL60 cells were sensitive to modulators of calcium, NO synthase, oxidant, and myeloperoxidase. Therefore, although oxidized guanine products were detected in all the human cells treated with arsenite, the pathways could be different in different cell types. Because the sensitivity and the mechanism of arsenic intoxication are cell specific, it is important that target tissues and target cells are used for investigations. It is also important that pathologically or pharmacologically meaningful concentrations of arsenic are used. This is because in most cases we are dealing with the chronic effect rather than acute toxicity. PMID:12426126

  8. Infrared laser effects at fluences used for treatment of dentin hypersensitivity on DNA repair in Escherichia coli and plasmids

    NASA Astrophysics Data System (ADS)

    Rocha Teixeira, Gleica; da Silva Marciano, Roberta; da Silva Sergio, Luiz Philippe; Castanheira Polignano, Giovanni Augusto; Roberto Guimarães, Oscar; Geller, Mauro; de Paoli, Flavia; de Souza da Fonseca, Adenilson

    2014-12-01

    Low-intensity infrared lasers are proposed in clinical protocols based on biostimulative effects, yet dosimetry is inaccurate and their effects on DNA at therapeutic doses are controversial. The aim of this work was to evaluate the effects of low-intensity infrared laser on survival and induction of filamentation of Escherichia coli cells, and induction of DNA lesions in bacterial plasmids. E. coli cultures were exposed to laser (808 nm, 100 mW, 40 and 60 J/cm2) to study bacterial survival and filamentation. Also, bacterial plasmids were exposed to laser to study DNA lesions by electrophoretic profile and action of DNA repair enzymes. Data indicate low-intensity infrared laser has no effect on survival of E. coli wild type and exonuclease III, but decreases the survival of formamidopyrimidine DNA glycosylase/MutM protein and endonuclease III deficient cells in stationary growth phase, induces bacterial filamentation, does not alter the electrophoretic profile of plasmids in agarose gels and does not alter the electrophoretic profile of plasmids incubated with endonuclease III, formamidopyrimidine DNA glycosylase/MutM protein and exonuclease III. Our findings show that low-intensity laser exposure causes DNA lesions at sub-lethal level and induces cellular mechanisms involved in repair of oxidative lesions in DNA. Studies about laser dosimetry and safety strategies are necessary for professionals and patients exposed to low-intensity lasers at therapeutic doses.

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

    PubMed Central

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

    2016-01-01

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

  10. Seasonal variations of DNA damage in human lymphocytes: correlation with different environmental variables.

    PubMed

    Giovannelli, Lisa; Pitozzi, Vanessa; Moretti, Silvia; Boddi, Vieri; Dolara, Piero

    2006-01-29

    Several types of DNA damage, including DNA breaks and DNA base oxidation, display a seasonal trend. In the present work, a sample of 79 healthy subjects living in the city of Florence, Italy, was used to analyse this effect. Three possible causative agents were taken into consideration: solar radiation, air temperature and air ozone level. DNA damage was measured in isolated human lymphocytes at different times during the year and the observed damage was correlated with the levels of these three agents in the days preceding blood sampling. Three time windows were chosen: 3, 7 and 30 days before blood sampling. DNA strand breaks and the oxidized purinic bases cleaved by the formamidopyrimidine glycosylase (FPG sites) were measured by means of the comet assay. The results of multivariate regression analysis showed a positive correlation between lymphocyte DNA damage and air temperature, and a less strong correlation with global solar radiation and air ozone levels. PMID:16095632

  11. Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM.

    PubMed

    Finney-Manchester, Shawn P; Maheshri, Narendra

    2013-05-01

    A major hurdle to evolutionary engineering approaches for multigenic phenotypes is the ability to simultaneously modify multiple genes rapidly and selectively. Here, we describe a method for in vivo-targeted mutagenesis in yeast, targeting glycosylases to embedded arrays for mutagenesis (TaGTEAM). By fusing the yeast 3-methyladenine DNA glycosylase MAG1 to a tetR DNA-binding domain, we are able to elevate mutation rates >800 fold in a specific ∼20-kb region of the genome or on a plasmid that contains an array of tetO sites. A wide spectrum of transitions, transversions and single base deletions are observed. We provide evidence that TaGTEAM generated point mutations occur through error-prone homologous recombination (HR) and depend on resectioning and the error-prone polymerase Pol ζ. We show that HR is error-prone in this context because of DNA damage checkpoint activation and base pair lesions and use this knowledge to shift the primary mutagenic outcome of targeted endonuclease breaks from HR-independent rearrangements to HR-dependent point mutations. The ability to switch repair in this way opens up the possibility of using targeted endonucleases in diverse organisms for in vivo-targeted mutagenesis. PMID:23470991

  12. OGG1 is essential in oxidative stress induced DNA demethylation.

    PubMed

    Zhou, Xiaolong; Zhuang, Ziheng; Wang, Wentao; He, Lingfeng; Wu, Huan; Cao, Yan; Pan, Feiyan; Zhao, Jing; Hu, Zhigang; Sekhar, Chandra; Guo, Zhigang

    2016-09-01

    DNA demethylation is an essential cellular activity to regulate gene expression; however, the mechanism that triggers DNA demethylation remains unknown. Furthermore, DNA demethylation was recently demonstrated to be induced by oxidative stress without a clear molecular mechanism. In this manuscript, we demonstrated that 8-oxoguanine DNA glycosylase-1 (OGG1) is the essential protein involved in oxidative stress-induced DNA demethylation. Oxidative stress induced the formation of 8-oxoguanine (8-oxoG). We found that OGG1, the 8-oxoG binding protein, promotes DNA demethylation by interacting and recruiting TET1 to the 8-oxoG lesion. Downregulation of OGG1 makes cells resistant to oxidative stress-induced DNA demethylation, while over-expression of OGG1 renders cells susceptible to DNA demethylation by oxidative stress. These data not only illustrate the importance of base excision repair (BER) in DNA demethylation but also reveal how the DNA demethylation signal is transferred to downstream DNA demethylation enzymes. PMID:27251462

  13. Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest

    SciTech Connect

    Liou, Jong-Shian; Wu, Yi-Chen; Yen, Wen-Yen; Tang, Yu-Shuan; Kakadiya, Rajesh B.; Su, Tsann-Long; Yih, Ling-Huei

    2014-08-01

    DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest. - Highlights: • Autophagy inhibitors enhanced the cytotoxicity of a DNA alkylating agent, BO-1012. • BO-1012-induced S phase arrest was a CHK1-dependent pro-survival response. • Autophagy inhibition enhanced BO-1012 cytotoxicity via disrupting the S phase arrest.

  14. The function of cux1 in oxidative dna damage repair is needed to prevent premature senescence of mouse embryo fibroblasts

    PubMed Central

    Ramdzan, Zubaidah M.; Pal, Ranjana; Kaur, Simran; Leduy, Lam; Bérubé, Ginette; Davoudi, Sayeh; Vadnais, Charles; Nepveu, Alain

    2015-01-01

    Despite having long telomeres, mouse embryo fibroblasts (MEFs) senesce more rapidly than human diploid fibroblasts because of the accumulation of oxidative DNA damage. The CUX1 homeodomain protein was recently found to prevent senescence in RAS-driven cancer cells that produce elevated levels of reactive-oxygen species. Here we show that Cux1−/− MEFs are unable to proliferate in atmospheric (20%) oxygen although they can proliferate normally in physiological (3%) oxygen levels. CUX1 contains three domains called Cut repeats. Structure/function analysis established that a single Cut repeat domain can stimulate the DNA binding, Schiff-base formation, glycosylase and AP-lyase activities of 8-oxoguanine DNA glycosylase 1, OGG1. Strikingly and in contrast to previous reports, OGG1 exhibits efficient AP-lyase activity in the presence of a Cut repeat. Repair of oxidative DNA damage and proliferation in 20% oxygen were both rescued in Cux1−/− MEFs by ectopic expression of CUX1 or of a recombinant Cut repeat protein that stimulates OGG1 but is devoid of transcription activation potential. These findings reinforce the causal link between oxidative DNA damage and cellular senescence and suggest that the role of CUX1 as an accessory factor in DNA repair will be critical in physiological situations that generate higher levels of reactive oxygen species. PMID:25682875

  15. NEIL1 Binding to DNA containing 2′-Fluorothymidine Glycol Stereoisomers and the Effect of Editing

    PubMed Central

    Onizuka, Kazumitsu; Yeo, Jongchan

    2012-01-01

    Thymine glycol (Tg), one of the oxidized bases formed in DNA by reactive oxygen species, is repaired by the DNA glycosylases such as NEIL1, NTH1 and Endo III. In our recent studies, we showed that NEIL1’s catalytic efficiency and lesion specificity are regulated by an RNA editing adenosine deamination reaction. In this study, we synthesized oligodeoxynucleotides containing 2′-fluorothymidine glycol with either ribo or arabino configuration and investigated the binding of these modified DNAs with the unedited and edited forms of human NEIL1 along with E. coli Endo III. For the two forms of hNEIL1, binding affinities to FTg-containing DNA were similar indicating the editing effect is more subtle than to simply alter substrate affinity. While the NEIL1 binding to FTg-containing DNAs was largely insensitive to C5 and 2′ stereochemistry, a preference was observed for the FTg-G pair over the FTg-A pair. In addition, we found that optimal binding is observed with Endo III and duplex DNA with riboFTg (5S) paired with dG. The modified DNAs reported here will provide useful tools for further characterizing the interaction between DNA repair glycosylases and thymine glycol containing DNA. PMID:22639086

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

    PubMed

    Nikiforov, Theo T; Roman, Steven

    2015-05-15

    Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3' ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5' phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5' extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido-pyrimidine-DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions). PMID:25728944

  17. Enzymatic DNA oxidation: mechanisms and biological significance

    PubMed Central

    Xu, Guo-Liang; Walsh, Colum P.

    2014-01-01

    DNA methylation at cytosines (5mC) is a major epigenetic modification involved in the regulation of multiple biological processes in mammals. How methylation is reversed was until recently poorly understood. The family of dioxygenases commonly known as Ten-eleven translocation (Tet) proteins are responsible for the oxidation of 5mC into three new forms, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Current models link Tet-mediated 5mC oxidation with active DNA demethylation. The higher oxidation products (5fC and 5caC) are recognized and excised by the DNA glycosylase TDG via the base excision repair pathway. Like DNA methyltransferases, Tet enzymes are important for embryonic development. We will examine the mechanism and biological significance of Tet-mediated 5mC oxidation in the context of pronuclear DNA demethylation in mouse early embryos. In contrast to its role in active demethylation in the germ cells and early embryo, a number of lines of evidence suggest that the intragenic 5hmC present in brain may act as a stable mark instead. This short review explores mechanistic aspects of TET oxidation activity, the impact Tet enzymes have on epigenome organization and their contribution to the regulation of early embryonic and neuronal development. [BMB Reports 2014; 47(11): 609-618] PMID:25341925

  18. Specificity of damage recognition and catalysis of DNA repair.

    PubMed

    Osman, R; Fuxreiter, M; Luo, N

    2000-05-01

    A common feature of DNA repair enzymes is their ability to recognize the damage independently of sequence in which they are found. The presence of a flipped out base inserted into the protein in several DNA-enzyme complexes suggests a contribution to enzyme specificity. Molecular simulations of damaged DNA indicate that the damage produces changes in DNA structure and changes the dynamics of DNA bending. The reduced bending force constant can be used by the enzyme to induce DNA bending and facilitate base flipping. We show that a thymine dimer (TD) containing DNA requires less energy to bend, lowering the barrier for base flipping. On the other hand, bending in DNA with U-G mismatch is affected only by a small amount and flipping is not enhanced significantly. T4 endonuclease V (endoV), which recognizes TD, utilizes the reduced barrier for flipping as a specific recognition element. In uracil DNA glycosylase (UDG), which recognizes U-G mismatches, base flipping is not enhanced and recognition is encoded in a highly specific binding pocket for the flipped base. Simulations of UDG and endoV in complex with damaged DNA provide insight into the essential elements of the catalytic mechanism. Calculations of pKas of active site residues in endoV and endoV-DNA complex show that the pKa, of the N-terminus is reduced from 8.01 to 6.52 while that of Glu-23 increases from 1.52 to 7.82. Thus, the key catalytic residues are in their neutral form. The simulations also show that Glu-23 is also H-bonded to O4' of the 5'-TD enhancing the nucleophilic attack on Cl and that Arg-26 enhances the hydrolysis by electrostatic stabilization but does not participate in proton transfer. In the enzyme-substrate complex of UDG, the role of electrostatic stabilization is played by His-268, whose pKa increases to 7.1 from 4.9 in the free enzyme. The pKa of Asp-145, the other important catalytic residue, remains around 4.2 in the free enzyme and in the complex. Thus, it can not act as a proton

  19. Repair of DNA-containing pyrimidine dimers

    SciTech Connect

    Grossman, L.; Caron, P.R.; Mazur, S.J.; Oh, E.Y.

    1988-08-01

    Ultraviolet light-induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. The most direct biochemical mechanism responding to this kind of genotoxicity involves direct photoreversal by flavin enzymes that specifically monomerize pyrimidine:pyrimidine dimers monophotonically in the presence of visible light. Incision reactions are catalyzed by a combined pyrimidine dimer DNA-glycosylase:apyrimidinic endonuclease found in some highly UV-resistant organisms. At a higher level of complexity, Escherichia coli has a uvr DNA repair system comprising the UvrA, UvrB, and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway, which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding is associated with localized topological unwinding of DNA. This same protein complex can catalyze an ATPase-dependent 5'----3'-directed strand displacement of D-loop DNA or short single strands annealed to a single-stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled excision-resynthesis step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress-induced protease that also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA, it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.21 references.

  20. Persistent damage induces mitochondrial DNA degradation

    PubMed Central

    Shokolenko, Inna N.; Wilson, Glenn L.; Alexeyev, Mikhail F.

    2013-01-01

    Considerable progress has been made recently toward understanding the processes of mitochondrial DNA (mtDNA) damage and repair. However, a paucity of information still exists regarding the physiological effects of persistent mtDNA damage. This is due, in part, to experimental difficulties associated with targeting mtDNA for damage, while sparing nuclear DNA. Here, we characterize two systems designed for targeted mtDNA damage based on the inducible (Tet-ON) mitochondrial expression of the bacterial enzyme, exonuclease III, and the human enzyme, uracil-N-glyosylase containing the Y147A mutation. In both systems, damage was accompanied by degradation of mtDNA, which was detectable by six hours after induction of mutant uracil-N-glycosylase and by twelve hours after induction of exoIII. Unexpectedly, increases in the steady-state levels of single-strand lesions, which led to degradation, were small in absolute terms indicating that both abasic sites and single-strand gaps may be poorly tolerated in mtDNA. mtDNA degradation was accompanied by the loss of expression of mtDNA-encoded COX2. After withdrawal of the inducer, recovery from mtDNA depletion occurred faster in the system expressing exonuclease III, but in both systems reduced mtDNA levels persisted longer than 144h after doxycycline withdrawal. mtDNA degradation was followed by reduction and loss of respiration, decreased membrane potential, reduced cell viability, reduced intrinsic reactive oxygen species production, slowed proliferation, and changes in mitochondrial morphology (fragmentation of the mitochondrial network, rounding and “foaming” of the mitochondria). The mutagenic effects of abasic sites in mtDNA were low, which indicates that damaged mtDNA molecules may be degraded if not rapidly repaired. This study establishes, for the first time, that mtDNA degradation can be a direct and immediate consequence of persistent mtDNA damage and that increased ROS production is not an invariant consequence

  1. Surviving the sun: Repair and bypass of DNA UV lesions

    PubMed Central

    Yang, Wei

    2011-01-01

    Structural studies of UV-induced lesions and their complexes with repair proteins reveal an intrinsic flexibility of DNA at lesion sites. Reduced DNA rigidity stems primarily from the loss of base stacking, which may manifest as bending, unwinding, base unstacking, or flipping out. The intrinsic flexibility at UV lesions allows efficient initial lesion recognition within a pool of millions to billions of normal DNA base pairs. To bypass the damaged site by translesion synthesis, the specialized DNA polymerase η acts like a molecular “splint” and reinforces B-form DNA by numerous protein–phosphate interactions. Photolyases and glycosylases that specifically repair UV lesions interact directly with UV lesions in bent DNA via surface complementation. UvrA and UvrB, which recognize a variety of lesions in the bacterial nucleotide excision repair pathway, appear to exploit hysteresis exhibited by DNA lesions and conduct an ATP-dependent stress test to distort and separate DNA strands. Similar stress tests are likely conducted in eukaryotic nucleotide excision repair. PMID:21898645

  2. Pro-oxidant Induced DNA Damage in Human Lymphoblastoid Cells: Homeostatic Mechanisms of Genotoxic Tolerance

    PubMed Central

    Seager, Anna L.

    2012-01-01

    Oxidative stress contributes to many disease etiologies including ageing, neurodegeneration, and cancer, partly through DNA damage induction (genotoxicity). Understanding the i nteractions of free radicals with DNA is fundamental to discern mutation risks. In genetic toxicology, regulatory authorities consider that most genotoxins exhibit a linear relationship between dose and mutagenic response. Yet, homeostatic mechanisms, including DNA repair, that allow cells to tolerate low levels of genotoxic exposure exist. Acceptance of thresholds for genotoxicity has widespread consequences in terms of understanding cancer risk and regulating human exposure to chemicals/drugs. Three pro-oxidant chemicals, hydrogen peroxide (H2O2), potassium bromate (KBrO3), and menadione, were examined for low dose-response curves in human lymphoblastoid cells. DNA repair and antioxidant capacity were assessed as possible threshold mechanisms. H2O2 and KBrO3, but not menadione, exhibited thresholded responses, containing a range of nongenotoxic low doses. Levels of the DNA glycosylase 8-oxoguanine glycosylase were unchanged in response to pro- oxidant stress. DNA repair–focused gene expression arrays reported changes in ATM and BRCA1, involved in double-strand break repair, in response to low-dose pro-oxidant exposure; however, these alterations were not substantiated at the protein level. Determination of oxidatively induced DNA damage in H2O2-treated AHH-1 cells reported accumulation of thymine glycol above the genotoxic threshold. Further, the H2O2 dose-response curve was shifted by modulating the antioxidant glutathione. Hence, observed pro- oxidant thresholds were due to protective capacities of base excision repair enzymes and antioxidants against DNA damage, highlighting the importance of homeostatic mechanisms in “genotoxic tolerance.” PMID:22539617

  3. DNA oxidation as triggered by H3K9me2 demethylation drives estrogen-induced gene expression.

    PubMed

    Perillo, Bruno; Ombra, Maria Neve; Bertoni, Alessandra; Cuozzo, Concetta; Sacchetti, Silvana; Sasso, Annarita; Chiariotti, Lorenzo; Malorni, Antonio; Abbondanza, Ciro; Avvedimento, Enrico V

    2008-01-11

    Modifications at the N-terminal tails of nucleosomal histones are required for efficient transcription in vivo. We analyzed how H3 histone methylation and demethylation control expression of estrogen-responsive genes and show that a DNA-bound estrogen receptor directs transcription by participating in bending chromatin to contact the RNA polymerase II recruited to the promoter. This process is driven by receptor-targeted demethylation of H3 lysine 9 at both enhancer and promoter sites and is achieved by activation of resident LSD1 demethylase. Localized demethylation produces hydrogen peroxide, which modifies the surrounding DNA and recruits 8-oxoguanine-DNA glycosylase 1 and topoisomeraseIIbeta, triggering chromatin and DNA conformational changes that are essential for estrogen-induced transcription. Our data show a strategy that uses controlled DNA damage and repair to guide productive transcription. PMID:18187655

  4. Circadian Modulation of 8-Oxoguanine DNA Damage Repair

    PubMed Central

    Manzella, Nicola; Bracci, Massimo; Strafella, Elisabetta; Staffolani, Sara; Ciarapica, Veronica; Copertaro, Alfredo; Rapisarda, Venerando; Ledda, Caterina; Amati, Monica; Valentino, Matteo; Tomasetti, Marco; Stevens, Richard G.; Santarelli, Lory

    2015-01-01

    The DNA base excision repair pathway is the main system involved in the removal of oxidative damage to DNA such as 8-Oxoguanine (8-oxoG) primarily via the 8-Oxoguanine DNA glycosylase (OGG1). Our goal was to investigate whether the repair of 8-oxoG DNA damage follow a circadian rhythm. In a group of 15 healthy volunteers, we found a daily variation of Ogg1 expression and activity with higher levels in the morning compared to the evening hours. Consistent with this, we also found lower levels of 8-oxoG in morning hours compared to those in the evening hours. Lymphocytes exposed to oxidative damage to DNA at 8:00 AM display lower accumulation of 8-oxoG than lymphocytes exposed at 8:00 PM. Furthermore, altered levels of Ogg1 expression were also observed in a group of shift workers experiencing a deregulation of circadian clock genes compared to a control group. Moreover, BMAL1 knockdown fibroblasts with a deregulated molecular clock showed an abolishment of circadian variation of Ogg1 expression and an increase of OGG1 activity. Our results suggest that the circadian modulation of 8-oxoG DNA damage repair, according to a variation of Ogg1 expression, could render humans less susceptible to accumulate 8-oxoG DNA damage in the morning hours. PMID:26337123

  5. Circadian Modulation of 8-Oxoguanine DNA Damage Repair.

    PubMed

    Manzella, Nicola; Bracci, Massimo; Strafella, Elisabetta; Staffolani, Sara; Ciarapica, Veronica; Copertaro, Alfredo; Rapisarda, Venerando; Ledda, Caterina; Amati, Monica; Valentino, Matteo; Tomasetti, Marco; Stevens, Richard G; Santarelli, Lory

    2015-01-01

    The DNA base excision repair pathway is the main system involved in the removal of oxidative damage to DNA such as 8-Oxoguanine (8-oxoG) primarily via the 8-Oxoguanine DNA glycosylase (OGG1). Our goal was to investigate whether the repair of 8-oxoG DNA damage follow a circadian rhythm. In a group of 15 healthy volunteers, we found a daily variation of Ogg1 expression and activity with higher levels in the morning compared to the evening hours. Consistent with this, we also found lower levels of 8-oxoG in morning hours compared to those in the evening hours. Lymphocytes exposed to oxidative damage to DNA at 8:00 AM display lower accumulation of 8-oxoG than lymphocytes exposed at 8:00 PM. Furthermore, altered levels of Ogg1 expression were also observed in a group of shift workers experiencing a deregulation of circadian clock genes compared to a control group. Moreover, BMAL1 knockdown fibroblasts with a deregulated molecular clock showed an abolishment of circadian variation of Ogg1 expression and an increase of OGG1 activity. Our results suggest that the circadian modulation of 8-oxoG DNA damage repair, according to a variation of Ogg1 expression, could render humans less susceptible to accumulate 8-oxoG DNA damage in the morning hours. PMID:26337123

  6. The induction of SCE and chromosomal aberrations with relation to specific base methylation of DNA in Chinese hamster cells by N-methyl-N-nitrosourea and dimethyl sulphate.

    PubMed

    Connell, J R; Medcalf, A S

    1982-01-01

    Chinese hamster cells (V79) were treated, either as exponentially proliferating cultures or under conditions where they were density-inhibited, with various doses of the potent carcinogen N-methyl-N-nitrosourea (MNU) or the relatively weak carcinogen dimethylsulphate (DMS). The colony forming ability of these cells and the induced frequencies of sister chromatid exchanges (SCEs) and chromosomal aberrations were assayed. Following the exposure of density-inhibited cells to radio-labelled methylating agents (labelled in the methyl group) these phenomena were related to the levels of 7-methylguanine (7-meGua), O6-methylguanine (O6-meGua) and 3-methyladenine (3-me-Ade) in the DNA. At equitoxic doses MNU and DMS induced similar frequencies of SCEs and chromosomal aberrations. Since, at equitoxic doses, MNU produces approximately 20 times more O6-meGua in V79 cell DNA than does DMS, this indicates that the formation of O6-meGua in DNA is not a major cause of SCEs and chromosomal aberrations. DMS-induced SCEs may be mediated via the production of both 3-meAde and 7-meGua in the DNA; these two methylated purines may also be responsible for MNU-induced SCEs. Therefore, no one specific methylated purine was identified as being solely accountable for the formation of SCEs. Also, the repair of lesions in the DNA of non-replicating V79 cells leads to a reduction in the SCE frequency on their subsequent release from the density-inhibited state, suggesting that repair is not intimately responsible for their formation. No association was discernable between chromosomal aberrations and any of the three methylated purines studied. PMID:7094205

  7. Bifilar enzyme-sensitive sites in ultraviolet-irradiated DNA are indicative of closely opposed cyclobutyl pyrimidine dimers.

    PubMed Central

    Lam, L H; Reynolds, R J

    1986-01-01

    Incubation of UV-irradiated DNA with pyrimidine dimer-DNA glycosylase in cell-free lysates prepared from Micrococcus luteus results in the appearance of double-strand breaks. It has previously been assumed that such double-strand breaks result from cleavage at closely opposed dimers. We have used hybrid molecules of bacteriophage T7 DNA comprised of two unirradiated strands, two UV-irradiated strands, or one unirradiated and one UV-irradiated strand to test this hypothesis. Bifilar cleavage was observed only with molecules consisting of two irradiated strands and no bifilar cleavage was observed after the monomerization of pyrimidine dimers by enzymatic photoreactivation. Our results indicate that at least 80% of the double-strand breaks result from cleavage at closely opposed dimers and that the induction of dimers in one strand does not influence the induction of dimers at closely opposed positions in the complementary strand of a DNA double helix. PMID:3527288

  8. The antileishmanial drug miltefosine (Impavido(®)) causes oxidation of DNA bases, apoptosis, and necrosis in mammalian cells.

    PubMed

    Castelo Branco, Patrícia Valéria; Soares, Rossy-Eric Pereira; de Jesus, Luís Cláudio Lima; Moreira, Vanessa Ribeiro; Alves, Hugo José; de Castro Belfort, Marta Regina; Silva, Vera Lucia Maciel; Ferreira Pereira, Silma Regina

    2016-08-01

    Miltefosine was developed to treat skin cancer; further studies showed that the drug also has activity against Leishmania. Miltefosine is the first oral agent for treating leishmaniasis. However, its mechanism of action is not completely understood. We have evaluated the induction of DNA damage by miltefosine. Cytotoxicity and genotoxicity (comet assay) tests were performed on human leukocytes exposed to the drug in vitro. Apoptosis and necrosis were also evaluated. In vivo tests were conducted in Swiss male mice (Mus musculus) treated orally with miltefosine. Oxidation of DNA bases in peripheral blood cells was measured using the comet assay followed by digestion with formamidopyrimidine glycosylase (FPG), which removes oxidized guanine bases. The micronucleus test was performed on bone marrow erythrocytes. Miltefosine caused DNA damage, apoptosis, and necrosis in vitro. Mice treated with miltefosine showed an increase in the DNA damage score, which was further increased following FPG digestion. The micronucleus test was also positive. PMID:27476333

  9. Modular Nuclease-Responsive DNA Three-Way Junction-Based Dynamic Assembly of a DNA Device and Its Sensing Application.

    PubMed

    Zhu, Jing; Wang, Lei; Xu, Xiaowen; Wei, Haiping; Jiang, Wei

    2016-04-01

    Here, we explored a modular strategy for rational design of nuclease-responsive three-way junctions (TWJs) and fabricated a dynamic DNA device in a "plug-and-play" fashion. First, inactivated TWJs were designed, which contained three functional domains: the inaccessible toehold and branch migration domains, the specific sites of nucleases, and the auxiliary complementary sequence. The actions of different nucleases on their specific sites in TWJs caused the close proximity of the same toehold and branch migration domains, resulting in the activation of the TWJs and the formation of a universal trigger for the subsequent dynamic assembly. Second, two hairpins (H1 and H2) were introduced, which could coexist in a metastable state, initially to act as the components for the dynamic assembly. Once the trigger initiated the opening of H1 via TWJs-driven strand displacement, the cascade hybridization of hairpins immediately switched on, resulting in the formation of the concatemers of H1/H2 complex appending numerous integrated G-quadruplexes, which were used to obtain label-free signal readout. The inherent modularity of this design allowed us to fabricate a flexible DNA dynamic device and detect multiple nucleases through altering the recognition pattern slightly. Taking uracil-DNA glycosylase and CpG methyltransferase M.SssI as models, we successfully realized the butt joint between the uracil-DNA glycosylase and M.SssI recognition events and the dynamic assembly process. Furthermore, we achieved ultrasensitive assay of nuclease activity and the inhibitor screening. The DNA device proposed here will offer an adaptive and flexible tool for clinical diagnosis and anticancer drug discovery. PMID:26943244

  10. Potentiation of cytotoxicity by 3-aminobenzamide in DNA repair-deficient human tumor cell lines following exposure to methylating agents or anti-neoplastic drugs.

    PubMed

    Babich, M A; Day, R S

    1988-04-01

    We studied the potentiation by 3-aminobenzamide (3AB) of killing of nine human cell lines exposed to alkylating agents. Cell lines included normal, transformed and DNA repair-proficient and -deficient phenotypes. 3AB potentiated cell killing by the methylating agents methylmethanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in all lines tested. The degree of potentiation ranged from 1.7- to 3.8-fold, based on the LD99. The average potentiation observed with MMS (2.7-fold) was greater than with MNNG (2.2-fold). On average the potentiation of MMS and MNNG killing of repair-deficient Mer- lines (2.4-fold) was similar to that of repair-proficient Mer+ lines. The degree of 3AB potentiation of MNNG killing (2.0-fold) was similar in Mer+ Rem- lines and in Mer+ Rem+ lines. Mer+ Rem+, Mer+ Rem-, Mer- Rem+, and Mer- Rem- strains all appeared proficient in a 3AB-sensitive DNA repair pathway. Within experimental error, 20 mM 3AB did not inhibit the removal of the MNNG-induced methylpurines 7-methylguanine, O6-methylguanine and 3-methyladenine from the DNA of repair-proficient Mer+ Rem+ HT29 cells, consistent with evidence that 3AB inhibits the ligation step of excision repair. 3AB potentiated cell killing by the bifunctional alkylating agents 1-(2-chlorethyl)-1-nitrosourea or busulfan, two anti-neoplastic drugs, by only 0.9- to 1.5-fold. These drugs therefore produce DNA damage which is not efficiently repaired by the pathways that repair methylated bases. PMID:3356063

  11. Atomic structure of the DNA repair [4Fe-4S] enzyme endonuclease III.

    PubMed

    Kuo, C F; McRee, D E; Fisher, C L; O'Handley, S F; Cunningham, R P; Tainer, J A

    1992-10-16

    The crystal structure of the DNA repair enzyme endonuclease III, which recognizes and cleaves DNA at damaged bases, has been solved to 2.0 angstrom resolution with an R factor of 0.185. This iron-sulfur [4Fe-4S] enzyme is elongated and bilobal with a deep cleft separating two similarly sized domains: a novel, sequence-continuous, six-helix domain (residues 22 to 132) and a Greek-key, four-helix domain formed by the amino-terminal and three carboxyl-terminal helices (residues 1 to 21 and 133 to 211) together with the [4Fe-4S] cluster. The cluster is bound entirely within the carboxyl-terminal loop with a ligation pattern (Cys-X6-Cys-X2-Cys-X5-Cys) distinct from all other known [4Fe-4S] proteins. Sequence conservation and the positive electrostatic potential of conserved regions identify a surface suitable for binding duplex B-DNA across the long axis of the enzyme, matching a 46 angstrom length of protected DNA. The primary role of the [4Fe-4S] cluster appears to involve positioning conserved basic residues for interaction with the DNA phosphate backbone. The crystallographically identified inhibitor binding region, which recognizes the damaged base thymine glycol, is a seven-residue beta-hairpin (residues 113 to 119). Location and side chain orientation at the base of the inhibitor binding site implicate Glu112 in the N-glycosylase mechanism and Lys120 in the beta-elimination mechanism. Overall, the structure reveals an unusual fold and a new biological function for [4Fe-4S] clusters and provides a structural basis for studying recognition of damaged DNA and the N-glycosylase and apurinic/apyrimidinic-lyase mechanisms. PMID:1411536

  12. RIP3 induces ischemic neuronal DNA degradation and programmed necrosis in rat via AIF

    PubMed Central

    Xu, Yang; Wang, Jingye; Song, Xinghui; Qu, Lindi; Wei, Ruili; He, Fangping; Wang, Kai; Luo, Benyan

    2016-01-01

    We have reported that nuclear translocation of Receptor-interacting protein 3 (RIP3) involves in neuronal programmed necrosis after 20-min global cerebral ischemia/reperfusion (I/R) injury. Herein, the underlying mechanisms and the nuclear role of RIP3 were investigated further. The necroptosis inhibitor necrostatin-1 (Nec-1), the autophagy inhibitor 3-methyladenine (3-MA), and the caspase-3 inhibitor acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspart-1-al (Ac-DMQD-CHO) were administered intracerebroventricularly 1 h before ischemia. Protein expression, location and interaction was determined by western blot, immunofluorescence or immunoprecipitation. Most CA1 neuronal death induced by 20-min global cerebral I/R injury was TUNEL-positive. Neuronal death and rat mortality rates were greatly inhibited by Nec-1 and 3-MA pre-treatment, but not by Ac-DMQD-CHO. And no activation of caspase-3 was detected after I/R injury. Caspase-8 was expressed richly in GFAP-positive astrocytes and Iba-1-positive microglia, but was not detected in Neun-positive neurons. The nuclear translocation and co-localization of RIP3 and AIF, and their interaction were detected after I/R injury. These processes were inhibited by Nec-1 and 3-MA pre-treatment, but not by Ac-DMQD-CHO. The formation of an RIP3-AIF complex and its nuclear translocation are critical to ischemic neuronal DNA degradation and programmed necrosis. Neurons are more likely to enter the programmed necrosis signal pathway for the loss of caspase-8 suppression. PMID:27377128

  13. DNA damage and repair kinetics of the Alternaria mycotoxins alternariol, altertoxin II and stemphyltoxin III in cultured cells.

    PubMed

    Fleck, Stefanie C; Sauter, Friederike; Pfeiffer, Erika; Metzler, Manfred; Hartwig, Andrea; Köberle, Beate

    2016-03-01

    The Alternaria mycotoxins alternariol (AOH) and altertoxin II (ATX II) have previously been shown to elicit mutagenic and genotoxic effects in bacterial and mammalian cells, although with vastly different activities. For example, ATX II was about 50 times more mutagenic than AOH. We now report that stemphyltoxin III (STTX III) is also highly mutagenic. The more pronounced effects of the perylene quinones ATX II and STTX III at lower concentrations compared to the dibenzo-α-pyrone AOH indicate a marked dependence of the genotoxic potential on the chemical structure and furthermore suggest that the underlying modes of action may be different. We have now further investigated the type of DNA damage induced by AOH, ATX II and STTX III, as well as the repair kinetics and their dependence on the status of nucleotide excision repair (NER). DNA double strand breaks induced by AOH due to poisoning of topoisomerase IIα were completely repaired in less than 2h. Under cell-free conditions, inhibition of topoisomerase IIα could also be measured for ATX II and STTX III at low concentrations, but the perylene quinones were catalytic inhibitors rather than topoisomerase poisons and did not induce DSBs. DNA strand breaks induced by ATX II and STTX III were more persistent and not completely repaired within 24h. A dependence of the repair rate on the NER status could only be demonstrated for STTX III, resulting in an accumulation of DNA damage in NER-deficient cells. Together with the finding that the DNA glycosylase formamidopyrimidine-DNA glycosylase (Fpg), but not T4 endonuclease V, is able to generate additional DNA strand breaks measurable by the alkaline unwinding assay, we conclude that the genotoxicity of the perylene quinones with an epoxide group is probably caused by the formation of DNA adducts which may be converted to Fpg sensitive sites. PMID:26994491

  14. Quantification of 8-oxodGuo lesions in double-stranded DNA using a photoelectrochemical DNA sensor.

    PubMed

    Zhang, Bintian; Guo, Liang-Hong; Greenberg, Marc M

    2012-07-17

    Exposure of DNA to oxidative stress conditions results in the generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). 8-OxodGuo is genotoxic if left unrepaired. We quantified 8-oxodGuo lesions in double-stranded DNA films by using a photoelectrochemical DNA sensor in conjunction with a specific covalent labeling method. A lesion-containing DNA film was assembled on a SnO(2) nanoparticle modified indium tin oxide electrode through layer-by-layer electrostatic adsorption. The lesions were covalently labeled with a biotin conjugated spermine derivative, and ruthenium tris(bipyridine) labeled streptavidin was introduced as the signal reporter molecule. Photocurrent increased with the number of lesions in the strand and decreased as the film was diluted with intact DNA. Quantification of 8-oxodGuo was achieved with an estimated detection limit of ∼1 lesion in 650 bases or 1.6 fmol of 8-oxodGuo on the electrode. Incubation of the film with a DNA base excision repair enzyme, E. coli formamidopyrimidine-DNA glycosylase (Fpg), resulted in complete loss of the signal, indicating efficient excision of the isolated lesions in the nucleotide. Oxidatively generated DNA damage to a double-stranded calf thymus DNA film by the Fenton reaction was then assessed. One 8-oxodGuo lesion in 520 bases was detected in DNA exposed to 50 μM Fe(2+)/200 μM H(2)O(2). Treatment with Fpg reduced the photocurrent by 50%, indicating only partial excision of 8-oxodGuo. This suggests that tandem lesions, which are resistant to Fpg excision, are generated by the Fenton reaction. Unlike repair enzyme dependent methods, the sensor recognizes 8-oxodGuo in tandem lesions and can avoid underestimating DNA damage. PMID:22746252

  15. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.

    PubMed

    Komor, Alexis C; Kim, Yongjoo B; Packer, Michael S; Zuris, John A; Liu, David R

    2016-05-19

    Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations. PMID:27096365

  16. Gadd45a promotes DNA demethylation through TDG.

    PubMed

    Li, Zheng; Gu, Tian-Peng; Weber, Alain R; Shen, Jia-Zhen; Li, Bin-Zhong; Xie, Zhi-Guo; Yin, Ruichuan; Guo, Fan; Liu, Xiaomeng; Tang, Fuchou; Wang, Hailin; Schär, Primo; Xu, Guo-Liang

    2015-04-30

    Growth arrest and DNA-damage-inducible protein 45 (Gadd45) family members have been implicated in DNA demethylation in vertebrates. However, it remained unclear how they contribute to the demethylation process. Here, we demonstrate that Gadd45a promotes active DNA demethylation through thymine DNA glycosylase (TDG) which has recently been shown to excise 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated in Ten-eleven-translocation (Tet)-initiated oxidative demethylation. The connection of Gadd45a with oxidative demethylation is evidenced by the enhanced activation of a methylated reporter gene in HEK293T cells expressing Gadd45a in combination with catalytically active TDG and Tet. Gadd45a interacts with TDG physically and increases the removal of 5fC and 5caC from genomic and transfected plasmid DNA by TDG. Knockout of both Gadd45a and Gadd45b from mouse ES cells leads to hypermethylation of specific genomic loci most of which are also targets of TDG and show 5fC enrichment in TDG-deficient cells. These observations indicate that the demethylation effect of Gadd45a is mediated by TDG activity. This finding thus unites Gadd45a with the recently defined Tet-initiated demethylation pathway. PMID:25845601

  17. Gadd45a promotes DNA demethylation through TDG

    PubMed Central

    Li, Zheng; Gu, Tian-Peng; Weber, Alain R.; Shen, Jia-Zhen; Li, Bin-Zhong; Xie, Zhi-Guo; Yin, Ruichuan; Guo, Fan; Liu, Xiaomeng; Tang, Fuchou; Wang, Hailin; Schär, Primo; Xu, Guo-Liang

    2015-01-01

    Growth arrest and DNA-damage-inducible protein 45 (Gadd45) family members have been implicated in DNA demethylation in vertebrates. However, it remained unclear how they contribute to the demethylation process. Here, we demonstrate that Gadd45a promotes active DNA demethylation through thymine DNA glycosylase (TDG) which has recently been shown to excise 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated in Ten-eleven-translocation (Tet)—initiated oxidative demethylation. The connection of Gadd45a with oxidative demethylation is evidenced by the enhanced activation of a methylated reporter gene in HEK293T cells expressing Gadd45a in combination with catalytically active TDG and Tet. Gadd45a interacts with TDG physically and increases the removal of 5fC and 5caC from genomic and transfected plasmid DNA by TDG. Knockout of both Gadd45a and Gadd45b from mouse ES cells leads to hypermethylation of specific genomic loci most of which are also targets of TDG and show 5fC enrichment in TDG-deficient cells. These observations indicate that the demethylation effect of Gadd45a is mediated by TDG activity. This finding thus unites Gadd45a with the recently defined Tet-initiated demethylation pathway. PMID:25845601

  18. Involvement of mammalian OGG1(MMH) in excision of the 8-hydroxyguanine residue in DNA.

    PubMed

    Nishimura, Susumu

    2002-05-01

    8-Hydroxyguanine (7,8-dihydro-8-oxoguanine, abbreviated as 8-OH-G or 8-oxoG) is the site of a frequent mutagenic DNA lesion produced by oxidative damage. MutM of E. coli and OGG1 of Saccharomyces cervisiae are known to possess 8-OH-G glycosylase and apurinic (AP) site lyase activity. cDNA clones of four isoforms (types 1a, 1b, 1c, and 2) of human OGG1 homologs (hMMH) were isolated. In order to examine whether expression of hMMH (hOGG1) protein actually occurs in human cells, we prepared type 1a specific antibody, and by using this antibody, we showed that type 1a protein isolated from HeLaS3 has 8-OH-G glycosylase/lyase activity. Furthermore, we showed that type 1a protein is a major enzyme for repair of the 8-OH-G lesion in human cells. In our second study, we generated a mouse line carrying an inactivated mutant Mmh allele by targeted gene disruption. Liver extracts of Mmh homozygous mutant mice were found to have loss of the nicking activity for the 8-OH-G site. In addition, the amount of endogenous 8-OH-G in liver DNA of the homozygous mice increased linearly with age, reaching 7-fold increase in 14 week old mice, over that of wild-type or heterozygous mice. Furthermore, when homozygous mice were fed the oxygen radical-forming agent KBrO3, to provide oxidative stress, the level of 8-OH-G in kidney DNA was tremendously increased: more than 200-fold as that of control mice without oxidative stress after 12 weeks of age. These results indicate that Ogg1/Mmh plays an essential role in the repair of the 8-OH-G residue in DNA produced by oxidative stress. PMID:11978483

  19. An immunochemical approach to the study of DNA damage and repair

    SciTech Connect

    Wallace, S.S.; Erlanger, B.F. . Dept. of Microbiology and Molecular Genetics; Columbia Univ., New York, NY . Dept. of Microbiology)

    1989-01-01

    The most studied radiation-induced modified DNA base is thymine glycol. Thymine glycols are produced in relatively high yields in irradiated DNA and are also formed as a consequence of oxidative stress. Thymine glycol has been shown to be an in vitro replicative block to DNA polymerases as well as a cytotoxic lesion. DNA glycosylases that remove thymine glycol from damaged DNA are found in both prokaryotes an deukaryotes. Thus this lesion provides a good model for studying the potential biological consequences of pyrimidine ring saturation products. In order to elicit antibodies that would react with unique modified base on a damaged DNA molecule, we have chosen to chemically synthesize the hapten of interest and conjugate it to a protein carrier. Thymine glycol monophosphate was synthesized, conjugated by the carbodiimide method to bovine serum albumin (BSA), and used as an immunogen. Initially, polyclonal antibodies were produced in rabbits. These antibodies had high affinity and specificity as measured by both albumin (RSA) and by enzyme immunoassay using either the conjugate or DNA oxidized inhibited by thymine glycol, thymidine glycol and thymine glycol monophosphate determinants for the phosphate group in addition to the thymidine glycol. In both the direct and competitive assays, this antibody reacts with osmium tetroxide-treated DNA containing cis-thymine glycols and DNA X-irradiated in vitro at a femtomole level of sensitivity. 24 refs.

  20. Low intensity infrared laser effects on Escherichia coli cultures and plasmid DNA

    NASA Astrophysics Data System (ADS)

    Fonseca, A. S.; Teixeira, A. F.; Presta, G. A.; Geller, M.; Valença, S. S.; Paoli, F.

    2012-10-01

    Biostimulative effect of low intensity laser in tissues has been described on a photobiological basis and clinical protocols are recommended for treatment of various diseases. The aim of this work was to evaluate effects of laser exposure on the survival of Escherichia coli cultures and plasmid topological forms. Escherichia coli cultures and plasmids were exposed to infrared laser to study bacterial survival and electrophoretic profile, respectively. Data indicate low intensity infrared laser: (i) had no effect on E. coli wild type, endonuclease IV, exonuclease III, formamidopyrimidine DNA glycosylase/MutM protein and endonuclease III deficient cultures, but decreased the survival of E. coli UvrA protein deficient cultures; (ii) there was no alteration in the electrophoretic profile of plasmids. Exposure to low intensity infrared laser decreases survival of Escherichia coli cultures deficient in nucleotide excision repair of DNA and this effect could depend on fluences, wavelength and tissues conditions.

  1. SUMO-modification and elimination of the active DNA demethylation enzyme TDG in cultured human cells.

    PubMed

    Moriyama, Taishi; Fujimitsu, Yuka; Yoshikai, Yushi; Sasano, Takashi; Yamada, Koji; Murakami, Masataka; Urano, Takeshi; Sugasawa, Kaoru; Saitoh, Hisato

    2014-05-01

    Thymine DNA glycosylase (TDG) is a base excision repair enzyme that interacts with the small ubiquitin-related modifier (SUMO)-targeted ubiquitin E3 ligase RNF4 and functions in the active DNA demethylation pathway. Here we showed that both SUMOylated and non-modified forms of endogenous TDG fluctuated during the cell cycle and in response to drugs that perturbed cell cycle progression, including hydroxyurea and nocodazole. Additionally, we detected a SUMOylation-independent association between TDG and RNF4 in vitro as well as in vivo, and observed that both forms of TDG were efficiently degraded in RNF4-depleted cells when arrested at S phase. Our findings provide insights into the in vivo dynamics of TDG SUMOylation and further clarify the TDG-RNF4 interaction. PMID:24727457

  2. Biomarkers of oxidative damage to DNA and repair.

    PubMed

    Loft, Steffen; Høgh Danielsen, Pernille; Mikkelsen, Lone; Risom, Lotte; Forchhammer, Lykke; Møller, Peter

    2008-10-01

    Oxidative-stress-induced damage to DNA includes a multitude of lesions, many of which are mutagenic and have multiple roles in cancer and aging. Many lesions have been characterized by MS-based methods after extraction and digestion of DNA. These preparation steps may cause spurious base oxidation, 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 DNA glycosylase 1), responsible for repair of 8-oxodG, by genotyping. Products of repair in DNA or the nucleotide pool, such as 8-oxodG, excreted into the urine can be assessed by MS-based methods and generally reflects the rate of damage. Experimental and population-based studies indicate that many environmental factors, including particulate air pollution, cause oxidative damage to DNA, whereas diets rich in fruit and vegetables or antioxidant supplements may reduce the levels and enhance repair. Urinary excretion of 8-oxodG, genotype and expression of OGG1 have been associated with risk of cancer in cohort settings, whereas altered levels of damage, repair or urinary excretion in case-control settings may be a consequence rather than the cause of the disease. PMID:18793191

  3. Use of the comet assay to measure DNA damage in cells exposed to photosensitizers and gamma radiation

    NASA Astrophysics Data System (ADS)

    Pouget, J.-P.; Ravanat, J.-L.; Douki, T.; Richard, M.-J.; Cadet, J.

    1999-01-01

    We used the comet assay associated with DNA-glycosylases to estimate DNA damage in cells exposed to gamma irradiation or photosensitized either with methylene blue or orange acridine. A calibration performed using irradiation allowed the measurement of the steady-state level and the yield of 8-oxodGuo as well as strand breaks and alkali-labile sites. Nous avons utilisé la méthode des comètes associée à des ADN-glycosylases, pour estimer les dommages de l'ADN dans des cellules après l'exposition à un rayonnement gamma ou après photosensibilisation par le bleu de méthylène ou l'acridine orange. Une calibration de la méthode des comètes a permis de mesurer le niveau basal et les taux de formation de 8-oxodGuo ainsi que le nombre de cassures de brins et de sites alcali labiles.

  4. The Role of Mitochondrial DNA in Mediating Alveolar Epithelial Cell Apoptosis and Pulmonary Fibrosis

    PubMed Central

    Kim, Seok-Jo; Cheresh, Paul; Jablonski, Renea P.; Williams, David B.; Kamp, David W.

    2015-01-01

    Convincing evidence has emerged demonstrating that impairment of mitochondrial function is critically important in regulating alveolar epithelial cell (AEC) programmed cell death (apoptosis) that may contribute to aging-related lung diseases, such as idiopathic pulmonary fibrosis (IPF) and asbestosis (pulmonary fibrosis following asbestos exposure). The mammalian mitochondrial DNA (mtDNA) encodes for 13 proteins, including several essential for oxidative phosphorylation. We review the evidence implicating that oxidative stress-induced mtDNA damage promotes AEC apoptosis and pulmonary fibrosis. We focus on the emerging role for AEC mtDNA damage repair by 8-oxoguanine DNA glycosylase (OGG1) and mitochondrial aconitase (ACO-2) in maintaining mtDNA integrity which is important in preventing AEC apoptosis and asbestos-induced pulmonary fibrosis in a murine model. We then review recent studies linking the sirtuin (SIRT) family members, especially SIRT3, to mitochondrial integrity and mtDNA damage repair and aging. We present a conceptual model of how SIRTs modulate reactive oxygen species (ROS)-driven mitochondrial metabolism that may be important for their tumor suppressor function. The emerging insights into the pathobiology underlying AEC mtDNA damage and apoptosis is suggesting novel therapeutic targets that may prove useful for the management of age-related diseases, including pulmonary fibrosis and lung cancer. PMID:26370974

  5. Ochratoxin A induces oxidative DNA damage in liver and kidney after oral dosing to rats.

    PubMed

    Kamp, Hennicke G; Eisenbrand, Gerhard; Janzowski, Christine; Kiossev, Jetchko; Latendresse, John R; Schlatter, Josef; Turesky, Robert J

    2005-12-01

    The nephrotoxic/carcinogenic mycotoxin ochratoxin A (OTA) occurs as a contaminant in food and feed and may be linked to human endemic Balkan nephropathy. The mechanism of OTA-derived carcinogenicity is still under debate, since reactive metabolites of OTA and DNA adducts have not been unambiguously identified. Oxidative DNA damage, however, has been observed in vitro after incubation of mammalian cells with OTA. In this study, we investigated whether OTA induces oxidative DNA damage in vivo as well. Male F344 rats were dosed with 0, 0.03, 0.1, 0.3 mg/kg bw per day OTA for 4 wk (gavage, 7 days/wk, five animals per dose group). Subsequently, oxidative DNA damage was determined in liver and kidney by the comet assay (single cell gel electrophoresis) with/without use of the repair enzyme formamido-pyrimidine-DNA-glycosylase (FPG). The administration of OTA had no effect on basic DNA damage (determined without FPG); however, OTA-mediated oxidative damage was detected with FPG treatment in kidney and liver DNA of all dose groups. Since the doses were in a range that had caused kidney tumors in a 2-year carcinogenicity study with rats, the oxidative DNA damage induced by OTA may help to explain its mechanism of carcinogenicity. For the selective induction of tumors in the kidney, increased oxidative stress in connection with severe cytotoxicity and increased cell proliferation might represent driving factors. PMID:16302199

  6. Sequence-specific DNA damage induced by ultraviolet A-irradiated folic acid via its photolysis product.

    PubMed

    Hirakawa, Kazutaka; Suzuki, Hiroyuki; Oikawa, Shinji; Kawanishi, Shosuke

    2003-02-15

    DNA damage mediated by photosensitizers participates in solar carcinogenesis. Fluorescence measurement and high-performance liquid chromatography analysis demonstrated that photoirradiated folic acid, one of the photosensitizers in cells, generates pterine-6-carboxylic acid (PCA). Experiments using 32P-labeled DNA fragments obtained from a human gene showed that ultraviolet A-irradiated folic acid or PCA caused DNA cleavage specifically at consecutive G residues in double-stranded DNA after Escherichia coli formamidopyrimidine-DNA glycosylase or piperidine treatment. The amount of 8-oxo-7,8-dihydro-2(')-deoxyguanosine formed through this DNA photoreaction in double-stranded DNA exceeded that in single-stranded DNA. Kinetic studies suggested that DNA damage is caused mainly by photoexcited PCA generated from folic acid rather than by folic acid itself. In conclusion, photoirradiated folic acid generates PCA, which induces DNA photooxidation specifically at consecutive G residues through electron transfer. Excess intake of folic acid supplements may increase a risk of skin cancer by solar ultraviolet light. PMID:12573286

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

    PubMed Central

    Cadet, J; Douki, T; Ravanat, J L

    1997-01-01

    In this paper we review recent aspects of the measurement of oxidized DNA bases, currently a matter of debate. There has long been an interest in the determination of the level of oxidized bases in cellular DNA under both normal and oxidative stress conditions. In this respect, the situation is confusing because variations that may be as large as two orders of magnitude have been reported for the yield of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) in similar DNA samples. However, recent findings clearly show that application of several assays like gas chromatography-mass spectrometry (GC-MS) and -32P--postlabeling may lead to a significant overestimation of the level of oxidized bases in cellular DNA. In particular, the silylation step, which is required to make the samples volatile for the GC-MS analysis, has been shown to induce oxidation of normal bases at the level of about one oxidized base per 10(4) normal bases. This has been found to be a general process that applies in particular to 8-oxoGua, 8-oxo-7, 8-dihydroadenine,5-hydroxycytosine, 5-(hydroxymethyl)uracil, and 5-formyluracil. Interestingly, prepurification of the oxidized bases from DNA hydrolysate prior to the derivatization reaction prevents artefactual oxidation. Under these conditions, the level of oxidized bases measured by GC-MS is similar to that obtained by HPLC associated with electrochemical detection (HPLC-EC). It should be added that the level of 8-oxo-7,8-dihydro-2;-deoxyguanosine in control cellular DNA has been found to be about fivefold lower than in earlier HPLC-EC measurements by using appropriate conditions of extraction and enzymatic digestion of DNA. Similar conclusions were reached by measuring formamidopyrimidine-DNA glycosylase sensitive sites as revealed by the single cell gel electrophoresis (comet) assay. Images Figure 1. PMID:9349826

  8. DNA damage induced by the direct effect of radiation

    NASA Astrophysics Data System (ADS)

    Yokoya, A.; Shikazono, N.; Fujii, K.; Urushibara, A.; Akamatsu, K.; Watanabe, R.

    2008-10-01

    We have studied the nature of DNA damage induced by the direct effect of radiation. The yields of single- (SSB) and double-strand breaks (DSB), base lesions and clustered damage were measured using the agarose gel electrophoresis method after exposing to various kinds of radiations to a simple model DNA molecule, fully hydrated closed-circular plasmid DNA (pUC18). The yield of SSB does not show significant dependence on linear energy transfer (LET) values. On the other hand, the yields of base lesions revealed by enzymatic probes, endonuclease III (Nth) and formamidopyrimidine DNA glycosylase (Fpg), which excise base lesions and leave a nick at the damage site, strongly depend on LET values. Soft X-ray photon (150 kVp) irradiation gives a maximum yield of the base lesions detected by the enzymatic probes as SSB and clustered damage, which is composed of one base lesion and proximate other base lesions or SSBs. The clustered damage is visualized as an enzymatically induced DSB. The yields of the enzymatically additional damages strikingly decrease with increasing levels of LET. These results suggest that in higher LET regions, the repair enzymes used as probes are compromised because of the dense damage clustering. The studies using simple plasmid DNA as a irradiation sample, however, have a technical difficulty to detect multiple SSBs in a plasmid DNA. To detect the additional SSBs induced in opposite strand of the first SSB, we have also developed a novel technique of DNA-denaturation assay. This allows us to detect multiply induced SSBs in both strand of DNA, but not induced DSB.

  9. Mechanism of maltal hydration catalyzed by. beta. -amylase: Role of protein structure in controlling the steric outcome of reactions catalyzed by a glycosylase

    SciTech Connect

    Kitahata, Sumio ); Chiba, S. ); Brewer, C.F.; Hehre, E.J. )

    1991-07-09

    Crystalline (monomeric) soybean and (tetrameric) sweet potato {beta}-amylase were shown to catalyze the cis hydration of maltal ({alpha}-D-glucopyranosyl-2-deoxy-D-arabino-hex-1-enitol) to form {beta}-2-deoxymaltose. As reported earlier with the sweet potato enzyme, maltal hydration in D{sub 2}O by soybean {beta}-amylase was found to exhibit an unusually large solvent deuterium kinetic isotope effect (V{sub H}/V{sub D}=6.5), a reaction rate linearly dependent on the mole fraction of deuterium, and 2-deoxy-(2(a)-{sup 2}H)maltose as product. These results indicate (for each {beta}-amylase) that protonation is the rate-limiting step in a reaction involving a nearly symmetric one-proton transition state and that maltal is specifically protonated from above the double bond. That maltal undergoes cis hydration provides evidence in support of a general-acid-catalyzed, carbonium ion mediated reaction. Of fundamental significance is that {beta}-amylase protonates maltal from a direction opposite that assumed for protonating strach, yet creates products of the same anomeric configuration from both. Such stereochemical dichotomy argues for the overriding role of protein structures is dictating the steric outcome of reactions catalyzed by a glycosylase, by limiting the approach and orientation of water or other acceptors to the reaction center.

  10. Beryllium chloride-induced oxidative DNA damage and alteration in the expression patterns of DNA repair-related genes.

    PubMed

    Attia, Sabry M; Harisa, Gamaleldin I; Hassan, Memy H; Bakheet, Saleh A

    2013-09-01

    Beryllium metal has physical properties that make its use essential for very specific applications, such as medical diagnostics, nuclear/fusion reactors and aerospace applications. Because of the widespread human exposure to beryllium metals and the discrepancy of the genotoxic results in the reported literature, detail assessments of the genetic damage of beryllium are warranted. Mice exposed to beryllium chloride at an oral dose of 23mg/kg for seven consecutive days exhibited a significant increase in the level of DNA-strand breaking and micronuclei formation as detected by a bone marrow standard comet assay and micronucleus test. Whereas slight beryllium chloride-induced oxidative DNA damage was detected following formamidopyrimidine DNA glycosylase digestion, digestion with endonuclease III resulted in considerable increases in oxidative DNA damage after the 11.5 and 23mg/kg/day treatment as detected by enzyme-modified comet assays. Increased 8-hydroxydeoxyguanosine was also directly correlated with increased bone marrow micronuclei formation and DNA strand breaks, which further confirm the involvement of oxidative stress in the induction of bone marrow genetic damage after exposure to beryllium chloride. Gene expression analysis on the bone marrow cells from beryllium chloride-exposed mice showed significant alterations in genes associated with DNA damage repair. Therefore, beryllium chloride may cause genetic damage to bone marrow cells due to the oxidative stress and the induced unrepaired DNA damage is probably due to the down-regulation in the expression of DNA repair genes, which may lead to genotoxicity and eventually cause carcinogenicity. PMID:23793613

  11. High-resolution Digital Mapping of N-Methylpurines in Human Cells Reveals Modulation of Their Induction and Repair by Nearest-neighbor Nucleotides.

    PubMed

    Li, Mingyang; Ko, Tengyu; Li, Shisheng

    2015-09-18

    N-Methylpurines (NMPs), including N(7)-methylguanine (7MeG) and N(3)-methyladenine (3MeA), can be induced by environmental methylating agents, chemotherapeutics, and natural cellular methyl donors. In human cells, NMPs are repaired by the multi-step base excision repair pathway initiated by human alkyladenine glycosylase. Repair of NMPs has been shown to be affected by DNA sequence contexts. However, the nature of the sequence contexts has been poorly understood. We developed a sensitive method, LAF-Seq (Lesion-Adjoining Fragment Sequencing), which allows nucleotide-resolution digital mapping of DNA damage and repair in multiple genomic fragments of interest in human cells. We also developed a strategy that allows accurate measurement of the excision kinetics of NMP bases in vitro. We demonstrate that 3MeAs are induced to a much lower level by the SN2 methylating agent dimethyl sulfate and repaired much faster than 7MeGs in human fibroblasts. Induction of 7MeGs by dimethyl sulfate is affected by nearest-neighbor nucleotides, being enhanced at sites neighbored by a G or T on the 3' side, but impaired at sites neighbored by a G on the 5' side. Repair of 7MeGs is also affected by nearest-neighbor nucleotides, being slow if the lesions are between purines, especially Gs, and fast if the lesions are between pyrimidines, especially Ts. Excision of 7MeG bases from the DNA backbone by human alkyladenine glycosylase in vitro is similarly affected by nearest-neighbor nucleotides, suggesting that the effect of nearest-neighbor nucleotides on repair of 7MeGs in the cells is primarily achieved by modulating the initial step of the base excision repair process. PMID:26240148

  12. Cleaving DNA with DNA

    NASA Astrophysics Data System (ADS)

    Carmi, Nir; Balkhi, Shameelah R.; Breaker, Ronald R.

    1998-03-01

    A DNA structure is described that can cleave single-stranded DNA oligonucleotides in the presence of ionic copper. This ``deoxyribozyme'' can self-cleave or can operate as a bimolecular complex that simultaneously makes use of duplex and triplex interactions to bind and cleave separate DNA substrates. Bimolecular deoxyribozyme-mediated strand scission proceeds with a kobs of 0.2 min-1, whereas the corresponding uncatalyzed reaction could not be detected. The duplex and triplex recognition domains can be altered, making possible the targeted cleavage of single-stranded DNAs with different nucleotide sequences. Several small synthetic DNAs were made to function as simple ``restriction enzymes'' for the site-specific cleavage of single-stranded DNA.

  13. Overproduction of stomatal lineage cells in Arabidopsis mutants defective in active DNA demethylation

    PubMed Central

    Yamamuro, Chizuko; Miki, Daisuke; Zheng, Zhimin; Ma, Jun; Wang, Jing; Yang, Zhenbiao; Dong, Juan; Zhu, Jian-Kang

    2014-01-01

    DNA methylation is a reversible epigenetic mark regulating genome stability and function in many eukaryotes. In Arabidopsis, active DNA demethylation depends on the function of the ROS1 subfamily of genes that encode 5-methylcytosine DNA glycosylases/lyases. ROS1-mediated DNA demethylation plays a critical role in the regulation of transgenes, transposable elements and some endogenous genes, but there have been no reports of clear developmental phenotypes in ros1 mutant plants. Here we report that, in the ros1 mutant, the promoter region of the peptide ligand gene EPF2 is hypermethylated, which greatly reduces EPF2 expression and thereby leads to a phenotype of overproduction of stomatal lineage cells. EPF2 gene expression in ros1 is restored and the defective epidermal cell patterning is suppressed by mutations in genes in the RNA-directed DNA methylation pathway. Our results show that active DNA demethylation combats the activity of RNA-directed DNA methylation to influence the initiation of stomatal lineage cells. PMID:24898766

  14. Dichromatic laser radiation effects on DNA of Escherichia coli and plasmids

    NASA Astrophysics Data System (ADS)

    Martins, W. A.; Polignano, G. A. C.; Guimarães, O. R.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2015-04-01

    Dichromatic and consecutive laser radiations have attracted increased attention for clinical applications as offering new tools for the treatment of dysfunctional tissues in situations where monochromatic radiation is not effective. This work evaluated the survival, filamentation and morphology of Escherichia coli cells, and the induction of DNA lesions, in plasmid DNA exposed to low-intensity consecutive dichromatic laser radiation. Exponential and stationary wild type and formamidopyrimidine DNA glycosylase/MutM protein deficient E. coli cultures were exposed to consecutive low-intensity dichromatic laser radiation (infrared laser immediately after red laser) to study the survival, filamentation and morphology of bacterial cells. Plasmid DNA samples were exposed to dichromatic radiation to study DNA lesions by electrophoretic profile. Dichromatic laser radiation affects the survival, filamentation and morphology of E. coli cultures depending on the growth phase and the functional repair mechanism of oxidizing lesions in DNA, but does not induce single/double strands breaks or alkali-labile DNA lesions. Results show that low-intensity consecutive dichromatic laser radiation induces biological effects that differ from those induced by monochromatic laser radiation, suggesting that other therapeutic effects could be obtained using dichromatic radiation.

  15. DNA damage in human skin keratinocytes caused by multiwalled carbon nanotubes with carboxylate functionalization.

    PubMed

    McShan, Danielle; Yu, Hongtao

    2014-07-01

    Water-soluble carbon nanotubes have been found to be one of the most promising nanomaterials in biological- and biomedical-based applications. However, there have been major concerns on their ability to cause cellular and DNA damages upon exposure. In this work, we explore the toxic effects of three multiwalled carbon nanotubes (MWCNTs: nonpurified, purified and carboxylate-functionalized) on human skin keratinocytes (HaCaT). Cytotoxicity tests using the conventional thiazolyl blue tetrazolium bromide (MTT) and the water-soluble tetrazolium (WST-1) assays for 0.5 or 24 h exposure to 20 μg/mL of MWCNTs show that all three caused minimum cytotoxicity that is generally not statistically significant. Assessment of direct and oxidative DNA damages using both alkaline Comet assay and formamidopyrimidine DNA glycosylase-modified Comet assay reveals that the treatment with 20 μg/mL of MWCNTs does not cause significant direct DNA damages, but causes great amount of oxidative DNA damages in HaCaT cells. The oxidative DNA damage reaches the maximum amount at 4 h of incubation in Dulbecco's minimum essential medium, but decreases to the minimum at 8 and 24 h of incubation, indicating repair of the oxidative damages by the intrinsic DNA repair mechanism of the cells. PMID:23012341

  16. DNA Damage Responses Are Induced by tRNA Anticodon Nucleases and Hygromycin B.

    PubMed

    Wemhoff, Sabrina; Klassen, Roland; Beetz, Anja; Meinhardt, Friedhelm

    2016-01-01

    Previous studies revealed DNA damage to occur during the toxic action of PaT, a fungal anticodon ribonuclease (ACNase) targeting the translation machinery via tRNA cleavage. Here, we demonstrate that other translational stressors induce DNA damage-like responses in yeast as well: not only zymocin, another ACNase from the dairy yeast Kluyveromyces lactis, but also translational antibiotics, most pronouncedly hygromycin B (HygB). Specifically, DNA repair mechanisms BER (base excision repair), HR (homologous recombination) and PRR (post replication repair) provided protection, whereas NHEJ (non-homologous end-joining) aggravated toxicity of all translational inhibitors. Analysis of specific BER mutants disclosed a strong HygB, zymocin and PaT protective effect of the endonucleases acting on apurinic sites. In cells defective in AP endonucleases, inactivation of the DNA glycosylase Ung1 increased tolerance to ACNases and HygB. In addition, Mag1 specifically contributes to the repair of DNA lesions caused by HygB. Consistent with DNA damage provoked by translation inhibitors, mutation frequencies were elevated upon exposure to both fungal ACNases and HygB. Since polymerase ζ contributed to toxicity in all instances, error-prone lesion-bypass probably accounts for the mutagenic effects. The finding that differently acting inhibitors of protein biosynthesis induce alike cellular responses in DNA repair mutants is novel and suggests the dependency of genome stability on translational fidelity. PMID:27472060

  17. Mitochondrial Targeted Endonuclease III DNA Repair Enzyme Protects against Ventilator Induced Lung Injury in Mice.

    PubMed

    Hashizume, Masahiro; Mouner, Marc; Chouteau, Joshua M; Gorodnya, Olena M; Ruchko, Mykhaylo V; Wilson, Glenn L; Gillespie, Mark N; Parker, James C

    2014-01-01

    The mitochondrial targeted DNA repair enzyme, 8-oxoguanine DNA glycosylase 1, was previously reported to protect against mitochondrial DNA (mtDNA) damage and ventilator induced lung injury (VILI). In the present study we determined whether mitochondrial targeted endonuclease III (EndoIII) which cleaves oxidized pyrimidines rather than purines from damaged DNA would also protect the lung. Minimal injury from 1 h ventilation at 40 cmH2O peak inflation pressure (PIP) was reversed by EndoIII pretreatment. Moderate lung injury due to ventilation for 2 h at 40 cmH2O PIP produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio, and marked increases in MIP-2 and IL-6. Oxidative mtDNA damage and decreases in the total tissue glutathione (GSH) and the GSH/GSSH ratio also occurred. All of these indices of injury were attenuated by mitochondrial targeted EndoIII. Massive lung injury caused by 2 h ventilation at 50 cmH2O PIP was not attenuated by EndoIII pretreatment, but all untreated mice died prior to completing the two hour ventilation protocol, whereas all EndoIII-treated mice lived for the duration of ventilation. Thus, mitochondrial targeted DNA repair enzymes were protective against mild and moderate lung damage and they enhanced survival in the most severely injured group. PMID:25153040

  18. DNA Damage Responses Are Induced by tRNA Anticodon Nucleases and Hygromycin B

    PubMed Central

    Beetz, Anja; Meinhardt, Friedhelm

    2016-01-01

    Previous studies revealed DNA damage to occur during the toxic action of PaT, a fungal anticodon ribonuclease (ACNase) targeting the translation machinery via tRNA cleavage. Here, we demonstrate that other translational stressors induce DNA damage-like responses in yeast as well: not only zymocin, another ACNase from the dairy yeast Kluyveromyces lactis, but also translational antibiotics, most pronouncedly hygromycin B (HygB). Specifically, DNA repair mechanisms BER (base excision repair), HR (homologous recombination) and PRR (post replication repair) provided protection, whereas NHEJ (non-homologous end-joining) aggravated toxicity of all translational inhibitors. Analysis of specific BER mutants disclosed a strong HygB, zymocin and PaT protective effect of the endonucleases acting on apurinic sites. In cells defective in AP endonucleases, inactivation of the DNA glycosylase Ung1 increased tolerance to ACNases and HygB. In addition, Mag1 specifically contributes to the repair of DNA lesions caused by HygB. Consistent with DNA damage provoked by translation inhibitors, mutation frequencies were elevated upon exposure to both fungal ACNases and HygB. Since polymerase ζ contributed to toxicity in all instances, error-prone lesion-bypass probably accounts for the mutagenic effects. The finding that differently acting inhibitors of protein biosynthesis induce alike cellular responses in DNA repair mutants is novel and suggests the dependency of genome stability on translational fidelity. PMID:27472060

  19. DNA polymerase X from Deinococcus radiodurans implicated in bacterial tolerance to DNA damage is characterized as a short patch base excision repair polymerase.

    PubMed

    Khairnar, Nivedita P; Misra, Hari S

    2009-09-01

    The Deinococcus radiodurans R1 genome encodes an X-family DNA repair polymerase homologous to eukaryotic DNA polymerase beta. The recombinant deinococcal polymerase X (PolX) purified from transgenic Escherichia coli showed deoxynucleotidyltransferase activity. Unlike the Klenow fragment of E. coli, this enzyme showed short patch DNA synthesis activity on heteropolymeric DNA substrate. The recombinant enzyme showed 5'-deoxyribose phosphate (5'-dRP) lyase activity and base excision repair function in vitro, with the help of externally supplied glycosylase and AP endonuclease functions. A polX disruption mutant of D. radiodurans expressing 5'-dRP lyase and a truncated polymerase domain was comparatively less sensitive to gamma-radiation than a polX deletion mutant. Both mutants showed higher sensitivity to hydrogen peroxide. Excision repair mutants of E. coli expressing this polymerase showed functional complementation of UV sensitivity. These results suggest the involvement of deinococcal polymerase X in DNA-damage tolerance of D. radiodurans, possibly by contributing to DNA double-strand break repair and base excision repair. PMID:19542005

  20. Manufactured silver nanoparticles of different sizes induced DNA strand breaks and oxidative DNA damage in hepatoma and leukaemia cells and in dermal and pulmonary fibroblasts.

    PubMed

    Ávalos, A; Haza, A I; Morales, P

    2015-01-01

    Many classes of silver nanoparticles (AgNPs) have been synthesized and widely applied, but no conclusive information on their potential cytotoxicity and genotoxicity mechanisms is available. Therefore, the purpose of this study was to compare the potential genotoxic effects (DNA strand breaks and oxidative DNA damage) of 4.7 nm coated and 42 nm uncoated AgNPs, using the comet assay, in four relevant human cell lines (hepatoma, leukaemia, and dermal and pulmonary fibroblasts) in order to understand the impact of such nanomaterials on cellular DNA. The results indicated that in all cell lines tested, 4.7 nm coated (0.1-1.6 μg ml⁻¹) and 42 nm uncoated (0.1-6.7 μg ml⁻¹) AgNPs increased DNA strand breaks in a dose- and size-dependent manner following 24 h treatment, the smaller AgNPs being more genotoxic. Human pulmonary fibroblasts showed the highest sensitivity to the AgNPs. A modified comet assay using endonuclease III and formamidopyrimidine- DNA glycosylase restriction enzymes showed that in tumoral and normal human dermal fibroblasts, pyrimidines and purines were oxidatively damaged by both AgNPs, but the damage was not size-dependent. However, in human pulmonary fibroblasts, no oxidative damage was observed after treatment with 42 nm AgNPs. In conclusion, both AgNP sizes induced DNA damage in human cells, and this damage could be related to oxidative stress. PMID:25958309

  1. Dynamics of uracil and 5-fluorouracil in DNA.

    PubMed

    Parker, Jared B; Stivers, James T

    2011-02-01

    The prodrug 5-fluorouracil (5-FU), after activation into 5-F-dUMP, is an extensively used anticancer agent that inhibits thymidylate synthase and leads to increases in dUTP and 5-F-dUTP levels in cells. One mechanism for 5-FU action involves DNA polymerase mediated incorporation of dUTP and 5-F-dUTP into genomic DNA leading to U/A, 5-FU/A, or 5-FU/G base pairs. These uracil-containing lesions are recognized and excised by several human uracil excision repair glycosylases (hUNG2, hSMUG2, and hTDG) leading to toxic abasic sites in DNA that may precipitate cell death. Each of these enzymes uses an extrahelical base recognition mechanism, and previous studies with UNG have shown that extrahelical recognition is facilitated by destabilized base pairs possessing kinetically enhanced base pair opening rates. Thus, the dynamic properties of base pairs containing 5-FU and U are an important unknown in understanding the role of these enzymes in damage recognition and prodrug activation. The pH dependence of the (19)F NMR chemical shift of 5-FU imbedded in a model trinucleotide was used to obtain a pK(a) = 8.1 for its imino proton (10 °C). This is about 1.5 units lower than the imino protons of uracil or thymine and indicates that at neutral pH 5-FU exists significantly as an ionized tautomer that can mispair with guanine during DNA replication. NMR imino proton exchange measurements show that U/A and 5-FU/A base pairs open with rate constants (k(op)) that are 6- and 13-fold faster than a T/A base pair in the same sequence context. In contrast, these same base pairs have apparent opening equilibrium constants (αK(op)) that differ by less than a factor of 2, indicating that the closing rates (k(cl)) are enhanced by nearly equal amounts as k(op). These dynamic measurements are consistent with the previously proposed kinetic trapping model for extrahelical recognition by UNG. In this model, the enhanced intrinsic opening rates of destabilized base pairs allow the bound

  2. Oxidative DNA damage and its repair in rat spleen following subchronic exposure to aniline

    SciTech Connect

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

    2008-12-01

    The mechanisms by which aniline exposure elicits splenotoxic response, especially the tumorigenic response, are not well-understood. Splenotoxicity of aniline is associated with iron overload and generation of reactive oxygen species (ROS) which can cause oxidative damage to DNA, proteins and lipids (oxidative stress). 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is one of the most abundant oxidative DNA lesions resulting from ROS, and 8-oxoguanine glycosylase 1 (OGG1), a specific DNA glycosylase/lyase enzyme, plays a key role in the removal of 8-OHdG adducts. This study focused on examining DNA damage (8-OHdG) and repair (OGG1) in the spleen in an experimental condition preceding a tumorigenic response. To achieve that, male Sprague-Dawley rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. Aniline treatment led to a significant increase in splenic oxidative DNA damage, manifested as a 2.8-fold increase in 8-OHdG levels. DNA repair activity, measured as OGG1 base excision repair (BER) activity, increased by {approx} 1.3 fold in the nuclear protein extracts (NE) and {approx} 1.2 fold in the mitochondrial protein extracts (ME) of spleens from aniline-treated rats as compared to the controls. Real-time PCR analysis for OGG1 mRNA expression in the spleen revealed a 2-fold increase in expression in aniline-treated rats than the controls. Likewise, OGG1 protein expression in the NEs of spleens from aniline-treated rats was {approx} 1.5 fold higher, whereas in the MEs it was {approx} 1.3 fold higher than the controls. Aniline treatment also led to stronger immunostaining for both 8-OHdG and OGG1 in the spleens, confined to the red pulp areas. It is thus evident from our studies that aniline-induced oxidative stress is associated with increased oxidative DNA damage. The BER pathway was also activated, but not enough to prevent the accumulation of oxidative DNA damage (8-OHdG). Accumulation of

  3. The cytosolic Fe-S cluster assembly component MET18 is required for the full enzymatic activity of ROS1 in active DNA demethylation

    PubMed Central

    Wang, Xiaokang; Li, Qi; Yuan, Wei; Cao, Zhendong; Qi, Bei; Kumar, Suresh; Li, Yan; Qian, Weiqiang

    2016-01-01

    DNA methylation patterns in plants are dynamically regulated by DNA methylation and active DNA demethylation in response to both environmental changes and development of plant. Beginning with the removal of methylated cytosine by ROS1/DME family of 5-methylcytosine DNA glycosylases, active DNA demethylation in plants occurs through base excision repair. So far, many components involved in active DNA demethylation remain undiscovered. Through a forward genetic screening of Arabidopsis mutants showing DNA hypermethylation at the EPF2 promoter region, we identified the conserved iron-sulfur cluster assembly protein MET18. MET18 dysfunction caused DNA hypermethylation at more than 1000 loci as well as the silencing of reporter genes and some endogenous genes. MET18 can directly interact with ROS1 in vitro and in vivo. ROS1 activity was reduced in the met18 mutant plants and point mutation in the conserved Fe-S cluster binding motif of ROS1 disrupted its biological function. Interestingly, a large number of DNA hypomethylated loci, especially in the CHH context, were identified from the met18 mutants and most of the hypo-DMRs were from TE regions. Our results suggest that MET18 can regulate both active DNA demethylation and DNA methylation pathways in Arabidopsis. PMID:27193999

  4. The cytosolic Fe-S cluster assembly component MET18 is required for the full enzymatic activity of ROS1 in active DNA demethylation.

    PubMed

    Wang, Xiaokang; Li, Qi; Yuan, Wei; Cao, Zhendong; Qi, Bei; Kumar, Suresh; Li, Yan; Qian, Weiqiang

    2016-01-01

    DNA methylation patterns in plants are dynamically regulated by DNA methylation and active DNA demethylation in response to both environmental changes and development of plant. Beginning with the removal of methylated cytosine by ROS1/DME family of 5-methylcytosine DNA glycosylases, active DNA demethylation in plants occurs through base excision repair. So far, many components involved in active DNA demethylation remain undiscovered. Through a forward genetic screening of Arabidopsis mutants showing DNA hypermethylation at the EPF2 promoter region, we identified the conserved iron-sulfur cluster assembly protein MET18. MET18 dysfunction caused DNA hypermethylation at more than 1000 loci as well as the silencing of reporter genes and some endogenous genes. MET18 can directly interact with ROS1 in vitro and in vivo. ROS1 activity was reduced in the met18 mutant plants and point mutation in the conserved Fe-S cluster binding motif of ROS1 disrupted its biological function. Interestingly, a large number of DNA hypomethylated loci, especially in the CHH context, were identified from the met18 mutants and most of the hypo-DMRs were from TE regions. Our results suggest that MET18 can regulate both active DNA demethylation and DNA methylation pathways in Arabidopsis. PMID:27193999

  5. DNA repair

    SciTech Connect

    Friedberg, E.C.; Hanawalt, P.C. )

    1988-01-01

    Topics covered in this book included: Eukaryote model systems for DNA repair study; Sensitive detection of DNA lesions and their repair; and Defined DNA sequence probes for analysis of mutagenesis and repair.

  6. Growth Arrest and DNA-Damage-Inducible, Beta (GADD45b)-Mediated DNA Demethylation in Major Psychosis

    PubMed Central

    Gavin, David P; Sharma, Rajiv P; Chase, Kayla A; Matrisciano, Francesco; Dong, Erbo; Guidotti, Alessandro

    2012-01-01

    Aberrant neocortical DNA methylation has been suggested to be a pathophysiological contributor to psychotic disorders. Recently, a growth arrest and DNA-damage-inducible, beta (GADD45b) protein-coordinated DNA demethylation pathway, utilizing cytidine deaminases and thymidine glycosylases, has been identified in the brain. We measured expression of several members of this pathway in parietal cortical samples from the Stanley Foundation Neuropathology Consortium (SFNC) cohort. We find an increase in GADD45b mRNA and protein in patients with psychosis. In immunohistochemistry experiments using samples from the Harvard Brain Tissue Resource Center, we report an increased number of GADD45b-stained cells in prefrontal cortical layers II, III, and V in psychotic patients. Brain-derived neurotrophic factor IX (BDNF IXabcd) was selected as a readout gene to determine the effects of GADD45b expression and promoter binding. We find that there is less GADD45b binding to the BDNF IXabcd promoter in psychotic subjects. Further, there is reduced BDNF IXabcd mRNA expression, and an increase in 5-methylcytosine and 5-hydroxymethylcytosine at its promoter. On the basis of these results, we conclude that GADD45b may be increased in psychosis compensatory to its inability to access gene promoter regions. PMID:22048458

  7. Molecular Dynamics of 8-oxoguanine Lesioned B-DNA Molecule — Structure and Energy Analysis

    NASA Astrophysics Data System (ADS)

    Pinak, M.; O'Neill, P.; Fujimoto, H.; Nemoto, T.

    2004-04-01

    The molecular dynamics (MD) simulation of DNA mutagenic oxidative lesion — 7,8-dihydro-8-oxoguanine (8-oxoG), complexed with the repair enzyme — human oxoguanine glycosylase 1 (hOGG1) was performed for 1 nanosecond (ns) in order to describe the dynamical process of DNA-enzyme complex formation. After 900 picoseconds of MD the lesioned DNA and enzyme formed a complex that lasted until the end of the simulation at 1 ns. The amino group of arginine 324 was located close to the phosphodiester bond of nucleotide with 8-oxoG enabling chemical reactions between amino acid and lesion. Phosphodiester bond at C5' of 8-oxoG was displaced to the position close to the amino group of arginine 324. In the background simulation of the identical molecular system with the native DNA, neither the complex nor the water mediated hydrogen bond network were observed. The electrostatic energy is supposed to be significant factor causing the disruption of DNA base stacking in DNA duplex and may also to serve as a signal toward the repair enzyme informing on the presence of the lesion.

  8. An enzymatic activity isolated from Brassica oleracea specific for UV-irradiated DNA

    SciTech Connect

    Gallagher, P.E.; Lenhart, J.R.; Weiss, R.B. )

    1991-03-11

    As a consequence of a breakdown in the ozone layer, an increase in the amount of DNA damage caused by ultraviolet irradiation can be expected. Organisms have evolved mechanisms to repair numerous types of DNA damages. While these DNA repair systems have been well characterized in bacteria and to a lesser extent in mammalian cells, surprisingly little is known about repair of potentially harmful DNA lesions in plants. An enzyme that recognizes and incises UV irradiated DNA has been partially purified from the leaf tissue of Brassica oleracea. Glycosylase-produced base loss sites were detected by a nitrocellulose filter-binding assay using UV-irradiated PM2 viral DNA as the substrate. The optimal temperature for maximal enzyme activity is 47C with a pH optimum between 7.0 and 7.5. In addition, the endonuclease is active in both Tris and phosphate buffers, although it is stimulated by phosphate concentrations up to 25 mM. Currently, a number of synthetic polynucleotides as well as DNAs of defined sequence are being employed as substrates to determine the nature of the UV-induced lesion and the precise mechanism of action of the enzyme.

  9. A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil

    PubMed Central

    Greagg, Martin A.; Fogg, Mark J.; Panayotou, George; Evans, Steven J.; Connolly, Bernard A.; Pearl, Laurence H.

    1999-01-01

    Deamination of cytosine to uracil is the most common promutagenic change in DNA, and it is greatly increased at the elevated growth temperatures of hyperthermophilic archaea. If not repaired to cytosine prior to replication, uracil in a template strand directs incorporation of adenine, generating a G⋅C → A⋅U transition mutation in half the progeny. Surprisingly, genomic analysis of archaea has so far failed to reveal any homologues of either of the known families of uracil-DNA glycosylases responsible for initiating the base-excision repair of uracil in DNA, which is otherwise universal. Here we show that DNA polymerases from several hyperthermophilic archaea (including Vent and Pfu) specifically recognize the presence of uracil in a template strand and stall DNA synthesis before mutagenic misincorporation of adenine. A specific template-checking function in a DNA polymerase has not been observed previously, and it may represent the first step in a pathway for the repair of cytosine deamination in archaea. PMID:10430892

  10. DNA repair defects sensitize cells to anticodon nuclease yeast killer toxins.

    PubMed

    Klassen, Roland; Wemhoff, Sabrina; Krause, Jens; Meinhardt, Friedhelm

    2011-03-01

    Killer toxins from Kluyveromyces lactis (zymocin) and Pichia acaciae (PaT) were found to disable translation in target cells by virtue of anticodon nuclease (ACNase) activities on tRNA(Glu) and tRNA(Gln), respectively. Surprisingly, however, ACNase exposure does not only impair translation, but also affects genome integrity and concomitantly DNA damage occurs. Previously, it was shown that homologous recombination protects cells from ACNase toxicity. Here, we have analyzed whether other DNA repair pathways are functional in conferring ACNase resistance as well. In addition to HR, base excision repair (BER) and postreplication repair (PRR) promote clear resistance to either, PaT and zymocin. Comparative toxin sensitivity analysis of BER mutants revealed that its ACNase protective function is due to the endonucleases acting on apurinic (AP) sites, whereas none of the known DNA glycosylases is involved. Because PaT and zymocin require the presence of the ELP3/TRM9-dependent wobble uridine modification 5-methoxy-carbonyl-methyl (mcm(5)) for tRNA cleavage, we analyzed toxin response in DNA repair mutants additionally lacking such tRNA modifications. ACNase resistance caused by elp3 or trm9 mutations was found to rescue hypersensitivity of DNA repair defects, consistent with DNA damage to occur as a consequence of tRNA cleavage. The obtained genetic evidence promises to reveal new aspects into the mechanism linking translational fidelity and genome surveillance. PMID:21188417

  11. A comet assay study reveals that aluminium induces DNA damage and inhibits the repair of radiation-induced lesions in human peripheral blood lymphocytes.

    PubMed

    Lankoff, Anna; Banasik, Anna; Duma, Anna; Ochniak, Edyta; Lisowska, Halina; Kuszewski, Tomasz; Góźdź, Stanisław; Wojcik, Andrzej

    2006-02-01

    Although it is known that many metals induce DNA damage and inhibit DNA repair, information regarding aluminium (Al) is scarce. The aim of this study was to analyze the level of DNA damage in human peripheral blood lymphocytes treated with Al and the impact of Al on the repair of DNA damage induced by ionizing radiation. Cells were treated with different doses of aluminium chloride (1, 2, 5, 10 and 25 microg/ml AlCl(3)) for 72 h. The level of DNA damage and of apoptosis was determined by the comet assay. The level of oxidative damage was determined by the application of endonuclease III and formamidopyrimidine DNA glycosylase. The results on apoptosis were confirmed by flow cytometry. Based on the fluorescence intensity, cells were divided into cohorts of different relative DNA content that corresponds to G(1), S and G(2) phases of the cell cycle. Our results revealed that Al induces DNA damage in a dose-dependent manner, however, at the dose of 25 microg/ml the level of damage declined. This decline was accompanied by a high level of apoptosis indicating selective elimination of damaged cells. Cells pre-treated with Al showed a decreased repair capacity indicating that Al inhibits DNA repair. The possible mechanisms by which Al induces DNA damage and inhibits the repair are discussed. PMID:16139969

  12. Base excision repair of ionizing radiation-induced DNA damage in G1 and G2 cell cycle phases

    PubMed Central

    Chaudhry, M Ahmad

    2007-01-01

    Background Major genomic surveillance mechanisms regulated in response to DNA damage exist at the G1/S and G2/M checkpoints. It is presumed that these delays provide time for the repair of damaged DNA. Cells have developed multiple DNA repair pathways to protect themselves from different types of DNA damage. Oxidative DNA damage is processed by the base excision repair (BER) pathway. Little is known about the BER of ionizing radiation-induced DNA damage and putative heterogeneity of BER in the cell cycle context. We measured the activities of three BER enzymes throughout the cell cycle to investigate the cell cycle-specific repair of ionizing radiation-induced DNA damage. We further examined BER activities in G2 arrested human cells after exposure to ionizing radiation. Results Using an in vitro incision assay involving radiolabeled oligonucleotides with specific DNA lesions, we examined the activities of several BER enzymes in the whole cell extracts prepared from synchronized human HeLa cells irradiated in G1 and G2 phase of the cell cycle. The activities of human endonuclease III (hNTH1), a glycosylase/lyase that removes several damaged bases from DNA including dihydrouracil (DHU), 8-oxoguanine-DNA glycosylase (hOGG1) that recognizes 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) lesion and apurinic/apyrimidinic endonuclease (hAPE1) that acts on abasic sites including synthetic analog furan were examined. Conclusion Overall the repair activities of hNTH1 and hAPE1 were higher in the G1 compared to G2 phase of the cell cycle. The percent cleavages of oligonucleotide substrate with furan were greater than substrate with DHU in both G1 and G2 phases. The irradiation of cells enhanced the cleavage of substrates with furan and DHU only in G1 phase. The activity of hOGG1 was much lower and did not vary within the cell cycle. These results demonstrate the cell cycle phase dependence on the BER of ionizing radiation-induced DNA damage. Interestingly no evidence of

  13. Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA.

    PubMed Central

    Livneh, Z; Elad, D; Sperling, J

    1979-01-01

    Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of lambda greater than 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 8-(2-hydroxy-2-propyl)adenine and 8-(2-hydroxy-2-propyl)guanine as the only detectable damage in the DNA. Using this specifically photoalkylated DNA as a substrate, we discovered in extracts of Micrococcus luteus an endonucleolytic activity that is directed towards 8-(2-hydroxy-2-propyl) purines in DNA. The activity is not a combination of a DNA-glycosylase and an apurinic site endonuclease. It is not inhibited by single-stranded DNA, by UV- or gamma-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA. however, gamma- or UV-(254 nm) irradiated double-stranded DNAs to inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNAs. Divalent cations are not required for the incising activity, and it is fully active in 1 mM EDTA, whereas caffeine and ATP cause inhibition. Extracts of mutant M. luteus lacking pyrimidine-dimer-directed endonucleases were found to contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, we could demonstrate the specific excision of the 8-alkylated purines from the damaged DNA. The special conformational consequences of the 8-alkylation of purines, at the nucleotide level, namely their nonregular syn conformation, suggest that it is the distortion in the DNA that is recognized by the endonuclease. PMID:293658

  14. Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA.

    PubMed

    Livneh, Z; Elad, D; Sperling, J

    1979-11-01

    Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of lambda greater than 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 8-(2-hydroxy-2-propyl)adenine and 8-(2-hydroxy-2-propyl)guanine as the only detectable damage in the DNA. Using this specifically photoalkylated DNA as a substrate, we discovered in extracts of Micrococcus luteus an endonucleolytic activity that is directed towards 8-(2-hydroxy-2-propyl) purines in DNA. The activity is not a combination of a DNA-glycosylase and an apurinic site endonuclease. It is not inhibited by single-stranded DNA, by UV- or gamma-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA. however, gamma- or UV-(254 nm) irradiated double-stranded DNAs to inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNAs. Divalent cations are not required for the incising activity, and it is fully active in 1 mM EDTA, whereas caffeine and ATP cause inhibition. Extracts of mutant M. luteus lacking pyrimidine-dimer-directed endonucleases were found to contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, we could demonstrate the specific excision of the 8-alkylated purines from the damaged DNA. The special conformational consequences of the 8-alkylation of purines, at the nucleotide level, namely their nonregular syn conformation, suggest that it is the distortion in the DNA that is recognized by the endonuclease. PMID:293658

  15. Mechanism of Werner DNA helicase: POT1 and RPA stimulates WRN to unwind beyond gaps in the translocating strand.

    PubMed

    Ahn, Byungchan; Lee, Jae Wan; Jung, Hana; Beck, Gad; Bohr, Vilhelm A

    2009-01-01

    WRN belongs to the RecQ family of DNA helicases and it plays a role in recombination, replication, telomere maintenance and long-patch base excision repair. Here, we demonstrate that WRN efficiently unwinds DNA substrates containing a 1-nucleotide gap in the translocating DNA strand, but when the gap size is increased to 3-nucleotides unwinding activity significantly declines. In contrast, E. coli UvrD (3'-->5' helicase), which recognizes nicks in DNA to initiate unwinding, does not unwind past a 1-nucleotide gap. This unique ability of WRN to bypass gaps supports its involvement in DNA replication and LP-BER where such gaps can be produced by glycosylases and the apurinic/apyrimidinic endonuclease 1 (APE1). Furthermore, we tested telomere repeat binding factor 2 (TRF2), both variants 1 and 2 of protector of telomeres 1 (POT1v1 and POT1v2) and RPA on telomeric DNA substrates containing much bigger gaps than 3-nucleotides in order to determine whether unwinding could be facilitated through WRN-protein interaction. Interestingly, POT1v1 and RPA are capable of stimulating WRN helicase on gapped DNA and 5'-overhang substrates, respectively. PMID:19262689

  16. Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications.

    PubMed

    Róna, Gergely; Scheer, Ildikó; Nagy, Kinga; Pálinkás, Hajnalka L; Tihanyi, Gergely; Borsos, Máté; Békési, Angéla; Vértessy, Beáta G

    2016-02-18

    The role of uracil in genomic DNA has been recently re-evaluated. It is now widely accepted to be a physiologically important DNA element in diverse systems from specific phages to antibody maturation and Drosophila development. Further relevant investigations would largely benefit from a novel reliable and fast method to gain quantitative and qualitative information on uracil levels in DNA both in vitro and in situ, especially since current techniques does not allow in situ cellular detection. Here, starting from a catalytically inactive uracil-DNA glycosylase protein, we have designed several uracil sensor fusion proteins. The designed constructs can be applied as molecular recognition tools that can be detected with conventional antibodies in dot-blot applications and may also serve as in situ uracil-DNA sensors in cellular techniques. Our method is verified on numerous prokaryotic and eukaryotic cellular systems. The method is easy to use and can be applied in a high-throughput manner. It does not require expensive equipment or complex know-how, facilitating its easy implementation in any basic molecular biology laboratory. Elevated genomic uracil levels from cells of diverse genetic backgrounds and/or treated with different drugs can be demonstrated also in situ, within the cell. PMID:26429970

  17. Enzymology of repair of DNA adducts produced by N-nitroso compounds

    SciTech Connect

    Setlow, R.B.; Cao, E.H.; Delihas, N.C.

    1983-01-01

    The biological effects of DNA adducts depend on their nature, and on their half-lives relative to the rates of DNA replication and transcription. Their half-lives are determined by the rates of spontaneous decay, such as depurination, and the rates of enzymatic repair of the adducts or their decay products. The principle modes of repair of methylating and ethylating agents are by glycosylase catalyzed depurination of 7-alkylguanine and 3-alkyladenine and by the dealkalation of O/sup 6/-alkylguanine. Repair by dealkylation cannot be detected by the standard methods used to measure DNA repair, but it is easy to estimate the acceptor activity in cell extracts by measuring the transfer of radioactive O/sup 6/-alkyl groups in an exogenous DNA to protein. In extracts of cells treated with alkylating agents the activity is depressed because the endogenous DNA is rapidly dealkylated, using up the acceptor activity. In many cell types the decrease in activity is followed by an increase to the normal constitutive level. In other cells there is no such adaptive response. Differences in constitutive levels of methyl accepting activity in extracts of human lymphocytes and on the acceptor activity in lung macrophages from smokers (low activity) and non-smokers (high activity) have been observed. 46 references.

  18. Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications

    PubMed Central

    Róna, Gergely; Scheer, Ildikó; Nagy, Kinga; Pálinkás, Hajnalka L.; Tihanyi, Gergely; Borsos, Máté; Békési, Angéla; Vértessy, Beáta G.

    2016-01-01

    The role of uracil in genomic DNA has been recently re-evaluated. It is now widely accepted to be a physiologically important DNA element in diverse systems from specific phages to antibody maturation and Drosophila development. Further relevant investigations would largely benefit from a novel reliable and fast method to gain quantitative and qualitative information on uracil levels in DNA both in vitro and in situ, especially since current techniques does not allow in situ cellular detection. Here, starting from a catalytically inactive uracil-DNA glycosylase protein, we have designed several uracil sensor fusion proteins. The designed constructs can be applied as molecular recognition tools that can be detected with conventional antibodies in dot-blot applications and may also serve as in situ uracil-DNA sensors in cellular techniques. Our method is verified on numerous prokaryotic and eukaryotic cellular systems. The method is easy to use and can be applied in a high-throughput manner. It does not require expensive equipment or complex know-how, facilitating its easy implementation in any basic molecular biology laboratory. Elevated genomic uracil levels from cells of diverse genetic backgrounds and/or treated with different drugs can be demonstrated also in situ, within the cell. PMID:26429970

  19. New approaches to DNA damage and repair: the ultraviolet light example

    SciTech Connect

    Haseltine, W.A.; Gordon, L.K.; Lindan, C.; Lippke, J.; Brash, D.; Lo, K.M.; Royer-Pokora, B.

    1982-01-01

    DNA fragments of defined sequence are used as probes to study DNA damage and repair. The case of ultraviolet light is presented and includes the following: (a) Description of the distribution of cyclobutane pyrimidine dimers within defined DNA sequences. Considerations of the effect of neighboring base composition, dose rate, and double- or single-stranded property of the DNA are discussed. (b) Dissection of the anatomy of the incision event and subsequent repair steps. A three-step incision model for repair of cyclobutane dimers by the Micrococcus luteus repair enzymes will be presented. The steps are (1) recognition of the lesion and N-glycosylase scission, (2) cleavage of the phosphodiester bond 3' to the newly created apyrimidinic site, and (3) scission of the apyrimidinic sugar on the 5' side. (c) Use of human alphoid sequences as indicators of DNA damage in intact human cells. (d) Biological significance of a novel ultraviolet light-induced photoproduct. This photoproduct occurs at pyrimidine-cytosine sequences and may have a significant biological role.

  20. HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins.

    PubMed

    Hrecka, Kasia; Hao, Caili; Shun, Ming-Chieh; Kaur, Sarabpreet; Swanson, Selene K; Florens, Laurence; Washburn, Michael P; Skowronski, Jacek

    2016-07-01

    HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) cytidine deaminases, and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase, which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA, and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr, found in all primate lentiviruses, and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx, hijack the CRL4(DCAF1) E3 ubiquitin ligase to alleviate some of these conditions, but the extent of their interactions with DNA repair proteins has not been thoroughly characterized. Here, we identify HLTF, a postreplication DNA repair helicase, as a common target of HIV-1/SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms CRL4(DCAF1) E3 to direct HLTF for proteasome-dependent degradation independent from previously reported Vpr interactions with base excision repair enzyme uracil DNA glycosylase (UNG2) and crossover junction endonuclease MUS81, which Vpr also directs for degradation via CRL4(DCAF1) E3. Thus, separate functions of HIV-1 Vpr usurp CRL4(DCAF1) E3 to remove key enzymes in three DNA repair pathways. In contrast, we find that HIV-2 Vpr is unable to efficiently program HLTF or UNG2 for degradation. Our findings reveal complex interactions between HIV-1 and the DNA repair machinery, suggesting that DNA repair plays important roles in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these viruses use different strategies to guard their genomes and facilitate their replication in the host. PMID:27335459

  1. An ECVAG† trial on assessment of oxidative damage to DNA measured by the comet assay

    PubMed Central

    Johansson, Clara; Møller, Peter; Forchhammer, Lykke; Loft, Steffen; Godschalk, Roger W. L.; Langie, Sabine A. S.; Lumeij, Stijn; Jones, George D. D.; Kwok, Rachel W. L.; Azqueta, Amaya; Phillips, David H.; Sozeri, Osman; Routledge, Michael N.; Charlton, Alexander J.; Riso, Patrizia; Porrini, Marisa; Allione, Alessandra; Matullo, Giuseppe; Palus, Jadwiga; Stepnik, Maciej; Collins, Andrew R.; Möller, Lennart

    2010-01-01

    The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due to differences in assay protocols (e.g. lysis conditions, enzyme treatment, the duration of the alkaline treatment and electrophoresis) and in the end points used for reporting results (e.g. %DNA in tail, arbitrary units, tail moment and tail length). One way to facilitate comparisons is to convert primary comet assay end points to number of lesions/106 bp by calibration with ionizing radiation. The aim of this study was to investigate the inter-laboratory variation in assessment of oxidatively damaged DNA by the comet assay in terms of oxidized purines converted to strand breaks with formamidopyrimidine DNA glycosylase (FPG). Coded samples with DNA oxidation damage induced by treatment with different concentrations of photosensitizer (Ro 19-8022) plus light and calibration samples irradiated with ionizing radiation were distributed to the 10 participating laboratories to measure DNA damage using their own comet assay protocols. Nine of 10 laboratories reported the same ranking of the level of damage in the coded samples. The variation in assessment of oxidatively damaged DNA was largely due to differences in protocols. After conversion of the data to lesions/106 bp using laboratory-specific calibration curves, the variation between the laboratories was reduced. The contribution of the concentration of photosensitizer to the variation in net FPG-sensitive sites increased from 49 to 73%, whereas the inter-laboratory variation decreased. The participating laboratories were successful in finding a dose–response of oxidatively damaged DNA in coded samples, but there remains a need to standardize the protocols to enable direct comparisons between laboratories. PMID:19948595

  2. Pathophysiology of Bronchoconstriction: Role of Oxidatively Damaged DNA Repair

    PubMed Central

    Bacsi, Attila; Pan, Lang; Ba, Xueqing; Boldogh, Istvan

    2016-01-01

    Purpose of review To provide an overview on the present understanding of roles of oxidative DNA damage repair in cell signaling underlying bronchoconstriction common to, but not restricted to various forms of asthma and chronic obstructive pulmonary disease Recent findings Bronchoconstriction is a tightening of smooth muscle surrounding the bronchi and bronchioles with consequent wheezing and shortness of breath. Key stimuli include air pollutants, viral infections, allergens, thermal and osmotic changes, and shear stress of mucosal epithelium, triggering a wide range of cellular, vascular and neural events. Although activation of nerve fibers, the role of G-proteins, protein kinases and Ca++, and molecular interaction within contracting filaments of muscle are well defined, the overarching mechanisms by which a wide range of stimuli initiate these events are not fully understood. Many, if not all, stimuli increase levels of reactive oxygen species (ROS), which are signaling and oxidatively modifying macromolecules, including DNA. The primary ROS target in DNA is guanine, and 8-oxoguanine is one of the most abundant base lesions. It is repaired by 8-oxoguanine DNA glycosylase1 (OGG1) during base excision repair processes. The product, free 8-oxoG base, is bound by OGG1 with high affinity, and the complex then functions as an activator of small GTPases, triggering pathways for inducing gene expression and contraction of intracellular filaments in mast and smooth muscle cells. Summary Oxidative DNA damage repair-mediated cell activation signaling result in gene expression that “primes” the mucosal epithelium and submucosal tissues to generate mediators of airway smooth muscle contractions. PMID:26694039

  3. The adaptive imbalance in base excision–repair enzymes generates microsatellite instability in chronic inflammation

    PubMed Central

    Hofseth, Lorne J.; Khan, Mohammed A.; Ambrose, Mark; Nikolayeva, Olga; Xu-Welliver, Meng; Kartalou, Maria; Hussain, S. Perwez; Roth, Richard B.; Zhou, Xiaoling; Mechanic, Leah E.; Zurer, Irit; Rotter, Varda; Samson, Leona D.; Harris, Curtis C.

    2003-01-01

    Chronic infection and associated inflammation are key contributors to human carcinogenesis. Ulcerative colitis (UC) is an oxyradical overload disease and is characterized by free radical stress and colon cancer proneness. Here we examined tissues from noncancerous colons of ulcerative colitis patients to determine (a) the activity of two base excision–repair enzymes , AAG, the major 3-methyladenine DNA glycosylase, and APE1, the major apurinic site endonuclease; and (b) the prevalence of microsatellite instability (MSI). AAG and APE1 were significantly increased in UC colon epithelium undergoing elevated inflammation and MSI was positively correlated with their imbalanced enzymatic activities. These latter results were supported by mechanistic studies using yeast and human cell models in which overexpression of AAG and/or APE1 was associated with frameshift mutations and MSI. Our results are consistent with the hypothesis that the adaptive and imbalanced increase in AAG and APE1 is a novel mechanism contributing to MSI in patients with UC and may extend to chronic inflammatory or other diseases with MSI of unknown etiology. PMID:14679184

  4. Mitochondrial DNA.

    ERIC Educational Resources Information Center

    Wright, Russell G.; Bottino, Paul J.

    1986-01-01

    Provides background information for teachers on mitochondrial DNA, pointing out that it may have once been a free-living organism. Includes a ready-to-duplicate exercise titled "Using Microchondrial DNA to Measure Evolutionary Distance." (JN)

  5. DNA Banking

    SciTech Connect

    Reilly, P.R. )

    1992-11-01

    The author is involved in the ethical, legal, and social issues of banking of DNA and data from DNA analysis. In his attempt to determine the extent of DNA banking in the U.S., the author surveyed some commercial companies performing DNA banking services. This article summarizes the results of that survey, with special emphasis on the procedures the companies use to protect the privacy of individuals. 4 refs.

  6. Regulation of mitochondrial genome replication by hypoxia: The role of DNA oxidation in D-loop region.

    PubMed

    Pastukh, Viktor M; Gorodnya, Olena M; Gillespie, Mark N; Ruchko, Mykhaylo V

    2016-07-01

    Mitochondria of mammalian cells contain multiple copies of mitochondrial (mt) DNA. Although mtDNA copy number can fluctuate dramatically depending on physiological and pathophysiologic conditions, the mechanisms regulating mitochondrial genome replication remain obscure. Hypoxia, like many other physiologic stimuli that promote growth, cell proliferation and mitochondrial biogenesis, uses reactive oxygen species as signaling molecules. Emerging evidence suggests that hypoxia-induced transcription of nuclear genes requires controlled DNA damage and repair in specific sequences in the promoter regions. Whether similar mechanisms are operative in mitochondria is unknown. Here we test the hypothesis that controlled oxidative DNA damage and repair in the D-loop region of the mitochondrial genome are required for mitochondrial DNA replication and transcription in hypoxia. We found that hypoxia had little impact on expression of mitochondrial proteins in pulmonary artery endothelial cells, but elevated mtDNA content. The increase in mtDNA copy number was accompanied by oxidative modifications in the D-loop region of the mitochondrial genome. To investigate the role of this sequence-specific oxidation of mitochondrial genome in mtDNA replication, we overexpressed mitochondria-targeted 8-oxoguanine glycosylase Ogg1 in rat pulmonary artery endothelial cells, enhancing the mtDNA repair capacity of transfected cells. Overexpression of Ogg1 resulted in suppression of hypoxia-induced mtDNA oxidation in the D-loop region and attenuation of hypoxia-induced mtDNA replication. Ogg1 overexpression also reduced binding of mitochondrial transcription factor A (TFAM) to both regulatory and coding regions of the mitochondrial genome without altering total abundance of TFAM in either control or hypoxic cells. These observations suggest that oxidative DNA modifications in the D-loop region during hypoxia are important for increased TFAM binding and ensuing replication of the mitochondrial

  7. Partial loss of the DNA repair scaffolding protein, Xrcc1, results in increased brain damage and reduced recovery from ischemic stroke in mice.

    PubMed

    Ghosh, Somnath; Canugovi, Chandrika; Yoon, Jeong Seon; Wilson, David M; Croteau, Deborah L; Mattson, Mark P; Bohr, Vilhelm A

    2015-07-01

    Oxidative DNA damage is mainly repaired by base excision repair (BER). Previously, our laboratory showed that mice lacking the BER glycosylases 8-oxoguanine glycosylase 1 (Ogg1) or nei endonuclease VIII-like 1 (Neil1) recover more poorly from focal ischemic stroke than wild-type mice. Here, a mouse model was used to investigate whether loss of 1 of the 2 alleles of X-ray repair cross-complementing protein 1 (Xrcc1), which encodes a nonenzymatic scaffold protein required for BER, alters recovery from stroke. Ischemia and reperfusion caused higher brain damage and lower functional recovery in Xrcc1(+/-) mice than in wild-type mice. Additionally, a greater percentage of Xrcc1(+/-) mice died as a result of the stroke. Brain samples from human individuals who died of stroke and individuals who died of non-neurological causes were assayed for various steps of BER. Significant losses of thymine glycol incision, abasic endonuclease incision, and single nucleotide incorporation activities were identified, as well as lower expression of XRCC1 and NEIL1 proteins in stroke brains compared with controls. Together, these results suggest that impaired BER is a risk factor in ischemic brain injury and contributes to its recovery. PMID:25971543

  8. Biochemical reconstitution of TET1–TDG–BER-dependent active DNA demethylation reveals a highly coordinated mechanism

    PubMed Central

    Weber, Alain R.; Krawczyk, Claudia; Robertson, Adam B.; Kuśnierczyk, Anna; Vågbø, Cathrine B.; Schuermann, David; Klungland, Arne; Schär, Primo

    2016-01-01

    Cytosine methylation in CpG dinucleotides is an epigenetic DNA modification dynamically established and maintained by DNA methyltransferases and demethylases. Molecular mechanisms of active DNA demethylation began to surface only recently with the discovery of the 5-methylcytosine (5mC)-directed hydroxylase and base excision activities of ten–eleven translocation (TET) proteins and thymine DNA glycosylase (TDG). This implicated a pathway operating through oxidation of 5mC by TET proteins, which generates substrates for TDG-dependent base excision repair (BER) that then replaces 5mC with C. Yet, direct evidence for a productive coupling of TET with BER has never been presented. Here we show that TET1 and TDG physically interact to oxidize and excise 5mC, and proof by biochemical reconstitution that the TET–TDG–BER system is capable of productive DNA demethylation. We show that the mechanism assures a sequential demethylation of symmetrically methylated CpGs, thereby avoiding DNA double-strand break formation but contributing to the mutability of methylated CpGs. PMID:26932196

  9. Biochemical reconstitution of TET1-TDG-BER-dependent active DNA demethylation reveals a highly coordinated mechanism.

    PubMed

    Weber, Alain R; Krawczyk, Claudia; Robertson, Adam B; Kuśnierczyk, Anna; Vågbø, Cathrine B; Schuermann, David; Klungland, Arne; Schär, Primo

    2016-01-01

    Cytosine methylation in CpG dinucleotides is an epigenetic DNA modification dynamically established and maintained by DNA methyltransferases and demethylases. Molecular mechanisms of active DNA demethylation began to surface only recently with the discovery of the 5-methylcytosine (5mC)-directed hydroxylase and base excision activities of ten-eleven translocation (TET) proteins and thymine DNA glycosylase (TDG). This implicated a pathway operating through oxidation of 5mC by TET proteins, which generates substrates for TDG-dependent base excision repair (BER) that then replaces 5mC with C. Yet, direct evidence for a productive coupling of TET with BER has never been presented. Here we show that TET1 and TDG physically interact to oxidize and excise 5mC, and proof by biochemical reconstitution that the TET-TDG-BER system is capable of productive DNA demethylation. We show that the mechanism assures a sequential demethylation of symmetrically methylated CpGs, thereby avoiding DNA double-strand break formation but contributing to the mutability of methylated CpGs. PMID:26932196

  10. Dna Sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  11. Oxidative DNA damage is instrumental in hyperreplication stress-induced inviability of Escherichia coli

    PubMed Central

    Charbon, Godefroid; Bjørn, Louise; Mendoza-Chamizo, Belén; Frimodt-Møller, Jakob; Løbner-Olesen, Anders

    2014-01-01

    In Escherichia coli, an increase in the ATP bound form of the DnaA initiator protein results in hyperinitiation and inviability. Here, we show that such replication stress is tolerated during anaerobic growth. In hyperinitiating cells, a shift from anaerobic to aerobic growth resulted in appearance of fragmented chromosomes and a decrease in terminus concentration, leading to a dramatic increase in ori/ter ratio and cessation of cell growth. Aerobic viability was restored by reducing the level of reactive oxygen species (ROS) or by deleting mutM (Fpg glycosylase). The double-strand breaks observed in hyperinitiating cells therefore results from replication forks encountering single-stranded DNA lesions generated while removing oxidized bases, primarily 8-oxoG, from the DNA. We conclude that there is a delicate balance between chromosome replication and ROS inflicted DNA damage so the number of replication forks can only increase when ROS formation is reduced or when the pertinent repair is compromised. PMID:25389264

  12. Mitochondrial ROS Induces Cardiac Inflammation via a Pathway through mtDNA Damage in a Pneumonia-Related Sepsis Model.

    PubMed

    Yao, Xiao; Carlson, Deborah; Sun, Yuxiao; Ma, Lisha; Wolf, Steven E; Minei, Joseph P; Zang, Qun S

    2015-01-01

    We have previously shown that mitochondria-targeted vitamin E (Mito-Vit-E), a mtROS specific antioxidant, improves cardiac performance and attenuates inflammation in a pneumonia-related sepsis model. In this study, we applied the same approaches to decipher the signaling pathway(s) of mtROS-dependent cardiac inflammation after sepsis. Sepsis was induced in Sprague Dawley rats by intratracheal injection of S. pneumoniae. Mito-Vit-E, vitamin E or vehicle was administered 30 minutes later. In myocardium 24 hours post-inoculation, Mito-Vit-E, but not vitamin E, significantly protected mtDNA integrity and decreased mtDNA damage. Mito-Vit-E alleviated sepsis-induced reduction in mitochondria-localized DNA repair enzymes including DNA polymerase γ, AP endonuclease, 8-oxoguanine glycosylase, and uracil-DNA glycosylase. Mito-Vit-E dramatically improved metabolism and membrane integrity in mitochondria, suppressed leakage of mtDNA into the cytoplasm, inhibited up-regulation of Toll-like receptor 9 (TLR9) pathway factors MYD88 and RAGE, and limited RAGE interaction with its ligand TFAM in septic hearts. Mito-Vit-E also deactivated NF-κB and caspase 1, reduced expression of the essential inflammasome component ASC, and decreased inflammatory cytokine IL-1β. In vitro, both Mito-Vit-E and TLR9 inhibitor OND-I suppressed LPS-induced up-regulation in MYD88, RAGE, ASC, active caspase 1, and IL-1β in cardiomyocytes. Since free mtDNA escaped from damaged mitochondria function as a type of DAMPs to stimulate inflammation through TLR9, these data together suggest that sepsis-induced cardiac inflammation is mediated, at least partially, through mtDNA-TLR9-RAGE. At last, Mito-Vit-E reduced the circulation of myocardial injury marker troponin-I, diminished apoptosis and amended morphology in septic hearts, suggesting that mitochondria-targeted antioxidants are a potential cardioprotective approach for sepsis. PMID:26448624

  13. Mitochondrial ROS Induces Cardiac Inflammation via a Pathway through mtDNA Damage in a Pneumonia-Related Sepsis Model

    PubMed Central

    Yao, Xiao; Carlson, Deborah; Sun, Yuxiao; Ma, Lisha; Wolf, Steven E.; Minei, Joseph P.; Zang, Qun S.

    2015-01-01

    We have previously shown that mitochondria-targeted vitamin E (Mito-Vit-E), a mtROS specific antioxidant, improves cardiac performance and attenuates inflammation in a pneumonia-related sepsis model. In this study, we applied the same approaches to decipher the signaling pathway(s) of mtROS-dependent cardiac inflammation after sepsis. Sepsis was induced in Sprague Dawley rats by intratracheal injection of S. pneumoniae. Mito-Vit-E, vitamin E or vehicle was administered 30 minutes later. In myocardium 24 hours post-inoculation, Mito-Vit-E, but not vitamin E, significantly protected mtDNA integrity and decreased mtDNA damage. Mito-Vit-E alleviated sepsis-induced reduction in mitochondria-localized DNA repair enzymes including DNA polymerase γ, AP endonuclease, 8-oxoguanine glycosylase, and uracil-DNA glycosylase. Mito-Vit-E dramatically improved metabolism and membrane integrity in mitochondria, suppressed leakage of mtDNA into the cytoplasm, inhibited up-regulation of Toll-like receptor 9 (TLR9) pathway factors MYD88 and RAGE, and limited RAGE interaction with its ligand TFAM in septic hearts. Mito-Vit-E also deactivated NF-κB and caspase 1, reduced expression of the essential inflammasome component ASC, and decreased inflammatory cytokine IL–1β. In vitro, both Mito-Vit-E and TLR9 inhibitor OND-I suppressed LPS-induced up-regulation in MYD88, RAGE, ASC, active caspase 1, and IL–1β in cardiomyocytes. Since free mtDNA escaped from damaged mitochondria function as a type of DAMPs to stimulate inflammation through TLR9, these data together suggest that sepsis-induced cardiac inflammation is mediated, at least partially, through mtDNA-TLR9-RAGE. At last, Mito-Vit-E reduced the circulation of myocardial injury marker troponin-I, diminished apoptosis and amended morphology in septic hearts, suggesting that mitochondria-targeted antioxidants are a potential cardioprotective approach for sepsis. PMID:26448624

  14. Simultaneous Detection of 3-Nitrotyrosine and 3-Nitro-4-hydroxyphenylacetic Acid in Human Urine by Online SPE LC-MS/MS and Their Association with Oxidative and Methylated DNA Lesions.

    PubMed

    Chao, Mu-Rong; Hsu, Yu-Wen; Liu, Hung-Hsin; Lin, Jia-Hong; Hu, Chiung-Wen

    2015-05-18

    Reactive nitrogen species (RNS) can modify proteins at tyrosine and tryptophan residues, and they are involved in the pathogenesis of various human diseases. In this study, we present the first liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method that enables the simultaneous measurement of urinary 3-nitrotyrosine (3-NTYR) and its metabolite 3-nitro-4-hydroxyphenylacetic acid (NHPA). After the addition of stable isotope-labeled internal standards, urine samples were purified and enriched using manual solid-phase extraction (SPE) and HPLC fractionation followed by online SPE LC-MS/MS analysis. The limits of quantification in urine were 3.1 and 2.5 pg/mL for 3-NTYR and NHPA, respectively. Inter- and intraday imprecision was <15%. The mean relative recoveries of 3-NTYR and NHPA in urine were 89-98% and 90-98%, respectively. We further applied this method to 65 urinary samples from healthy subjects. Urinary samples were also analyzed for N-nitrosodimethylamine (NDMA) as well as oxidative and methylated DNA lesions, namely, 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), N7-methylguanine (N7-MeG), and N3-methyladenine (N3-MeA), using reported LC-MS/MS methods. Urinary 3-NTYR and NHPA levels were measured at concentrations of 63.2 ± 51.5 and 77.4 ± 60.8 pg/mL, respectively. Urinary 3-NTYR and NHPA levels were highly correlated with each other and with 8-oxoGua and 8-oxodGuo. Our findings demonstrated that a relationship exists between oxidative and nitrative stress. However, 3-NTYR and NHPA were correlated with N7-MeG and N3-MeA but not with NDMA, suggesting that NDMA may not be a representative biomarker of N-nitroso compounds that are induced by RNS. PMID:25825822

  15. The aminoglycoside antibiotic kanamycin damages DNA bases in Escherichia coli: caffeine potentiates the DNA-damaging effects of kanamycin while suppressing cell killing by ciprofloxacin in Escherichia coli and Bacillus anthracis.

    PubMed

    Kang, Tina Manzhu; Yuan, Jessica; Nguyen, Angelyn; Becket, Elinne; Yang, Hanjing; Miller, Jeffrey H

    2012-06-01

    The distribution of mutants in the Keio collection of Escherichia coli gene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ, holC, holD, and priA knockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded by recA, recB, and recC, among others. Additionally, caffeine partially protects cells of both Escherichia coli and Bacillus anthracis from killing by the widely used fluoroquinolone antibiotic ciprofloxacin. PMID:22391551

  16. The Aminoglycoside Antibiotic Kanamycin Damages DNA Bases in Escherichia coli: Caffeine Potentiates the DNA-Damaging Effects of Kanamycin while Suppressing Cell Killing by Ciprofloxacin in Escherichia coli and Bacillus anthracis

    PubMed Central

    Kang, Tina Manzhu; Yuan, Jessica; Nguyen, Angelyn; Becket, Elinne; Yang, Hanjing

    2012-01-01

    The distribution of mutants in the Keio collection of Escherichia coli gene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ, holC, holD, and priA knockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded by recA, recB, and recC, among others. Additionally, caffeine partially protects cells of both Escherichia coli and Bacillus anthracis from killing by the widely used fluoroquinolone antibiotic ciprofloxacin. PMID:22391551

  17. Age-related increases in human lymphocyte DNA damage: is there a role of aerobic fitness?

    PubMed

    Soares, Jorge Pinto; Mota, Maria Paula; Duarte, José Alberto; Collins, Andrew; Gaivão, Isabel

    2013-12-01

    Oxidative stress has been advanced as one of the major causes of damage to DNA and other macromolecules. Although physical exercise may also increase oxidative stress, an important role has been recognized for regular exercise in improving the overall functionality of the body, as indicated by an increase in maximal aerobic uptake ((V)O2max), and in resistance to cell damage. The aims of this study were 1) to evaluate the association between DNA damage in human lymphocytes and age and 2) to evaluate the association between DNA damage in human lymphocytes and ((V)O2max. The sample was composed of 36 healthy and nonsmoking males, aged from 20 to 84 years. ((V)O2max was evaluated through the Bruce protocol with direct measurement of oxygen consumption. The comet assay was used to evaluate the DNA damage, strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites. We found a positive correlation of age with DNA strand breaks but not with FPG-sensitive sites. ((V)O2max was significantly inversely related with DNA strand breaks, but this relation disappeared when adjusted for age. A significantly positive relation between ((V)O2max and FPG-sensitive sites was verified. In conclusion, our results showed that younger subjects have lower DNA strand breaks and higher (V)O2max compared with older subjects and FPG-sensitive sites are positively related with ((V)O2max, probably as transient damage due to the acute effects of daily physical activity. PMID:24446564

  18. Exposure to Ultrafine Particles from Ambient Air and Oxidative Stress–Induced DNA Damage

    PubMed Central

    Bräuner, Elvira Vaclavik; Forchhammer, Lykke; Møller, Peter; Simonsen, Jacob; Glasius, Marianne; Wåhlin, Peter; Raaschou-Nielsen, Ole; Loft, Steffen

    2007-01-01

    Background Particulate matter, especially ultrafine particles (UFPs), may cause health effects through generation of oxidative stress, with resulting damage to DNA and other macromolecules. Objective We investigated oxidative damage to DNA and related repair capacity in peripheral blood mononuclear cells (PBMCs) during controlled exposure to urban air particles with assignment of number concentration (NC) to four size modes with average diameters of 12, 23, 57, and 212 nm. Design Twenty-nine healthy adults participated in a randomized, two-factor cross-over study with or without biking exercise for 180 min and with exposure to particles (NC 6169-15362/cm3) or filtered air (NC 91-542/cm3) for 24 hr. Methods The levels of DNA strand breaks (SBs), oxidized purines as formamidopyrimidine DNA glycolase (FPG) sites, and activity of 7,8-dihydro-8-oxoguanine-DNA glycosylase (OGG1) in PBMCs were measured by the Comet assay. mRNA levels of OGG1, nucleoside diphosphate linked moiety X-type motif 1 (NUDT1), and heme oxygenase-1 (HO1) were determined by real-time reverse transcriptase–polymerase chain reaction. Results Exposure to UFPs for 6 and 24 hr significantly increased the levels of SBs and FPG sites, with a further insignificant increase after physical exercise. The OGG1 activity and expression of OGG1, NUDT1, and HO1 were unaltered. There was a significant dose–response relationship between NC and DNA damage, with the 57-nm mode as the major contributor to effects. Concomitant exposure to ozone, nitrogen oxides, and carbon monoxide had no influence. Conclusion Our results indicate that UFPs, especially the 57-nm soot fraction from vehicle emissions, causes systemic oxidative stress with damage to DNA and no apparent compensatory up-regulation of DNA repair within 24 hr. PMID:17687444

  19. Genome analysis of DNA repair genes in the alpha proteobacterium Caulobacter crescentus

    PubMed Central

    Martins-Pinheiro, Marinalva; Marques, Regina CP; Menck, Carlos FM

    2007-01-01

    Background The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria. Results In this study, we employed a bioinformatic approach, to analyse and describe the open reading frames potentially related to DNA repair from the genome of the alpha-proteobacterium Caulobacter crescentus. This was performed by comparison with known DNA repair related genes found in public databases. As expected, although C. crescentus and E. coli bacteria belong to separate phylogenetic groups, many of their DNA repair genes are very similar. However, some important DNA repair genes are absent in the C. crescentus genome and other interesting functionally related gene duplications are present, which do not occur in E. coli. These include DNA ligases, exonuclease III (xthA), endonuclease III (nth), O6-methylguanine-DNA methyltransferase (ada gene), photolyase-like genes, and uracil-DNA-glycosylases. On the other hand, the genes imuA and imuB, which are involved in DNA damage induced mutagenesis, have recently been described in C. crescentus, but are absent in E. coli. Particularly interesting are the potential atypical phylogeny of one of the photolyase genes in alpha-proteobacteria, indicating an origin by horizontal transfer, and the duplication of the Ada orthologs, which have diverse structural configurations, including one that is still unique for C. crescentus. Conclusion The absence and the presence of certain genes are discussed and predictions are made considering the particular aspects of the C. crescentus

  20. DNA Immunization

    PubMed Central

    Wang, Shixia; Lu, Shan

    2013-01-01

    DNA immunization was discovered in early 1990s and its use has been expanded from vaccine studies to a broader range of biomedical research, such as the generation of high quality polyclonal and monoclonal antibodies as research reagents. In this unit, three common DNA immunization methods are described: needle injection, electroporation and gene gun. In addition, several common considerations related to DNA immunization are discussed. PMID:24510291

  1. 8-Oxo-7,8-dihydroguanine in DNA does not constitute a barrier to transcription, but is converted into transcription-blocking damage by OGG1

    PubMed Central

    Kitsera, Nataliya; Stathis, Dimitrios; Lühnsdorf, Bork; Müller, Heiko; Carell, Thomas; Epe, Bernd; Khobta, Andriy

    2011-01-01

    The common DNA base modification 8-oxo-7,8-dihydroguanine (8-oxo-G) affects the efficiency and fidelity of transcription. We constructed plasmid substrates carrying single 8-oxo-G residues, specifically positioned in the transcribed or the non-transcribed DNA strands, to investigate their effects on the expression of an EGFP reporter gene and to explore the role of base excision repair in the mechanism of transcription inhibition. We report that 8-oxo-G does not directly block transcription in cells, since a single 8-oxo-G in the transcribed DNA strand did not reduce the EGFP expression levels in repair-deficient (OGG1-null) mouse embryonic fibroblast cell lines. Rather, inhibition of transcription by 8-oxo-G fully depends on 8-oxoguanine DNA glycosylase (OGG1) and, at the same time, does not require the localization of the lesion in the transcribed DNA strand. We propose that the interruption of transcription is induced by base excision repair intermediates and, therefore, could be a common consequence of various DNA base modifications. Concordantly, the non-blocking DNA modification uracil was also found to inhibit transcription, but in an OGG1-independent manner. PMID:21441539

  2. Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage

    PubMed Central

    Jacob, Kimberly D.; Hooten, Nicole Noren; Tadokoro, Takashi; Lohani, Althaf; Barnes, Janice; Evans, Michele K.

    2013-01-01

    Brain tissues from Alzheimer’s Disease (AD) patients show increased levels of oxidative DNA damage and 7,8-dihydro-8-oxoguanine (8-oxoG) accumulation. In humans, the base excision repair protein 8-oxoguanine-DNA glycosylase (OGG1) is the major enzyme that recognizes and excises the mutagenic DNA base lesion 8-oxoG. Recently, two polymorphisms of OGG1, A53T and A288V, have been identified in brain tissues of AD patients, but little is known about how these polymorphisms may contribute to AD. We characterized the A53T and A288V polymorphic variants and detected a significant reduction in the catalytic activity for both proteins in vitro and in cells. Additionally, the A53T polymorphism has decreased substrate binding, while the A288V polymorphism has reduced AP lyase activity. Both variants have decreased binding to known OGG1 binding partners PARP-1 and XRCC1. We found that OGG1−/− cells expressing A53T and A288V OGG1 were significantly more sensitive to DNA damage and had significantly decreased survival. Our results provide both biochemical and cellular evidence that A53T and A288V polymorphic proteins have deficiencies in catalytic and protein binding activities that could be related to the increase in oxidative damage to DNA found in AD brains. PMID:23684897

  3. Modulating effects of pycnogenol® on oxidative stress and DNA damage induced by sepsis in rats.

    PubMed

    Taner, Gökçe; Aydın, Sevtap; Bacanlı, Merve; Sarıgöl, Zehra; Sahin, Tolga; Başaran, A Ahmet; Başaran, Nurşen

    2014-11-01

    The aim of this study was to evaluate the protective effects of Pycnogenol® (Pyc), a complex plant extract from the bark of French maritime pine, on oxidative stress parameters (superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities and total glutathione (GSH) and malondialdehyde (MDA) levels), an inflammatory cytokine (tumor necrosis factor alpha (TNF-α) level) and also DNA damage in Wistar albino rats. Rats were treated with 100 mg/kg intraperitonally Pyc following the induction of sepsis by cecal ligation and puncture. The decreases in MDA levels and increases in GSH levels, and SOD and GPx activities were observed in the livers and kidneys of Pyc-treated septic rats. Plasma TNF-α level was found to be decreased in the Pyc-treated septic rats. In the lymphocytes, kidney, and liver tissue cells of the sepsis-induced rats, Pyc treatment significantly decreased the DNA damage and oxidative base damage using standard alkaline assay and formamidopyrimidine DNA glycosylase-modified comet assay, respectively. In conclusion, Pyc treatment might have a role in the prevention of sepsis-induced oxidative damage not only by decreasing DNA damage but also increasing the antioxidant status and DNA repair capacity in rats. PMID:24919414

  4. The source and significance of DNA damage in human spermatozoa; a commentary on diagnostic strategies and straw man fallacies.

    PubMed

    Aitken, R J; Bronson, R; Smith, T B; De Iuliis, G N

    2013-08-01

    This article considers the origins of DNA damage in human spermatozoa, the methods that are available to monitor this aspect of semen quality and the clinical significance of such measurements. DNA damage in spermatozoa appears to be largely oxidative in nature, inversely correlated with levels of nuclear protamination and frequently associated with the activation of a truncated apoptotic pathway. DNA base adducts formed as a result of oxidative attack are released from the spermatozoa into the extracellular space through the action of a glycosylase, OGG1. This creates an abasic site, which is not resolved until fertilization because spermatozoa do not possess the molecular machinery needed to continue the base excision repair pathway. The abasic sites so generated in human spermatozoa are readily detected by SCSA or the Comet assay; however, no signal is detectable with TUNEL. This is because spermatozoa lack the enzyme (APE1) needed to create the free 3' hydroxyl groups required by this detection system. Nevertheless, spermatozoa do eventually become TUNEL positive as they enter the perimortem. The American Society of Reproductive Medicine Practice Committee has suggested that DNA damage in spermatozoa should not be assessed because the correlation with pregnancy is inconsistent across independent studies. However, this is a straw man argument. The reason why such assays should be undertaken is not just that they reflect the underlying quality of spermatogenesis but, more importantly, that the DNA damage they reveal may have detrimental effects on the developmental normality of the embryo and the health of possible future children. PMID:23548339

  5. Oxidative DNA damage induced by hair dye components ortho-phenylenediamines and the enhancement by superoxide dismutase.

    PubMed

    Murata, Mariko; Nishimura, Tomoko; Chen, Fang; Kawanishi, Shosuke

    2006-09-01

    There is an association between occupational exposure to hair dyes and incidence of cancers. Permanent oxidant hair dyes are consisted of many chemical components including ortho-phenylenediamines. To clarify the mechanism of carcinogenesis by hair dyes, we examined DNA damage induced by mutagenic ortho-phenylenediamine (o-PD) and its derivatives, 4-chloro-ortho-phenylenediamine (Cl-PD) and 4-nitro-ortho-phenylenediamine (NO(2)-PD), using (32)P-labeled DNA fragments obtained from the human p16 and the p53 tumor suppressor gene. We also measured the content of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a marker of oxidative DNA damage, in calf thymus DNA with an electrochemical detector coupled to a high performance liquid chromatograph. Carcinogenic o-PD and Cl-PD caused Cu(II)-mediated DNA damage, including 8-oxodG formation, and antioxidant enzyme superoxide dismutase (SOD) enhanced DNA damage. o-PD and Cl-PD caused piperidine-labile and formamidopyrimidine-DNA glycosylase-sensitive lesions at cytosine and guanine residues respectively in the 5'-ACG-3' sequence, complementary to codon 273, a well-known hotspot of the human p53 tumor suppressor gene. UV-vis spectroscopic studies showed that the spectral change of o-PD and Cl-PD required Cu(II), and addition of SOD enhanced it. This suggested that SOD enhanced the rate of Cu(II)-mediated autoxidation of o-PD and Cl-PD, leading to enhancement of DNA damage. On the other hand, mutagenic but non-carcinogenic NO(2)-PD induced no DNA damage. These results suggest that carcinogenicity of ortho-phenylenediamines is associated with ability to cause oxidative DNA damage rather than bacterial mutagenicity. PMID:16798066

  6. Myricetin, quercetin, (+)-catechin and (-)-epicatechin protect against N-nitrosamines-induced DNA damage in human hepatoma cells.

    PubMed

    Delgado, M E; Haza, A I; García, A; Morales, P

    2009-10-01

    The aim of this study was to investigate the protective effect of myricetin, quercetin, (+)-catechin and (-)-epicatechin, against N-nitrosodibutylamine (NDBA) and N-nitrosopiperidine (NPIP)-induced DNA damage in human hepatoma cells (HepG2). DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single-cell gel electrophoresis or Comet assay. (+)-Catechin at the lowest concentration (10 microM) showed the maximum reduction of DNA strand breaks (23%), the formation of endonuclease III (Endo III, 19-21%) and formamidopyrimidine-DNA glycosylase (Fpg, 28-40%) sensitive sites induced by NDBA or NPIP. (-)-Epicatechin also decreased DNA strand breaks (10 microM, 20%) and the oxidized pyrimidines/purines (33-39%) induced by NDBA or NPIP, respectively. DNA strand breaks induced by NDBA or NPIP were weakly reduced by myricetin at the lowest concentration (0.1 microM, 10-19%, respectively). Myricetin also reduced the oxidized purines (0.1 microM, 17%) and pyrimidines (0.1 microM, 15%) induced by NDBA, but not the oxidized pyrimidines induced by NPIP. Quercetin did not protect against NDBA-induced DNA damage, but it reduced the formation of Endo III and Fpg sensitive sites induced by NPIP (0.1 microM, 17-20%, respectively). In conclusion, our results indicate that (+)-catechin and (-)-epicatechin at the concentrations tested protect human derived cells against oxidative DNA damage effects of NDBA and NPIP. However, myricetin at the concentrations tested only protects human cells against oxidative DNA damage induced by NDBA and quercetin against oxidative DNA damage induced by NPIP. PMID:19628030

  7. DNA ALTERATIONS

    EPA Science Inventory

    The exposure of an organism to genotoxic chemicals may induce a cascade of genetic events. nitially, structural alterations to DNA are formed. ext, the DNA damage is processed and subsequently expressed in mutant gene products. inally, diseases result from the genetic damage. he ...

  8. Low-Resolution Structure of Vaccinia Virus DNA Replication Machinery

    PubMed Central

    Sèle, Céleste; Gabel, Frank; Gutsche, Irina; Ivanov, Ivan; Burmeister, Wim P.

    2013-01-01

    Smallpox caused by the poxvirus variola virus is a highly lethal disease that marked human history and was eradicated in 1979 thanks to a worldwide mass vaccination campaign. This virus remains a significant threat for public health due to its potential use as a bioterrorism agent and requires further development of antiviral drugs. The viral genome replication machinery appears to be an ideal target, although very little is known about its structure. Vaccinia virus is the prototypic virus of the Orthopoxvirus genus and shares more than 97% amino acid sequence identity with variola virus. Here we studied four essential viral proteins of the replication machinery: the DNA polymerase E9, the processivity factor A20, the uracil-DNA glycosylase D4, and the helicase-primase D5. We present the recombinant expression and biochemical and biophysical characterizations of these proteins and the complexes they form. We show that the A20D4 polymerase cofactor binds to E9 with high affinity, leading to the formation of the A20D4E9 holoenzyme. Small-angle X-ray scattering yielded envelopes for E9, A20D4, and A20D4E9. They showed the elongated shape of the A20D4 cofactor, leading to a 150-Å separation between the polymerase active site of E9 and the DNA-binding site of D4. Electron microscopy showed a 6-fold rotational symmetry of the helicase-primase D5, as observed for other SF3 helicases. These results favor a rolling-circle mechanism of vaccinia virus genome replication similar to the one suggested for tailed bacteriophages. PMID:23175373

  9. Enzymatic Excision of Uracil Residues in Nucleosomes Depends on Local DNA Structure and Dynamics†

    PubMed Central

    Ye, Yu; Stahley, Mary R.; Xu, Jianqing; Friedman, Joshua I.; Sun, Yan; McKnight, Jeffrey N.; Gray, Jeffrey J.; Bowman, Gregory D.; Stivers, James T.

    2012-01-01

    The excision of uracil bases from DNA is accomplished by the enzyme uracil DNA glycosylase (UNG). Recognition of uracil bases in free DNA is facilitated by uracil base pair dynamics, but it is not known whether this same mechanistic feature is relevant for detection and excision of uracil residues embedded in nucleosomes. Here we investigate this question using nucleosome core particles (NCPs) generated from X. laevis histones and the high-affinity “Widom 601” positioning sequence. The reactivity of uracil residues in NCPs under steady-state multiple turnover conditions was generally decreased as compared to free 601 DNA, mostly due to anticipated steric effects of histones. However, some sites in NCPs had equal or even greater reactivity than free DNA, and the observed reactivities were not readily explained by simple steric considerations, or by global DNA unwrapping models for nucleosome invasion. In particular, some reactive uracils were found in occluded positions, while some unreactive uracils were found in exposed positions. One feature of many exposed reactive sites is a wide DNA minor groove, which allows penetration of a key active site loop of the enzyme. In single-turnover kinetic measurements, multi-phasic reaction kinetics were observed for several uracil sites, where each kinetic transient was independent of the UNG concentration. These kinetic measurements, and supporting structural analyses, support a mechanism where some uracils are transiently exposed to UNG by local, rate-limiting nucleosome conformational dynamics, followed by rapid trapping of the exposed state by the enzyme. We present structural models and plausible reaction mechanisms for the reaction of UNG at three distinct uracil sites in the NCP. PMID:22784353

  10. DNA repair by MGMT, but not AAG, causes a threshold in alkylation-induced colorectal carcinogenesis.

    PubMed

    Fahrer, Jörg; Frisch, Janina; Nagel, Georg; Kraus, Alexander; Dörsam, Bastian; Thomas, Adam D; Reißig, Sonja; Waisman, Ari; Kaina, Bernd

    2015-10-01

    Epidemiological studies indicate that N-nitroso compounds (NOC) are causally linked to colorectal cancer (CRC). NOC induce DNA alkylations, including O (6)-methylguanine (O (6)-MeG) and N-methylated purines, which are repaired by O (6)-MeG-DNA methyltransferase (MGMT) and N-alkyladenine-DNA glycosylase (AAG)-initiated base excision repair, respectively. In view of recent evidence of nonlinear mutagenicity for NOC-like compounds, the question arises as to the existence of threshold doses in CRC formation. Here, we set out to determine the impact of DNA repair on the dose-response of alkylation-induced CRC. DNA repair proficient (WT) and deficient (Mgmt (-/-), Aag (-/-) and Mgmt (-/-)/Aag (-/-)) mice were treated with azoxymethane (AOM) and dextran sodium sulfate to trigger CRC. Tumors were quantified by non-invasive mini-endoscopy. A non-linear increase in CRC formation was observed in WT and Aag (-/-) mice. In contrast, a linear dose-dependent increase in tumor frequency was found in Mgmt (-/-) and Mgmt (-/-)/Aag (-/-) mice. The data were corroborated by hockey stick modeling, yielding similar carcinogenic thresholds for WT and Aag (-/-) and no threshold for MGMT lacking mice. O (6)-MeG levels and depletion of MGMT correlated well with the observed dose-response in CRC formation. AOM induced dose-dependently DNA double-strand breaks in colon crypts including Lgr5-positive colon stem cells, which coincided with ATR-Chk1-p53 signaling. Intriguingly, Mgmt (-/-) mice displayed significantly enhanced levels of γ-H2AX, suggesting the usefulness of γ-H2AX as an early genotoxicity marker in the colorectum. This study demonstrates for the first time a non-linear dose-response for alkylation-induced colorectal carcinogenesis and reveals DNA repair by MGMT, but not AAG, as a key node in determining a carcinogenic threshold. PMID:26243310

  11. Sensitive and selective real-time electrochemical monitoring of DNA repair (Presentation Recording)

    NASA Astrophysics Data System (ADS)

    Slinker, Jason D.; McWilliams, Marc; Anka, Fadwa; Balkus, Kenneth

    2015-10-01

    Unrepaired DNA damage can lead to mutation, cancer, and death of cells or organisms. However, due to the subtlety of DNA damage, it is difficult to sense the repair of damage products with high selectivity and sensitivity. Here, we show sensitive and selective electrochemical sensing of the repair activity of 8-oxoguanine and uracil glycosylases within DNA monolayers on gold by multiplexed analysis with silicon chips and low-cost electrospun nanofibers. Our approach involves comparing the electrochemical signal of redox probe modified monolayers containing the defect versus the rational control of defect-free monolayers. We find sequence-specific sensitivity thresholds on the order of femtomoles of proteins and dynamic ranges of over two orders of magnitude for each target. For 8-oxoguanine repair, temperature-dependent kinetics are extracted, showing exponential signal loss with time constants of seconds. Electrospun fibers are shown to behave similarly to conventional gold-on-silicon devices, showing the potential of these low-cost devices for sensing applications.

  12. [DNA computing].

    PubMed

    Błasiak, Janusz; Krasiński, Tadeusz; Popławski, Tomasz; Sakowski, Sebastian

    2011-01-01

    Biocomputers can be an alternative for traditional "silicon-based" computers, which continuous development may be limited due to further miniaturization (imposed by the Heisenberg Uncertainty Principle) and increasing the amount of information between the central processing unit and the main memory (von Neuman bottleneck). The idea of DNA computing came true for the first time in 1994, when Adleman solved the Hamiltonian Path Problem using short DNA oligomers and DNA ligase. In the early 2000s a series of biocomputer models was presented with a seminal work of Shapiro and his colleguas who presented molecular 2 state finite automaton, in which the restriction enzyme, FokI, constituted hardware and short DNA oligomers were software as well as input/output signals. DNA molecules provided also energy for this machine. DNA computing can be exploited in many applications, from study on the gene expression pattern to diagnosis and therapy of cancer. The idea of DNA computing is still in progress in research both in vitro and in vivo and at least promising results of these research allow to have a hope for a breakthrough in the computer science. PMID:21735816

  13. Genotoxic Potential of Reactive Oxygen Species (Ros), Lipid Peroxidation and DNA Repair Enzymes (Fpg and Endo III) in Alloxan Injected Diabetic Rats.

    PubMed

    Yaduvanshi, Santosh Kumar; Srivastava, Nalini; Prasad, G B K S; Yadav, Mukesh; Jain, Shalini; Yadav, Hariom

    2012-11-28

    Diabetes is a chronic metabolic syndrome due to insulin deficiency and is associated with increased oxidative stress in vivo. Oxidative stress including, increased production of reactive oxygen species (ROS) in vivo, can lead to cellular biomolecule damage. Such damage has been suggested to contribute to the pathogenesis of diabetes mellitus. Genotoxicity induced by ROS in diabetic rats, was estimated by measuring DNA single strand breaks and double strand breaks by standard comet assay/ single cell gel electrophoresis (SCGE). To find out whether DNA lesions were caused due to oxidative stress, combination of bacterial DNA repair enzymes which convert base damage to breaks are used. Formamidoaminopyrimidine glycosylase (Fpg) and Endonuclese (Endo III)] recognize oxidized purines and oxidized pyrimidines, respectively, were used in modified comet assay. Significant increase in DNA strand breaks in terms of DNA damage index were observed in diabetic rat lymphocytes in modified comet assay. The involvement of oxidative stress was also examined by estimation of thiobarbituric acid reactive substances (TBARS) in diabetic rats. The levels of TBARS, reactive oxygen species (ROS) namely, hydrogen peroxide, superoxide and nitrate/nitrite anion were also increased in diabetic rats that further shows the involvement of oxidative stress in ROS induced DNA damage. The results of the present study show genotoxic potential of ROS in diabetic rats. PMID:23210730

  14. MET18 Connects the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Active DNA Demethylation in Arabidopsis

    PubMed Central

    Tang, Kai; Zhang, Huiming; Mangrauthia, Satendra K.; Lei, Mingguang; Hsu, Chuan-Chih; Hou, Yueh-Ju; Wang, Chunguo; Li, Yan; Tao, W. Andy; Zhu, Jian-Kang

    2015-01-01

    DNA demethylation mediated by the DNA glycosylase ROS1 helps determine genomic DNA methylation patterns and protects active genes from being silenced. However, little is known about the mechanism of regulation of ROS1 enzymatic activity. Using a forward genetic screen, we identified an anti-silencing (ASI) factor, ASI3, the dysfunction of which causes transgene promoter hyper-methylation and silencing. Map-based cloning identified ASI3 as MET18, a component of the cytosolic iron-sulfur cluster assembly (CIA) pathway. Mutation in MET18 leads to hyper-methylation at thousands of genomic loci, the majority of which overlap with hypermethylated loci identified in ros1 and ros1dml2dml3 mutants. Affinity purification followed by mass spectrometry indicated that ROS1 physically associates with MET18 and other CIA components. Yeast two-hybrid and split luciferase assays showed that ROS1 can directly interact with MET18 and another CIA component, AE7. Site-directed mutagenesis of ROS1 indicated that the conserved iron-sulfur motif is indispensable for ROS1 enzymatic activity. Our results suggest that ROS1-mediated active DNA demethylation requires MET18-dependent transfer of the iron-sulfur cluster, highlighting an important role of the CIA pathway in epigenetic regulation. PMID:26492035

  15. Methylperoxyl radicals as intermediates in the damage to DNA irradiated in aqueous dimethyl sulfoxide with gamma rays

    SciTech Connect

    Milligan, J.R.; Ng, J.Y.Y.; Wu, C.C.L.

    1996-10-01

    Using agarose gel electrophoresis, we have measured the yields of DNA single-strand breaks (SSBs) for plasmid DNA {gamma}-irradiated in aerobic aqueous solution. Incubation after irradiation with the base damage repair endonucleases formamidopyrimidine-DNA N-glycosylase (FPG) or endonuclease III (endo III) results in an increase in the yield of SSBs. In the absence of dimethyl sulfoxide (DMSO) during irradiation, this increase is consistent with the yields of known substrates for FPG and endo III as determined by gas chromatography/mass spectrometry. After irradiation in the presence of 1 mol dm{sup {minus}3} DMSO, the increase in the yield of SSBs after enzyme incubation was further enhanced by a factor of about 5 to 7. The magnitude of this effect, the inability of acrylamide or oxygen to suppress it, and its attenuation by N,N,N{prime}, N{prime}-tetramethylphenylenediamine (TMPD) or glycerol all suggest that the methylperoxyl radical (derived from DMSO) is involved as an intermediate. Reactions of the methylperoxyl radical (or some other species derived from it) do not result in strand break damage, but are responsible for DNA base damages which which are recognized by FPG and endo III. 41 refs., 5 figs.

  16. MET18 Connects the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Active DNA Demethylation in Arabidopsis.

    PubMed

    Duan, Cheng-Guo; Wang, Xingang; Tang, Kai; Zhang, Huiming; Mangrauthia, Satendra K; Lei, Mingguang; Hsu, Chuan-Chih; Hou, Yueh-Ju; Wang, Chunguo; Li, Yan; Tao, W Andy; Zhu, Jian-Kang

    2015-10-01

    DNA demethylation mediated by the DNA glycosylase ROS1 helps determine genomic DNA methylation patterns and protects active genes from being silenced. However, little is known about the mechanism of regulation of ROS1 enzymatic activity. Using a forward genetic screen, we identified an anti-silencing (ASI) factor, ASI3, the dysfunction of which causes transgene promoter hyper-methylation and silencing. Map-based cloning identified ASI3 as MET18, a component of the cytosolic iron-sulfur cluster assembly (CIA) pathway. Mutation in MET18 leads to hyper-methylation at thousands of genomic loci, the majority of which overlap with hypermethylated loci identified in ros1 and ros1dml2dml3 mutants. Affinity purification followed by mass spectrometry indicated that ROS1 physically associates with MET18 and other CIA components. Yeast two-hybrid and split luciferase assays showed that ROS1 can directly interact with MET18 and another CIA component, AE7. Site-directed mutagenesis of ROS1 indicated that the conserved iron-sulfur motif is indispensable for ROS1 enzymatic activity. Our results suggest that ROS1-mediated active DNA demethylation requires MET18-dependent transfer of the iron-sulfur cluster, highlighting an important role of the CIA pathway in epigenetic regulation. PMID:26492035

  17. Dancing DNA.

    ERIC Educational Resources Information Center

    Pennisi, Elizabeth

    1991-01-01

    An imaging technique that uses fluorescent dyes and allows scientists to track DNA as it moves through gels or in solution is described. The importance, opportunities, and implications of this technique are discussed. (KR)

  18. Origin of the Heterogeneous Distribution of the Yield of Guanyl Radical in UV Laser Photolyzed DNA

    PubMed Central

    Angelov, Dimitar; Beylot, Benedicte; Spassky, Annick

    2005-01-01

    Oxidative guanine lesions were analyzed, at the nucleotide level, within DNA exposed to nanosecond ultraviolet (266 nm) laser pulses of variable intensity (0.002–0.1 J/cm2). Experiments were carried out, at room temperature, in TE buffer (20 mM Tris-HCl, pH 7.5; 1 mM EDTA) containing 35 mM NaCl, on 5′-end radioactively labeled double-stranded and single-stranded oligomer DNA at a size of 33–37 nucleobases. Lesions were analyzed on polyacrylamide gel electrophoresis by taking advantage of the specific removal of 8-oxodG from DNA by the formamidopyrimidine DNA glycosylase (Fpg protein) and of the differential sensitivity of 8-oxodG and oxazolone to piperidine. The quantum yields of lesions at individual sites, determined from the normalized intensities of bands, were plotted against the irradiation energy levels. Simplified model fitting of the experimental data enabled to evaluate the spectroscopic parameters characterizing excitation and photoionization processes. Results show that the distribution of guanine residues, excited to the lowest triplet state or photoionized, is heterogeneous and depends on the primary and secondary DNA structure. These findings are generalized in terms of excitation energy and charge-migration mediated biphotonic ionization. On the basis of the changes in the yield of the guanyl radical resulting from local helical perturbations in the DNA π-stack, it can be assessed that the distance range of migration is <6–8 bp. PMID:15613625

  19. A simpler, more robust method for the analysis of 8-oxoguanine in DNA.

    PubMed

    Beckman, K B; Saljoughi, S; Mashiyama, S T; Ames, B N

    2000-08-01

    The oxidized DNA base 8-oxoguanine has been commonly measured by enzymatic digestion of DNA to nucleosides followed by high-performance liquid chromatography (HPLC) separation of the adduct 8-oxodeoxyguanosine. There has recently been an enormous debate surrounding the validity of this approach, from which it has become clear that artifactual oxidation of the native base to 8-oxoguanine can occur at numerous stages in sample preparation. Hence, we have designed an alternative protocol to traditional enzymatic digestion of DNA which (i) limits the potential for artifactual oxidation, (ii) speeds up the assay markedly, (iii) increases the assay's sensitivity moderately, and (iv) addresses criticisms that have been raised concerning the efficiency of DNA digestion by nucleases. In short, we use the Escherichia coli repair enzyme formamidopyrimidine (Fapy) glycosylase to release the base 8-oxoguanine from full-length DNA, then separate 8-oxoguanine from high molecular weight molecules by ultrafiltration (10,000 Da exclusion) and analyze the base adduct by reverse-phase HPLC. Benefits of this approach include (i) rapid removal of the roughly million-fold molar excess of unaltered bases from the sample, (ii) reduction in the length of enzymatic incubations and the number of steps, (iii) elimination of high temperature incubation, (iv) a very clean chromatographic separation, and (v) rapid elution of the analyte and correspondingly greater throughput. Using this improved method, we have followed the induction of 8-oxoguanine in the DNA of peroxide-treated HeLa cells, an experiment that had proved cumbersome with traditional methods. PMID:11035265

  20. Incorporation of deoxyuridine monophosphate into DNA increases the sister-chromatid exchange yield

    SciTech Connect

    Pardo, E.G.; Hernandez, P.; Gutierrez, C.

    1987-02-01

    The effect of a treatment with 5-fluoro-2'-deoxyuridine (FdUrd) in combination with 2'-deoxyuridine (dUrd) on cell proliferation, incorporation of DNA precursors into DNA and sister-chromatid exchanges (SCEs) has been analyzed in Allium cepa meristem cells. FdUrd in the range 10/sup -9/-5 x 10/sup -7/ M produced a dose- and time-dependent decrease in the amount of cells in mitosis. This inhibitory effect could be reversed by 70-80% in short-term (6 h) experiments, by exogenously supplied dUrd at a concentration of 10/sup -1/ M. However, at the highest FdUrd dose tested (10/sup -7/ M), 10/sup -4/ M dUrd could not reverse the FdUrd effect in long-term experiments as shown by analyzing the kinetics of synchronous cell populations. DNA extracted from cells pulsed with (6-/sup 3/H)dUrd in the presence of FdUrd and 6-amino-uracil (6-AU), an inhibitor of uracil-DNA glycosylase, contained a small amount of label in the form of (6-/sup 3/H)dUMP. Thus the authors conclude that under the experimental conditions, exogenously supplied dUrd may be metabolized intracellularly to 2'-deoxyuridine triphosphate (dUTP) and that this deoxynucleotide may eventually be mis-incorporated into DNA. By analyzing SCE levels in third division chromosomes of cells treated with FdUrd and dUrd during their second cycle, they has scored a 6-fold increase in the reciprocal SCE level which demonstrates that the replication of a dUMP-containing DNA template leads to a higher SCE yield.

  1. Unravelling DNA

    NASA Astrophysics Data System (ADS)

    Conroy, Rs; Danilowicz, C.

    2004-04-01

    The forces involved in the biology of life are carefully balanced between stopping thermal fluctuations ripping our DNA apart and having bonds weak enough to allow enzymes to function. The application of recently developed techniques for measuring piconewton forces and imaging at the nanometre scale on a molecule-by-molecule basis has dramatically increased the impact of single-molecule biophysics. This article describes the most commonly used techniques for imaging and manipulating single biomolecules. Using these techniques, the mechanical properties of DNA can be investigated, for example through measurements of the forces required to stretch and unzip the DNA double helix. These properties determine the ease with which DNA can be folded into the cell nucleus and the size and complexity of the accompanying cellular machinery. Part of this cellular machinery is enzymes, which manipulate, repair and transcribe the DNA helix. Enzymatic function is increasingly being investigated at the single molecule level to give better understanding of the forces and processes involved in the genetic cycle. One of the challenges is to transfer this understanding of single molecules into living systems. Already there have been some notable successes, such as the development of techniques for gene expression through the application of mechanical forces to cells, and the imaging and control of viral infection of a cell. This understanding and control of DNA has also been used to design molecules, which can self-assemble into a range of structures.

  2. Efficiency of Base Excision Repair of Oxidative DNA Damage and Its Impact on the Risk of Colorectal Cancer in the Polish Population

    PubMed Central

    Kabzinski, J.; Mucha, B.; Cuchra, M.; Markiewicz, L.; Przybylowska, K.; Dziki, A.; Dziki, L.; Majsterek, I.

    2016-01-01

    DNA oxidative lesions are widely considered as a potential risk factor for colorectal cancer development. The aim of this work was to determine the role of the efficiency of base excision repair, both in lymphocytes and in epithelial tissue, in patients with CRC and healthy subjects. SNPs were identified within genes responsible for steps following glycosylase action in BER, and patients and healthy subjects were genotyped. A radioisotopic BER assay was used for assessing repair efficiency and TaqMan for genotyping. Decreased BER activity was observed in lymphocyte extract from CRC patients and in cancer tissue extract, compared to healthy subjects. In addition, polymorphisms of EXO1, LIG3, and PolB may modulate the risk of colorectal cancer by decreasing (PolB) or increasing (LIG3 and EXO1) the chance of malignant transformation. PMID:26649135

  3. What Is Mitochondrial DNA?

    MedlinePlus

    ... DNA What is mitochondrial DNA? What is mitochondrial DNA? Although most DNA is packaged in chromosomes within ... proteins. For more information about mitochondria and mitochondrial DNA: Molecular Expressions, a web site from the Florida ...

  4. Ancient DNA

    PubMed Central

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    In the past two decades, ancient DNA research has progressed from the retrieval of small fragments of mitochondrial DNA from a few late Holocene specimens, to large-scale studies of ancient populations, phenotypically important nuclear loci, and even whole mitochondrial genome sequences of extinct species. However, the field is still regularly marred by erroneous reports, which underestimate the extent of contamination within laboratories and samples themselves. An improved understanding of these processes and the effects of damage on ancient DNA templates has started to provide a more robust basis for research. Recent methodological advances have included the characterization of Pleistocene mammal populations and discoveries of DNA preserved in ancient sediments. Increasingly, ancient genetic information is providing a unique means to test assumptions used in evolutionary and population genetics studies to reconstruct the past. Initial results have revealed surprisingly complex population histories, and indicate that modern phylogeographic studies may give misleading impressions about even the recent evolutionary past. With the advent and uptake of appropriate methodologies, ancient DNA is now positioned to become a powerful tool in biological research and is also evolving new and unexpected uses, such as in the search for extinct or extant life in the deep biosphere and on other planets. PMID:15875564

  5. DNA vaccines

    NASA Astrophysics Data System (ADS)

    Gregersen, Jens-Peter

    2001-12-01

    Immunization by genes encoding immunogens, rather than with the immunogen itself, has opened up new possibilities for vaccine research and development and offers chances for new applications and indications for future vaccines. The underlying mechanisms of antigen processing, immune presentation and regulation of immune responses raise high expectations for new and more effective prophylactic or therapeutic vaccines, particularly for vaccines against chronic or persistent infectious diseases and tumors. Our current knowledge and experience of DNA vaccination is summarized and critically reviewed with particular attention to basic immunological mechanisms, the construction of plasmids, screening for protective immunogens to be encoded by these plasmids, modes of application, pharmacokinetics, safety and immunotoxicological aspects. DNA vaccines have the potential to accelerate the research phase of new vaccines and to improve the chances of success, since finding new immunogens with the desired properties is at least technically less demanding than for conventional vaccines. However, on the way to innovative vaccine products, several hurdles have to be overcome. The efficacy of DNA vaccines in humans appears to be much less than indicated by early studies in mice. Open questions remain concerning the persistence and distribution of inoculated plasmid DNA in vivo, its potential to express antigens inappropriately, or the potentially deleterious ability to insert genes into the host cell's genome. Furthermore, the possibility of inducing immunotolerance or autoimmune diseases also needs to be investigated more thoroughly, in order to arrive at a well-founded consensus, which justifies the widespread application of DNA vaccines in a healthy population.

  6. AP endonuclease 1 prevents the extension of a T/G mismatch by DNA polymerase β to prevent mutations in CpGs during base excision repair.

    PubMed

    Lai, Yanhao; Jiang, Zhongliang; Zhou, Jing; Osemota, Emmanuel; Liu, Yuan

    2016-07-01

    Dynamics of DNA methylation and demethylation at CpG clusters are involved in gene regulation. CpG clusters have been identified as hot spots of mutagenesis because of their susceptibility to oxidative DNA damage. Damaged Cs and Gs at CpGs can disrupt a normal DNA methylation pattern through modulation of DNA methylation and demethylation, leading to mutations and deregulation of gene expression. DNA base excision repair (BER) plays a dual role of repairing oxidative DNA damage and mediating an active DNA demethylation pathway on CpG clusters through removal of a T/G mismatch resulting from deamination of a 5mC adjacent to a guanine that can be simultaneously damaged by oxidative stress. However, it remains unknown how BER processes clustered lesions in CpGs and what are the consequences from the repair of these lesions. In this study, we examined BER of an abasic lesion next to a DNA demethylation intermediate, the T/G mismatch in a CpG dinucleotide, and its effect on the integrity of CpGs. Surprisingly, we found that the abasic lesion completely abolished the activity of thymine DNA glycosylase (TDG) for removing the mismatched T. However, we found that APE1 could still efficiently incise the abasic lesion leaving a 3-terminus mismatched T, which was subsequently extended by pol β. This in turn resulted in a C to T transition mutation. Interestingly, we also found that APE1 3'-5' exonuclease activity efficiently removed the mismatched T, thereby preventing pol β extension of the mismatched nucleotide and the resulting mutation. Our results demonstrate a crucial role of APE1 3'-5' exonuclease activity in combating mutations in CpG clusters caused by an intermediate of DNA demethylation during BER. PMID:27183823

  7. Whole transcriptome analysis reveals a role for OGG1-initiated DNA repair signaling in airway remodeling

    PubMed Central

    Aguilera-Aguirre, Leopoldo; Hosoki, Koa; Bacsi, Attila; Radák, Zsolt; Sur, Sanjiv; Hegde, Muralidhar L.; Tian, Bing; Saavedra-Molina, Alfredo; Brasier, Allan R.; Ba, Xueqing; Boldogh, Istvan

    2016-01-01

    Reactive oxygen species (ROS) generated by environmental exposures, and endogenously as by-products of respiration, oxidatively modify biomolecules including DNA. Accumulation of ROS-induced DNA damage has been implicated in various diseases that involve inflammatory processes, and efficient DNA repair is considered critical in preventing such diseases. One of the most abundant DNA base lesions is 7,8-dihydro-8-oxoguanine (8-oxoG), which is repaired by the 8-oxoguanine DNA glycosylase 1 (OGG1)-initiated base-excision repair (OGG1-BER) pathway. Recent studies have shown that the OGG1-BER byproduct 8-oxoG base forms a complex with cytosolic OGG1, activating small GTPases and downstream cell signaling in cultured cells and lungs. This implies that persistent OGG1-BER could result in signaling leading to histological changes in airways. To test this, we mimicked OGG1-BER by repeatedly challenging airways with its repair product 8-oxoG base. Gene expression was analyzed by RNA sequencing (RNA-Seq) and qRT-PCR, and datasets were evaluated by gene ontology and statistical tools. RNA-Seq analysis identified 3252 differentially expressed transcripts (2435 up- and 817 downregulated, Z3-fold change). Among the upregulated transcripts, 2080 mRNAs were identified whose encoded protein products were involved in modulation of the actin family cytoskeleton, extracellular matrix, cell adhesion, cadherin, and cell junctions, affecting biological processes such as tissue development, cell-to-cell adhesion, cell communication, and the immune system. These data are supported by histological observations showing epithelial alterations, subepithelial fibrosis, and collagen deposits in the lungs. These data imply that continuous challenge by the environment and consequent OGG1-BER-driven signaling trigger gene expression consistent with airway remodeling. PMID:26187872

  8. Modulation of the DNA scanning activity of the Micrococcus luteus UV endonuclease

    SciTech Connect

    Hamilton, R.W.; Lloyd, R.S. )

    1989-10-15

    Micrococcus luteus UV endonuclease incises DNA at the sites of ultraviolet (UV) light-induced pyrimidine dimers. The mechanism of incision has been previously shown to be a glycosylic bond cleavage at the 5'-pyrimidine of the dimer followed by an apyrimidine endonuclease activity which cleaves the phosphodiester backbone between the pyrimidines. The process by which M. luteus UV endonuclease locates pyrimidine dimers within a population of UV-irradiated plasmids was shown to occur, in vitro, by a processive or sliding mechanism on non-target DNA as opposed to a distributive or random hit mechanism. Form I plasmid DNA containing 25 dimers per molecule was incubated with M. luteus UV endonuclease in time course reactions. The three topological forms of plasmid DNA generated were analyzed by agarose gel electrophoresis. When the enzyme encounters a pyrimidine dimer, it is significantly more likely to make only the glycosylase cleavage as opposed to making both the glycosylic and phosphodiester bond cleavages. Thus, plasmids are accumulated with many alkaline-labile sites relative to single-stranded breaks. In addition, reactions were performed at both pH 8.0 and pH 6.0, in the absence of NaCl, as well as 25,100, and 250 mM NaCl. The efficiency of the DNA scanning reaction was shown to be dependent on both the ionic strength and pH of the reaction. At low ionic strengths, the reaction was shown to proceed by a processive mechanism and shifted to a distributive mechanism as the ionic strength of the reaction increased. Processivity at pH 8.0 is shown to be more sensitive to increases in ionic strength than reactions performed at pH 6.0.

  9. Cerebellar Oxidative DNA Damage and Altered DNA Methylation in the BTBR T+tf/J Mouse Model of Autism and Similarities with Human Post Mortem Cerebellum

    PubMed Central

    Shpyleva, Svitlana; Ivanovsky, Samuil; de Conti, Aline; Melnyk, Stepan; Tryndyak, Volodymyr; Beland, Frederick A.; James, S. Jill; Pogribny, Igor P.

    2014-01-01

    The molecular pathogenesis of autism is complex and involves numerous genomic, epigenomic, proteomic, metabolic, and physiological alterations. Elucidating and understanding the molecular processes underlying the pathogenesis of autism is critical for effective clinical management and prevention of this disorder. The goal of this study is to investigate key molecular alterations postulated to play a role in autism and their role in the pathophysiology of autism. In this study we demonstrate that DNA isolated from the cerebellum of BTBR T+tf/J mice, a relevant mouse model of autism, and from human post-mortem cerebellum of individuals with autism, are both characterized by an increased levels of 8-oxo-7-hydrodeoxyguanosine (8-oxodG), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC). The increase in 8-oxodG and 5mC content was associated with a markedly reduced expression of the 8-oxoguanine DNA-glycosylase 1 (Ogg1) and increased expression of de novo DNA methyltransferases 3a and 3b (Dnmt3a and Dnmt3b). Interestingly, a rise in the level of 5hmC occurred without changes in the expression of ten-eleven translocation expression 1 (Tet1) and Tet2 genes, but significantly correlated with the presence of 8-oxodG in DNA. This finding and similar elevation in 8-oxodG in cerebellum of individuals with autism and in the BTBR T+tf/J mouse model warrant future large-scale studies to specifically address the role of OGG1 alterations in pathogenesis of autism. PMID:25423485

  10. DNA codes

    SciTech Connect

    Torney, D. C.

    2001-01-01

    We have begun to characterize a variety of codes, motivated by potential implementation as (quaternary) DNA n-sequences, with letters denoted A, C The first codes we studied are the most reminiscent of conventional group codes. For these codes, Hamming similarity was generalized so that the score for matched letters takes more than one value, depending upon which letters are matched [2]. These codes consist of n-sequences satisfying an upper bound on the similarities, summed over the letter positions, of distinct codewords. We chose similarity 2 for matches of letters A and T and 3 for matches of the letters C and G, providing a rough approximation to double-strand bond energies in DNA. An inherent novelty of DNA codes is 'reverse complementation'. The latter may be defined, as follows, not only for alphabets of size four, but, more generally, for any even-size alphabet. All that is required is a matching of the letters of the alphabet: a partition into pairs. Then, the reverse complement of a codeword is obtained by reversing the order of its letters and replacing each letter by its match. For DNA, the matching is AT/CG because these are the Watson-Crick bonding pairs. Reversal arises because two DNA sequences form a double strand with opposite relative orientations. Thus, as will be described in detail, because in vitro decoding involves the formation of double-stranded DNA from two codewords, it is reasonable to assume - for universal applicability - that the reverse complement of any codeword is also a codeword. In particular, self-reverse complementary codewords are expressly forbidden in reverse-complement codes. Thus, an appropriate distance between all pairs of codewords must, when large, effectively prohibit binding between the respective codewords: to form a double strand. Only reverse-complement pairs of codewords should be able to bind. For most applications, a DNA code is to be bi-partitioned, such that the reverse-complementary pairs are separated

  11. Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins.

    PubMed

    Zdżalik, Daria; Domańska, Anna; Prorok, Paulina; Kosicki, Konrad; van den Born, Erwin; Falnes, Pål Ø; Rizzo, Carmelo J; Guengerich, F Peter; Tudek, Barbara

    2015-06-01

    AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N(2)-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. PMID:25797601

  12. Inhalative Exposure to Vanadium Pentoxide Causes DNA Damage in Workers: Results of a Multiple End Point Study

    PubMed Central

    Ehrlich, Veronika A.; Nersesyan, Armen K.; Hoelzl, Christine; Ferk, Franziska; Bichler, Julia; Valic, Eva; Schaffer, Andreas; Schulte-Hermann, Rolf; Fenech, Michael; Wagner, Karl-Heinz; Knasmüller, Siegfried

    2008-01-01

    Background Inhalative exposure to vanadium pentoxide (V2O5) causes lung cancer in rodents. Objective The aim of the study was to investigate the impact of V2O5 on DNA stability in workers from a V2O5 factory. Methods We determined DNA strand breaks in leukocytes of 52 workers and controls using the alkaline comet assay. We also investigated different parameters of chromosomal instability in lymphocytes of 23 workers and 24 controls using the cytokinesis-block micronucleus (MN) cytome method. Results Seven of eight biomarkers were increased in blood cells of the workers, and vanadium plasma concentrations in plasma were 7-fold higher than in the controls (0.31 μg/L). We observed no difference in DNA migration under standard conditions, but we found increased tail lengths due to formation of oxidized purines (7%) and pyrimidines (30%) with lesion-specific enzymes (formamidopyrimidine glycosylase and endonuclease III) in the workers. Bleomycin-induced DNA migration was higher in the exposed group (25%), whereas the repair of bleomycin-induced lesions was reduced. Workers had a 2.5-fold higher MN frequency, and nucleoplasmic bridges (NPBs) and nuclear buds (Nbuds) were increased 7-fold and 3-fold, respectively. Also, apoptosis and necrosis rates were higher, but only the latter parameter reached statistical significance. Conclusions V2O5 causes oxidation of DNA bases, affects DNA repair, and induces formation of MNs, NPBs, and Nbuds in blood cells, suggesting that the workers are at increased risk for cancer and other diseases that are related to DNA instability. PMID:19079721

  13. DNA computing.

    PubMed

    Gibbons, A; Amos, M; Hodgson, D

    1997-02-01

    DNA computation is a novel and exciting recent development at the interface of computer science and molecular biology. We describe the current activity in this field following the seminal work of Adleman, who recently showed how techniques of molecular biology may be applied to the solution of a computationally intractable problem. PMID:9013647

  14. DNA Music.

    ERIC Educational Resources Information Center

    Miner, Carol; della Villa, Paula

    1997-01-01

    Describes an activity in which students reverse-translate proteins from their amino acid sequences back to their DNA sequences then assign musical notes to represent the adenine, guanine, cytosine, and thymine bases. Data is obtained from the National Institutes of Health (NIH) on the Internet. (DDR)

  15. DNA Investigations.

    ERIC Educational Resources Information Center

    Mayo, Ellen S.; Bertino, Anthony J.

    1991-01-01

    Presents a simulation activity that allow students to work through the exercise of DNA profiling and to grapple with some analytical and ethical questions involving a couple arranging with a surrogate mother to have a baby. Can be used to teach the principles of restriction enzyme digestion, gel electrophoresis, and probe hybridization. (MDH)

  16. DNA Methylation

    PubMed Central

    Marinus, M.G.; Løbner-Olesen, A.

    2014-01-01

    The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholerae, Caulobacter crescentus) adenine methylation is essential and in C. crescentus, it is important for temporal gene expression which, in turn, is required for coordinating chromosome initiation, replication and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage; decrease transformation frequency in certain bacteria; decrease the stability of short direct repeats; are necessary for site-directed mutagenesis; and to probe eukaryotic structure and function. PMID:26442938

  17. First-In-Class Small Molecule Inhibitors of the Single-Strand DNA Cytosine Deaminase APOBEC3G

    SciTech Connect

    Li, Ming; Shandilya, Shivender M.D.; Carpenter, Michael A.; Rathore, Anurag; Brown, William L.; Perkins, Angela L.; Harki, Daniel A.; Solberg, Jonathan; Hook, Derek J.; Pandey, Krishan K.; Parniak, Michael A.; Johnson, Jeffrey R.; Krogan, Nevan J.; Somasundaran, Mohan; Ali, Akbar; Schiffer, Celia A.; Harris, Reuben S.

    2012-04-04

    APOBEC3G is a single-stranded DNA cytosine deaminase that comprises part of the innate immune response to viruses and transposons. Although APOBEC3G is the prototype for understanding the larger mammalian polynucleotide deaminase family, no specific chemical inhibitors exist to modulate its activity. High-throughput screening identified 34 compounds that inhibit APOBEC3G catalytic activity. Twenty of 34 small molecules contained catechol moieties, which are known to be sulfhydryl reactive following oxidation to the orthoquinone. Located proximal to the active site, C321 was identified as the binding site for the inhibitors by a combination of mutational screening, structural analysis, and mass spectrometry. Bulkier substitutions C321-to-L, F, Y, or W mimicked chemical inhibition. A strong specificity for APOBEC3G was evident, as most compounds failed to inhibit the related APOBEC3A enzyme or the unrelated enzymes E. coli uracil DNA glycosylase, HIV-1 RNase H, or HIV-1 integrase. Partial, but not complete, sensitivity could be conferred to APOBEC3A by introducing the entire C321 loop from APOBEC3G. Thus, a structural model is presented in which the mechanism of inhibition is both specific and competitive, by binding a pocket adjacent to the APOBEC3G active site, reacting with C321, and blocking access to substrate DNA cytosines.

  18. Mitochondria-targeted Ogg1 and Aconitase-2 Prevent Oxidant-induced Mitochondrial DNA Damage in Alveolar Epithelial Cells*

    PubMed Central

    Kim, Seok-Jo; Cheresh, Paul; Williams, David; Cheng, Yuan; Ridge, Karen; Schumacker, Paul T.; Weitzman, Sigmund; Bohr, Vilhelm A.; Kamp, David W.

    2014-01-01

    Mitochondria-targeted human 8-oxoguanine DNA glycosylase (mt-hOgg1) and aconitase-2 (Aco-2) each reduce oxidant-induced alveolar epithelial cell (AEC) apoptosis, but it is unclear whether protection occurs by preventing AEC mitochondrial DNA (mtDNA) damage. Using quantitative PCR-based measurements of mitochondrial and nuclear DNA damage, mtDNA damage was preferentially noted in AEC after exposure to oxidative stress (e.g. amosite asbestos (5–25 μg/cm2) or H2O2 (100–250 μm)) for 24 h. Overexpression of wild-type mt-hOgg1 or mt-long α/β 317–323 hOgg1 mutant incapable of DNA repair (mt-hOgg1-Mut) each blocked A549 cell oxidant-induced mtDNA damage, mitochondrial p53 translocation, and intrinsic apoptosis as assessed by DNA fragmentation and cleaved caspase-9. In contrast, compared with controls, knockdown of Ogg1 (using Ogg1 shRNA in A549 cells or primary alveolar type 2 cells from ogg1−/− mice) augmented mtDNA lesions and intrinsic apoptosis at base line, and these effects were increased further after exposure to oxidative stress. Notably, overexpression of Aco-2 reduced oxidant-induced mtDNA lesions, mitochondrial p53 translocation, and apoptosis, whereas siRNA for Aco-2 (siAco-2) enhanced mtDNA damage, mitochondrial p53 translocation, and apoptosis. Finally, siAco-2 attenuated the protective effects of mt-hOgg1-Mut but not wild-type mt-hOgg1 against oxidant-induced mtDNA damage and apoptosis. Collectively, these data demonstrate a novel role for mt-hOgg1 and Aco-2 in preserving AEC mtDNA integrity, thereby preventing oxidant-induced mitochondrial dysfunction, p53 mitochondrial translocation, and intrinsic apoptosis. Furthermore, mt-hOgg1 chaperoning of Aco-2 in preventing oxidant-mediated mtDNA damage and apoptosis may afford an innovative target for the molecular events underlying oxidant-induced toxicity. PMID:24429287

  19. Protection from palmitate-induced mitochondrial DNA damage prevents from mitochondrial oxidative stress, mitochondrial dysfunction, apoptosis, and impaired insulin signaling in rat L6 skeletal muscle cells.

    PubMed

    Yuzefovych, Larysa V; Solodushko, Viktoriya A; Wilson, Glenn L; Rachek, Lyudmila I

    2012-01-01

    Saturated free fatty acids have been implicated in the increase of oxidative stress, mitochondrial dysfunction, apoptosis, and insulin resistance seen in type 2 diabetes. The purpose of this study was to determine whether palmitate-induced mitochondrial DNA (mtDNA) damage contributed to increased oxidative stress, mitochondrial dysfunction, apoptosis, impaired insulin signaling, and reduced glucose uptake in skeletal muscle cells. Adenoviral vectors were used to deliver the DNA repair enzyme human 8-oxoguanine DNA glycosylase/(apurinic/apyrimidinic) lyase (hOGG1) to mitochondria in L6 myotubes. After palmitate exposure, we evaluated mtDNA damage, mitochondrial function, production of mitochondrial reactive oxygen species, apoptosis, insulin signaling pathways, and glucose uptake. Protection of mtDNA from palmitate-induced damage by overexpression of hOGG1 targeted to mitochondria significantly diminished palmitate-induced mitochondrial superoxide production, restored the decline in ATP levels, reduced activation of c-Jun N-terminal kinase (JNK) kinase, prevented cells from entering apoptosis, increased insulin-stimulated phosphorylation of serine-threonine kinase (Akt) (Ser473) and tyrosine phosphorylation of insulin receptor substrate-1, and thereby enhanced glucose transporter 4 translocation to plasma membrane, and restored insulin signaling. Addition of a specific inhibitor of JNK mimicked the effect of mitochondrial overexpression of hOGG1 and partially restored insulin sensitivity, thus confirming the involvement of mtDNA damage and subsequent increase of oxidative stress and JNK activation in insulin signaling in L6 myotubes. Our results are the first to report that mtDNA damage is the proximal cause in palmitate-induced mitochondrial dysfunction and impaired insulin signaling and provide strong evidence that targeting DNA repair enzymes into mitochondria in skeletal muscles could be a potential therapeutic treatment for insulin resistance. PMID:22128025

  20. The eucalyptus oil ingredient 1,8-cineol induces oxidative DNA damage.

    PubMed

    Dörsam, Bastian; Wu, Ching-Fen; Efferth, Thomas; Kaina, Bernd; Fahrer, Jörg

    2015-05-01

    The natural compound 1,8-cineol, also known as eucalyptol, is a major constituent of eucalyptus oil. This epoxy-monoterpene is used as flavor and fragrance in consumer goods as well as medical therapies. Due to its anti-inflammatory properties, 1,8-cineol is also applied to treat upper and lower airway diseases. Despite its widespread use, only little is known about the genotoxicity of 1,8-cineol in mammalian cells. This study investigates the genotoxicity and cytotoxicity of 1,8-cineol in human and hamster cells. First, we observed a significant and concentration-dependent increase in oxidative DNA damage in human colon cancer cells, as detected by the Formamidopyrimidine-DNA glycosylase (Fpg)-modified alkaline comet assay. Pre-treatment of cells with the antioxidant N-acetylcysteine prevented the formation of Fpg-sensitive sites after 1,8-cineol treatment, supporting the notion that 1,8-cineol induces oxidative DNA damage. In the dose range of DNA damage induction, 1,8-cineol did neither reduce the viability of colon cancer cells nor affected their cell cycle distribution, suggesting that cells tolerate 1,8-cineol-induced oxidative DNA damage by engaging DNA repair. To test this hypothesis, hamster cell lines with defects in BRCA2 and Rad51, which are essentials players of homologous recombination (HR)-mediated repair, were treated with 1,8-cineol. The monoterpene induced oxidative DNA damage and subsequent DNA double-strand breaks in the hamster cell lines tested. Intriguingly, we detected a significant concentration-dependent decrease in viability of the HR-defective cells, whereas the corresponding wild-type cell lines with functional HR were not affected. Based on these findings, we conclude that 1,8-cineol is weakly genotoxic, inducing primarily oxidative DNA damage, which is most likely tolerated in DNA repair proficient cells without resulting in cell cycle arrest and cell death. However, cells with deficiency in HR were compromised after 1,8-cineol

  1. Influence of DNA torsional rigidity on excision of 7,8-dihydro-8-oxo-2′-deoxyguanosine in the presence of opposing abasic sites by human OGG1 protein

    PubMed Central

    Barone, F.; Dogliotti, E.; Cellai, L.; Giordano, C.; Bjørås, M.; Mazzei, F.

    2003-01-01

    The human protein OGG1 (hOGG1) targets the highly mutagenic base 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) and shows a high specificity for the opposite DNA base. Abasic sites can arise in DNA in close opposition to 8-oxodG either during repair of mismatched bases (i.e. 8-oxodG/A mismatches) or, more frequently, as a consequence of ionizing radiation exposure. Bistranded DNA lesions may remain unrepaired and lead to cell death via double-strand break formation. In order to explore the role of damaged-DNA dynamics in recognition/excision by the hOGG1 repair protein, specific oligonucleotides containing an 8-oxodG opposite an abasic site, at different relative distances on the complementary strand, were synthesized. Rotational dynamics were studied by means of fluorescence polarization anisotropy decay experiments and the torsional elastic constant as well as the hydrodynamic radius of the DNA fragments were evaluated. Efficiency of excision of 8-oxodG was tested using purified human glycosylase. A close relation between the twisting flexibility of the DNA fragment and the excision efficiency of the oxidative damage by hOGG1 protein within a cluster was found. PMID:12655006

  2. Role of Bacillus subtilis Error Prevention Oxidized Guanine System in Counteracting Hexavalent Chromium-Promoted Oxidative DNA Damage

    PubMed Central

    Santos-Escobar, Fernando; Gutiérrez-Corona, J. Félix

    2014-01-01

    Chromium pollution is potentially detrimental to bacterial soil communities, compromising carbon and nitrogen cycles that are essential for life on earth. It has been proposed that intracellular reduction of hexavalent chromium [Cr(VI)] to trivalent chromium [Cr(III)] may cause bacterial death by a mechanism that involves reactive oxygen species (ROS)-induced DNA damage; the molecular basis of the phenomenon was investigated in this work. Here, we report that Bacillus subtilis cells lacking a functional error prevention oxidized guanine (GO) system were significantly more sensitive to Cr(VI) treatment than cells of the wild-type (WT) strain, suggesting that oxidative damage to DNA is involved in the deleterious effects of the oxyanion. In agreement with this suggestion, Cr(VI) dramatically increased the ROS concentration and induced mutagenesis in a GO-deficient B. subtilis strain. Alkaline gel electrophoresis (AGE) analysis of chromosomal DNA of WT and ΔGO mutant strains subjected to Cr(VI) treatment revealed that the DNA of the ΔGO strain was more susceptible to DNA glycosylase Fpg attack, suggesting that chromium genotoxicity is associated with 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-G) lesions. In support of this notion, specific monoclonal antibodies detected the accumulation of 8-oxo-G lesions in the chromosomes of B. subtilis cells subjected to Cr(VI) treatment. We conclude that Cr(VI) promotes mutagenesis and cell death in B. subtilis by a mechanism that involves radical oxygen attack of DNA, generating 8-oxo-G, and that such effects are counteracted by the prevention and repair GO system. PMID:24973075

  3. Role of Bacillus subtilis error prevention oxidized guanine system in counteracting hexavalent chromium-promoted oxidative DNA damage.

    PubMed

    Santos-Escobar, Fernando; Gutiérrez-Corona, J Félix; Pedraza-Reyes, Mario

    2014-09-01

    Chromium pollution is potentially detrimental to bacterial soil communities, compromising carbon and nitrogen cycles that are essential for life on earth. It has been proposed that intracellular reduction of hexavalent chromium [Cr(VI)] to trivalent chromium [Cr(III)] may cause bacterial death by a mechanism that involves reactive oxygen species (ROS)-induced DNA damage; the molecular basis of the phenomenon was investigated in this work. Here, we report that Bacillus subtilis cells lacking a functional error prevention oxidized guanine (GO) system were significantly more sensitive to Cr(VI) treatment than cells of the wild-type (WT) strain, suggesting that oxidative damage to DNA is involved in the deleterious effects of the oxyanion. In agreement with this suggestion, Cr(VI) dramatically increased the ROS concentration and induced mutagenesis in a GO-deficient B. subtilis strain. Alkaline gel electrophoresis (AGE) analysis of chromosomal DNA of WT and ΔGO mutant strains subjected to Cr(VI) treatment revealed that the DNA of the ΔGO strain was more susceptible to DNA glycosylase Fpg attack, suggesting that chromium genotoxicity is associated with 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-G) lesions. In support of this notion, specific monoclonal antibodies detected the accumulation of 8-oxo-G lesions in the chromosomes of B. subtilis cells subjected to Cr(VI) treatment. We conclude that Cr(VI) promotes mutagenesis and cell death in B. subtilis by a mechanism that involves radical oxygen attack of DNA, generating 8-oxo-G, and that such effects are counteracted by the prevention and repair GO system. PMID:24973075

  4. Porphyromonas gingivalis mutY is involved in the repair of oxidative stress-induced DNA mispairing.

    PubMed

    Robles, A G; Reid, K; Roy, F; Fletcher, H M

    2011-06-01

    The ability for DNA mismatch repair, after oxidative stress-induced DNA damage, is critical for the persistence of Porphyromonas gingivalis in the inflammatory environment of the periodontal pocket. Our previous report demonstrated that, in contrast to other organisms, the repair of oxidative stress-induced DNA damage involving 8-oxo-7,8-dihydroguanine (8-oxoG) may occur by a yet-to-be described mechanism in P. gingivalis. 8-oxoG does not block DNA replication; rather, it mispairs with adenine, which can be repaired by the MutY glycosylase. To determine the function of the P. gingivalis MutY homologue in DNA repair, it was insertionally inactivated using the ermF-ermAM antibiotic cassette and used to create a mutY-deficient mutant (FLL147) by allelic exchange mutagenesis. FLL147 had an increased rate of spontaneous mutation and was more sensitive to hydrogen peroxide compared with the wild-type W83 strain. DNA oligomers containing a site-specific 8-oxoG:A mispair was repaired similarly in both the P. gingivalis mutY-defective mutant and wild-type strains. The P. gingivalis mutY homologue was shown to complement the mutY mutation in Escherichia coli. In a gel mobility shift assay, the purified recombinant MutY is able to bind an oligo containing an 8-oxoG:A mispair. Taken together, MutY may play the expected role in oxidative stress resistance in P. gingivalis. However, there may exist other redundant mechanism(s) for the removal of 8-oxoG:A mismatch in this organism. PMID:21545695

  5. DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Nguyen, C.; Gidrol, X.

    Genomics has revolutionised biological and biomedical research. This revolution was predictable on the basis of its two driving forces: the ever increasing availability of genome sequences and the development of new technology able to exploit them. Up until now, technical limitations meant that molecular biology could only analyse one or two parameters per experiment, providing relatively little information compared with the great complexity of the systems under investigation. This gene by gene approach is inadequate to understand biological systems containing several thousand genes. It is essential to have an overall view of the DNA, RNA, and relevant proteins. A simple inventory of the genome is not sufficient to understand the functions of the genes, or indeed the way that cells and organisms work. For this purpose, functional studies based on whole genomes are needed. Among these new large-scale methods of molecular analysis, DNA microarrays provide a way of studying the genome and the transcriptome. The idea of integrating a large amount of data derived from a support with very small area has led biologists to call these chips, borrowing the term from the microelectronics industry. At the beginning of the 1990s, the development of DNA chips on nylon membranes [1, 2], then on glass [3] and silicon [4] supports, made it possible for the first time to carry out simultaneous measurements of the equilibrium concentration of all the messenger RNA (mRNA) or transcribed RNA in a cell. These microarrays offer a wide range of applications, in both fundamental and clinical research, providing a method for genome-wide characterisation of changes occurring within a cell or tissue, as for example in polymorphism studies, detection of mutations, and quantitative assays of gene copies. With regard to the transcriptome, it provides a way of characterising differentially expressed genes, profiling given biological states, and identifying regulatory channels.

  6. Wrinkled DNA.

    PubMed Central

    Arnott, S; Chandrasekaran, R; Puigjaner, L C; Walker, J K; Hall, I H; Birdsall, D L; Ratliff, R L

    1983-01-01

    The B form of poly d(GC):poly d(GC) in orthorhombic microcrystallites in oriented fibers has a secondary structure in which a dinucleotide is the repeated motif rather than a mononucleotide as in standard, smooth B DNA. One set of nucleotides (probably GpC) has the same conformations as the smooth form but the alternate (CpG) nucleotides have a different conformation at C3'-O3'. This leads to a distinctive change in the orientation of the phosphate groups. Similar perturbations can be detected in other poly d(PuPy):poly d(PuPy) DNAs such as poly d(IC):poly d(IC) and poly d(AT):poly d(AT) in their D forms which have tetragonal crystal environments. This suggests that such perturbations are intrinsic to all stretches of duplex DNA where purines and pyrimidines alternate and may play a role in the detection and exploitation of such sequences by regulatory proteins. Images PMID:6572358

  7. Optical DNA

    NASA Astrophysics Data System (ADS)

    Vijaywargi, Deepak; Lewis, Dave; Kirovski, Darko

    A certificate of authenticity (COA) is an inexpensive physical object with a random and unique structure S which is hard to near-exactly replicate. An inexpensive device should be able to scan object’s physical “fingerprint,” a set of features that represents S. In this paper, we explore one set of requirements that optical media such as DVDs should satisfy, to be considered as COAs. As manufacturing of such media produces inevitable errors, we use the locations and count of these errors as a “fingerprint” for each optical disc: its optical DNA. The “fingerprint” is signed using publisher’s private-key and the resulting signature is stored onto the optical medium using a post-production process. Standard DVD players with altered firmware that includes publisher’s public-key, should be able to verify the authenticity of DVDs protected with optical DNA. Our key finding is that for the proposed protocol, only DVDs with exceptional wear-and-tear characteristics would result in an inexpensive and viable anti-counterfeiting technology.

  8. Isolated and clustered DNA lesions induced by high-energy iron and carbon ions

    NASA Astrophysics Data System (ADS)

    Ide, H.; Tanaka, R.; Nakaarai, Y.; Terato, H.; Furusawa, Y.

    total clustered damage was 39% (gamma), 66% (C), and 68% (Fe). The overall yield of clustered damage (DSB + clustered base lesions) that may be pertinent to cell killing and mutations was decreased with increasing LET [relative yield: 1 (gamma) > 0.77 (C) > 0.69 (Fe)]. This result is in contrast to the higher biological effectiveness (e.g. cell killing) of high-energy Fe and C ions than gamma-rays, suggesting a role of more complex damage clusters that cannot be distinguished by simple analysis of direct and DNA glycosylase-induced DSB.

  9. Methanol exposure does not produce oxidatively damaged DNA in lung, liver or kidney of adult mice, rabbits or primates

    SciTech Connect

    McCallum, Gordon P.; Siu, Michelle; Sweeting, J. Nicole; Wells, Peter G.

    2011-01-15

    In vitro and in vivo genotoxicity tests indicate methanol (MeOH) is not mutagenic, but carcinogenic potential has been claimed in one controversial long-term rodent cancer bioassay that has not been replicated. To determine whether MeOH could indirectly damage DNA via reactive oxygen species (ROS)-mediated mechanisms, we treated male CD-1 mice, New Zealand white rabbits and cynomolgus monkeys with MeOH (2.0 g/kg ip) and 6 h later assessed oxidative damage to DNA, measured as 8-oxo-2'-deoxyguanosine (8-oxodG) by HPLC with electrochemical detection. We found no MeOH-dependent increases in 8-oxodG in lung, liver or kidney of any species. Chronic treatment of CD-1 mice with MeOH (2.0 g/kg ip) daily for 15 days also did not increase 8-oxodG levels in these organs. These results were corroborated in DNA repair-deficient oxoguanine glycosylase 1 (Ogg1) knockout (KO) mice, which accumulated 8-oxodG in lung, kidney and liver with age, but exhibited no increase following MeOH, despite a 2-fold increase in renal 8-oxodG in Ogg1 KO mice following treatment with a ROS-initiating positive control, the renal carcinogen potassium bromate (KBrO{sub 3}; 100 mg/kg ip). These observations suggest that MeOH exposure does not promote the accumulation of oxidatively damaged DNA in lung, kidney or liver, and that environmental exposure to MeOH is unlikely to initiate carcinogenesis in these organs by DNA oxidation.

  10. Ebselen attenuates oxidative DNA damage and enhances its repair activity in the thalamus after focal cortical infarction in hypertensive rats.

    PubMed

    He, Meixia; Xing, Shihui; Yang, Bo; Zhao, Liqun; Hua, Haiying; Liang, Zhijian; Zhou, Wenliang; Zeng, Jinsheng; Pei, Zhong

    2007-11-21

    Oxidative DNA damage has been proposed to be a major contributor to focal cerebral ischemic injury. However, little is known about the role of oxidative DNA damage in remote damage secondary to the primary infarction. In the present study, we investigated oxidative damage within the ventroposterior nucleus (VPN) after distal middle cerebral artery occlusion (MCAO) in hypertensive rats. We also examined the possible protective effect of ebselen, one glutathione peroxidase mimic, on delayed degeneration in the VPN after distal MCAO. Neuronal damage in the ipsilateral VPN was examined by Nissl staining. Oxidative DNA damage and base repair enzyme activity were assessed by analyzing immunoreactivity of 8-hydroxy-2'-deoxyguanosine (8-ohdG) and 8-oxoguanine DNA glycosylase (OGG1), respectively. The number of intact neurons in the ipsilateral VPN decreased by 52% compared to the contralateral side in ischemia group 2 weeks after distal cerebral cortical infarction. The immunoreactivity of 8-ohdG significantly increased while OGG1 immunoreactivity significantly decreased in the ipsilateral VPN 2 weeks after distal cortical infarction (all p<0.01). Compared with vehicle treatment, ebselen significantly attenuated the neuron loss, ameliorated ischemia-induced increase in 8-ohdG level as well as decrease in OGG1 level within the ipsilateral VPN (all p<0.01). OGG1 was further demonstrated to mainly express in neurons. These findings strongly suggest that oxidative DNA damage may be involved in the delayed neuronal death in the VPN region following distal MCAO. Furthermore, ebselen protects against the delayed damage in the VPN when given at 24 h following distal MCAO. PMID:17920569

  11. Benzene-derived N2-(4-hydroxyphenyl)-deoxyguanosine adduct: UvrABC incision and its conformation in DNA

    SciTech Connect

    Hang, Bo; Rodriguez, Ben; Yang, Yanu; Guliaev, Anton B.; Chenna, Ahmed

    2010-06-14

    Benzene, a ubiquitous human carcinogen, forms DNA adducts through its metabolites such as p-benzoquinone (p-BQ) and hydroquinone (HQ). N(2)-(4-Hydroxyphenyl)-2'-deoxyguanosine (N(2)-4-HOPh-dG) is the principal adduct identified in vivo by (32)P-postlabeling in cells or animals treated with p-BQ or HQ. To study its effect on repair specificity and replication fidelity, we recently synthesized defined oligonucleotides containing a site-specific adduct using phosphoramidite chemistry. We here report the repair of this adduct by Escherichia coli UvrABC complex, which performs the initial damage recognition and incision steps in the nucleotide excision repair (NER) pathway. We first showed that the p-BQ-treated plasmid was efficiently cleaved by the complex, indicating the formation of DNA lesions that are substrates for NER. Using a 40-mer substrate, we found that UvrABC incises the DNA strand containing N(2)-4-HOPh-dG in a dose- and time-dependent manner. The specificity of such repair was also compared with that of DNA glycosylases and damage-specific endonucleases of E. coli, both of which were found to have no detectable activity toward N(2)-4-HOPh-dG. To understand why this adduct is specifically recognized and processed by UvrABC, molecular modeling studies were performed. Analysis of molecular dynamics trajectories showed that stable G:C-like hydrogen bonding patterns of all three Watson-Crick hydrogen bonds are present within the N(2)-4-HOPh-G:C base pair, with the hydroxyphenyl ring at an almost planar position. In addition, N(2)-4-HOPh-dG has a tendency to form more stable stacking interactions than a normal G in B-type DNA. These conformational properties may be critical in differential recognition of this adduct by specific repair enzymes.

  12. DNA mimicry by proteins.

    PubMed

    Dryden, D T F; Tock, M R

    2006-04-01

    It has been discovered recently, via structural and biophysical analyses, that proteins can mimic DNA structures in order to inhibit proteins that would normally bind to DNA. Mimicry of the phosphate backbone of DNA, the hydrogen-bonding properties of the nucleotide bases and the bending and twisting of the DNA double helix are all present in the mimics discovered to date. These mimics target a range of proteins and enzymes such as DNA restriction enzymes, DNA repair enzymes, DNA gyrase and nucleosomal and nucleoid-associated proteins. The unusual properties of these protein DNA mimics may provide a foundation for the design of targeted inhibitors of DNA-binding proteins. PMID:16545103

  13. Chemical repair of base lesions, AP-sites, and strand breaks on plasmid DNA in dilute aqueous solution by ascorbic acid

    SciTech Connect

    Hata, Kuniki; Urushibara, Ayumi; Yamashita, Shinichi; Shikazono, Naoya; Yokoya, Akinari; Katsumura, Yosuke

    2013-05-03

    Highlights: •We report a novel mechanism of radiation protection of DNA by chemical activity of ascorbic acid. •The “chemical repair” of DNA damage was revealed using biochemical assay and chemical kinetics analysis. •We found that ascorbic acid significantly repairs precursors of nucleobase lesions and abasic sites. •However, ascorbic acid seldom repairs precursors of DNA-strand breaks. -- Abstract: We quantified the damage yields produced in plasmid DNA by γ-irradiation in the presence of low concentrations (10–100 μM) of ascorbic acid, which is a major antioxidant in living systems, to clarify whether it chemically repairs radiation damage in DNA. The yield of DNA single strand breaks induced by irradiation was analyzed with agarose gel electrophoresis as conformational changes in closed circular plasmids. Base lesions and abasic sites were also observed as additional conformational changes by treating irradiated samples with glycosylase proteins. By comparing the suppression efficiencies to the induction of each DNA lesion, in addition to scavenging of the OH radicals derived from water radiolysis, it was found that ascorbic acid promotes the chemical repair of precursors of AP-sites and base lesions more effectively than those of single strand breaks. We estimated the efficiency of the chemical repair of each lesion using a kinetic model. Approximately 50–60% of base lesions and AP-sites were repaired by 10 μM ascorbic acid, although strand breaks were largely unrepaired by ascorbic acid at low concentrations. The methods in this study will provide a route to understanding the mechanistic aspects of antioxidant activity in living systems.

  14. The LSH/DDM1 Homolog MUS-30 Is Required for Genome Stability, but Not for DNA Methylation in Neurospora crassa

    PubMed Central

    Basenko, Evelina Y.; Kamei, Masayuki; Ji, Lexiang; Schmitz, Robert J.; Lewis, Zachary A.

    2016-01-01

    LSH/DDM1 enzymes are required for DNA methylation in higher eukaryotes and have poorly defined roles in genome maintenance in yeast, plants, and animals. The filamentous fungus Neurospora crassa is a tractable system that encodes a single LSH/DDM1 homolog (NCU06306). We report that the Neurospora LSH/DDM1 enzyme is encoded by mutagen sensitive-30 (mus-30), a locus identified in a genetic screen over 25 years ago. We show that MUS-30-deficient cells have normal DNA methylation, but are hypersensitive to DNA damaging agents. MUS-30 is a nuclear protein, consistent with its predicted role as a chromatin remodeling enzyme, and levels of MUS-30 are increased following DNA damage. MUS-30 co-purifies with Neurospora WDR76, a homolog of yeast Changed Mutation Rate-1 and mammalian WD40 repeat domain 76. Deletion of wdr76 rescued DNA damage-hypersensitivity of Δmus-30 strains, demonstrating that the MUS-30-WDR76 interaction is functionally important. DNA damage-sensitivity of Δmus-30 is partially suppressed by deletion of methyl adenine glycosylase-1, a component of the base excision repair machinery (BER); however, the rate of BER is not affected in Δmus-30 strains. We found that MUS-30-deficient cells are not defective for DSB repair, and we observed a negative genetic interaction between Δmus-30 and Δmei-3, the Neurospora RAD51 homolog required for homologous recombination. Together, our findings suggest that MUS-30, an LSH/DDM1 homolog, is required to prevent DNA damage arising from toxic base excision repair intermediates. Overall, our study provides important new information about the functions of the LSH/DDM1 family of enzymes. PMID:26771905

  15. Low-level laser irradiation alters mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts

    NASA Astrophysics Data System (ADS)

    Trajano, L. A. S. N.; Sergio, L. P. S.; Silva, C. L.; Carvalho, L.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

    2016-07-01

    Low-level lasers are used for the treatment of diseases in soft and bone tissues, but few data are available regarding their effects on genomic stability. In this study, we investigated mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts exposed to low-level infrared laser. C2C12 myoblast cultures in different fetal bovine serum concentrations were exposed to low-level infrared laser (10, 35 and 70 J cm‑2), and collected for the evaluation of DNA repair gene expression. Laser exposure increased gene expression related to base excision repair (8-oxoguanine DNA glycosylase and apurinic/apyrimidinic endonuclease 1), nucleotide excision repair (excision repair cross-complementation group 1 and xeroderma pigmentosum C protein) and genomic stabilization (ATM serine/threonine kinase and tumor protein p53) in normal and low fetal bovine serum concentrations. Results suggest that genomic stability could be part of a biostimulation effect of low-level laser therapy in injured muscles.

  16. Mitochondrial DNA replacement versus nuclear DNA persistence

    NASA Astrophysics Data System (ADS)

    Serva, Maurizio

    2006-10-01

    In this paper we consider two populations whose generations are not overlapping and whose size is large. The number of males and females in both populations is constant. Any generation is replaced by a new one and any individual has two parents concerning nuclear DNA and a single one (the mother) concerning mtDNA. Moreover, at any generation some individuals migrate from the first population to the second. In a finite random time T, the mtDNA of the second population is completely replaced by the mtDNA of the first. In the same time, the nuclear DNA is not completely replaced and a fraction F of the ancient nuclear DNA persists. We compute both T and F. Since this study shows that complete replacement of mtDNA in a population is compatible with the persistence of a large fraction of nuclear DNA, it may have some relevance for the 'out of Africa'/multiregional debate in palaeoanthropology.

  17. DNA modifications: Another stable base in DNA

    NASA Astrophysics Data System (ADS)

    Brazauskas, Pijus; Kriaucionis, Skirmantas

    2014-12-01

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

  18. Synthesis of DNA

    DOEpatents

    Mariella, Jr., Raymond P.

    2008-11-18

    A method of synthesizing a desired double-stranded DNA of a predetermined length and of a predetermined sequence. Preselected sequence segments that will complete the desired double-stranded DNA are determined. Preselected segment sequences of DNA that will be used to complete the desired double-stranded DNA are provided. The preselected segment sequences of DNA are assembled to produce the desired double-stranded DNA.

  19. Sperm DNA oxidative damage and DNA adducts.

    PubMed

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

    2015-12-01

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

  20. Establishment of a non-radioactive cleavage assay to assess the DNA repair capacity towards oxidatively damaged DNA in subcellular and cellular systems and the impact of copper.

    PubMed

    Hamann, Ingrit; Schwerdtle, Tanja; Hartwig, Andrea

    2009-10-01

    Oxidative stress is involved in many diseases, and the search for appropriate biomarkers is one major focus in molecular epidemiology. 8-Oxoguanine (8-oxoG), a potentially mutagenic DNA lesion, is considered to be a sensitive biomarker for oxidative stress. Another approach consists in assessing the repair capacity towards 8-oxoG, mediated predominantly by the human 8-oxoguanine DNA glycosylase 1 (hOGG1). With respect to the latter, during the last few years so-called cleavage assays have been described, investigating the incision of (32)P-labelled and 8-oxoG damaged oligonucleotides by cell extracts. Within the present study, a sensitive non-radioactive test system based on a Cy5-labelled oligonucleotide has been established. Sources of incision activity are isolated proteins or extracts prepared from cultured cells and peripheral blood mononuclear cells (PBMC). After comparing different oligonucleotide structures, a hairpin-like structure was selected which was not degraded by cell extracts. Applying this test system the impact of copper on the activity of isolated hOGG1 and on hOGG activity in A549 cells was examined, showing a distinct inhibition of the isolated protein at low copper concentration as compared to a modest inhibition of hOGG activity in cells at beginning cytotoxic concentrations. For investigating PBMC, all reaction conditions, including the amounts of oligonucleotide and cell extract as well as the reaction time have been optimized. The incision activities of PBMC protein extracts obtained from different donors have been investigated, and inter-individual differences have been observed. In summary, the established method is as sensitive and even faster than the radioactive technique, and additionally, offers the advantage of reduced costs and low health risk. PMID:19505484

  1. DNA damage in wounded, hypoxic and acidotic human skin fibroblast cell cultures after low laser irradiation

    NASA Astrophysics Data System (ADS)

    Hawkins Evans, D.; Mbene, A.; Zungu, I.; Houreld, N.; Abrahamse, H.

    2009-02-01

    Phototherapy has become more popular and widely used in the treatment of a variety of medical conditions. To ensure sound results as evidence of its effectiveness, well designed experiments must be conducted when determining the effect of phototherapy. Cell culture models such as hypoxic, acidotic and wounded cell cultures simulating different disease conditions including ischemic heart disease, diabetes and wound healing were used to determine the effect of laser irradiation on the genetic integrity of the cell. Even though phototherapy has been found to be beneficial in a wide spectrum of conditions, it has been shown to induce DNA damage. However, this damage appears to be repairable. The risk lies in the fact that phototherapy may help the medical condition initially but damage DNA at the same time leaving undetected damage that may result in late onset, more severe, induced medical conditions including cancer. Human skin fibroblasts were cultured and used to induce a wound (by the central scratch model), hypoxic (by incubation in an anaerobic jar, 95% N2 and 5% O2) and acidotic (reducing the pH of the media to 6.7) conditions. Different models were irradiated using a Helium-Neon (632.8 nm) laser with a power density of 2.07 mW/cm2 and a fluence of 5 J/cm2 or 16 J/cm2. The effect of the irradiation was determined using the Comet assay 1 and 24 h after irradiation. In addition, the Comet assay was performed with the addition of formamidopyrimidine glycosylase (FPG) obviating strand brakes in oxidized bases at a high fluence of 16 J/cm2. A significant increase in DNA damage was seen in all three injured models at both 1 and 24 h post-irradiation when compared to the normal un-injured cells. However, when compared to non-irradiated controls the acidotic model showed a significant decrease in DNA damage 24 h after irradiation indicating the possible induction of cellular DNA repair mechanisms. When wounded cells were irradiated with higher fluences of 16 J/cm2

  2. DNA encoding a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-08-15

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  3. DNA polymerases and cancer

    PubMed Central

    Lange, Sabine S.; Takata, Kei-ichi; Wood, Richard D.

    2013-01-01

    There are fifteen different DNA polymerases encoded in mammalian genomes, which are specialized for replication, repair or the tolerance of DNA damage. New evidence is emerging for lesion-specific and tissue-specific functions of DNA polymerases. Many point mutations that occur in cancer cells arise from the error-generating activities of DNA polymerases. However, the ability of some of these enzymes to bypass DNA damage may actually defend against chromosome instability in cells and at least one DNA polymerase, POLζ, is a suppressor of spontaneous tumorigenesis. Because DNA polymerases can help cancer cells tolerate DNA damage, some of these enzymes may be viable targets for therapeutic strategies. PMID:21258395

  4. Genetic analysis of repair and damage tolerance mechanisms for DNA-protein cross-links in Escherichia coli.

    PubMed

    Salem, Amir M H; Nakano, Toshiaki; Takuwa, Minako; Matoba, Nagisa; Tsuboi, Tomohiro; Terato, Hiroaki; Yamamoto, Kazuo; Yamada, Masami; Nohmi, Takehiko; Ide, Hiroshi

    2009-09-01

    DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. We have recently shown that nucleotide excision repair (NER) and RecBCD-dependent homologous recombination (HR) collaboratively alleviate the lethal effect of DPCs in Escherichia coli. In this study, to gain further insight into the damage-processing mechanism for DPCs, we assessed the sensitivities of a panel of repair-deficient E. coli mutants to DPC-inducing agents, including formaldehyde (FA) and 5-azacytidine (azaC). We show here that the damage tolerance mechanism involving HR and subsequent replication restart (RR) provides the most effective means of cell survival against DPCs. Translesion synthesis does not serve as an alternative damage tolerance mechanism for DPCs in cell survival. Elimination of DPCs from the genome relies primarily on NER, which provides a second and moderately effective means of cell survival against DPCs. Interestingly, Cho rather than UvrC seems to be an effective nuclease for the NER of DPCs. Together with the genes responsible for HR, RR, and NER, the mutation of genes involved in several aspects of DNA repair and transactions, such as recQ, xth nfo, dksA, and topA, rendered cells slightly but significantly sensitive to FA but not azaC, possibly reflecting the complexity of DPCs or cryptic lesions induced by FA. UvrD may have an additional role outside NER, since the uvrD mutation conferred a slight azaC sensitivity on cells. Finally, DNA glycosylases mitigate azaC toxicity, independently of the repair of DPCs, presumably by removing 5-azacytosine or its degradation product from the chromosome. PMID:19617358

  5. DNA Nanotechnology-- Architectures Designed with DNA

    NASA Astrophysics Data System (ADS)

    Han, Dongran

    As the genetic information storage vehicle, deoxyribonucleic acid (DNA) molecules are essential to all known living organisms and many viruses. It is amazing that such a large amount of information about how life develops can be stored in these tiny molecules. Countless scientists, especially some biologists, are trying to decipher the genetic information stored in these captivating molecules. Meanwhile, another group of researchers, nanotechnologists in particular, have discovered that the unique and concise structural features of DNA together with its information coding ability can be utilized for nano-construction efforts. This idea culminated in the birth of the field of DNA nanotechnology which is the main topic of this dissertation. The ability of rationally designed DNA strands to self-assemble into arbitrary nanostructures without external direction is the basis of this field. A series of novel design principles for DNA nanotechnology are presented here, from topological DNA nanostructures to complex and curved DNA nanostructures, from pure DNA nanostructures to hybrid RNA/DNA nanostructures. As one of the most important and pioneering fields in controlling the assembly of materials (both DNA and other materials) at the nanoscale, DNA nanotechnology is developing at a dramatic speed and as more and more construction approaches are invented, exciting advances will emerge in ways that we may or may not predict.

  6. No oxidative stress or DNA damage in peripheral blood mononuclear cells after exposure to particles from urban street air in overweight elderly

    PubMed Central

    Hemmingsen, Jette Gjerke; Jantzen, Kim; Møller, Peter; Loft, Steffen

    2015-01-01

    Exposure to traffic-related particulate matter (PM) has been associated with increased risk of lung disease, cancer and cardiovascular disease especially in elderly and overweight subjects. The proposed mechanisms involve intracellular production of reactive oxygen species (ROS), inflammation and oxidation-induced DNA damage studied mainly in young normal-weight subjects. We performed a controlled cross-over, randomised, single-blinded, repeated-measure study where 60 healthy subjects (25 males and 35 females) with age 55–83 years and body mass index above 25kg/m2 were exposed for 5h to either particle-filtered or sham-filtered air from a busy street with number of concentrations and PM2.5 levels of 1800/cm3 versus 23 000/cm3 and 3 µg/m3 versus 24 µg/m3, respectively. Peripheral blood mononuclear cells (PBMCs) were collected and assayed for production of ROS with and without ex vivo exposure to nanosized carbon black as well as expression of genes related to inflammation (chemokine (C-C motif) ligand 2, interleukin-8 and tumour necrosis factor), oxidative stress response (heme oxygenase (decycling)-1) and DNA repair (oxoguanine DNA glycosylase). DNA strand breaks and oxidised purines were assayed by the alkaline comet assay. No statistically significant differences were found for any biomarker immediately after exposure to PM from urban street air although strand breaks and oxidised purines combined were significantly associated with the particle number concentration during exposure. In conclusion, 5h of controlled exposure to PM from urban traffic did not change the gene expression related to inflammation, oxidative stress or DNA repair, ROS production or oxidatively damaged DNA in PBMCs from elderly overweight human subjects. PMID:25904586

  7. Characterization of Family IV UDG from Aeropyrum pernix and Its Application in Hot-Start PCR by Family B DNA Polymerase

    PubMed Central

    Liu, Xi-Peng; Liu, Jian-Hua

    2011-01-01

    Recombinant uracil-DNA glycosylase (UDG) from Aeropyrum pernix (A. pernix) was expressed in E. coli. The biochemical characteristics of A. pernix UDG (ApeUDG) were studied using oligonucleotides carrying a deoxyuracil (dU) base. The optimal temperature range and pH value for dU removal by ApeUDG were 55–65°C and pH 9.0, respectively. The removal of dU was inhibited by the divalent ions of Zn, Cu, Co, Ni, and Mn, as well as a high concentration of NaCl. The opposite base in the complementary strand affected the dU removal by ApeUDG as follows: U/C≈U/G>U/T≈U/AP≈U/->U/U≈U/I>U/A. The phosphorothioate around dU strongly inhibited dU removal by ApeUDG. Based on the above biochemical characteristics and the conservation of amino acid residues, ApeUDG was determined to belong to the IV UDG family. ApeUDG increased the yield of PCR by Pfu DNA polymerase via the removal of dU in amplified DNA. Using the dU-carrying oligonucleotide as an inhibitor and ApeUDG as an activator of Pfu DNA polymerase, the yield of undesired DNA fragments, such as primer-dimer, was significantly decreased, and the yield of the PCR target fragment was increased. This strategy, which aims to amplify the target gene with high specificity and yield, can be applied to all family B DNA polymerases. PMID:22087273

  8. No oxidative stress or DNA damage in peripheral blood mononuclear cells after exposure to particles from urban street air in overweight elderly.

    PubMed

    Hemmingsen, Jette Gjerke; Jantzen, Kim; Møller, Peter; Loft, Steffen

    2015-09-01

    Exposure to traffic-related particulate matter (PM) has been associated with increased risk of lung disease, cancer and cardiovascular disease especially in elderly and overweight subjects. The proposed mechanisms involve intracellular production of reactive oxygen species (ROS), inflammation and oxidation-induced DNA damage studied mainly in young normal-weight subjects. We performed a controlled cross-over, randomised, single-blinded, repeated-measure study where 60 healthy subjects (25 males and 35 females) with age 55-83 years and body mass index above 25 kg/m(2) were exposed for 5h to either particle-filtered or sham-filtered air from a busy street with number of concentrations and PM2.5 levels of 1800/cm(3) versus 23 000/cm(3) and 3 µg/m(3) versus 24 µg/m(3), respectively. Peripheral blood mononuclear cells (PBMCs) were collected and assayed for production of ROS with and without ex vivo exposure to nanosized carbon black as well as expression of genes related to inflammation (chemokine (C-C motif) ligand 2, interleukin-8 and tumour necrosis factor), oxidative stress response (heme oxygenase (decycling)-1) and DNA repair (oxoguanine DNA glycosylase). DNA strand breaks and oxidised purines were assayed by the alkaline comet assay. No statistically significant differences were found for any biomarker immediately after exposure to PM from urban street air although strand breaks and oxidised purines combined were significantly associated with the particle number concentration during exposure. In conclusion, 5h of controlled exposure to PM from urban traffic did not change the gene expression related to inflammation, oxidative stress or DNA repair, ROS production or oxidatively damaged DNA in PBMCs from elderly overweight human subjects. PMID:25904586

  9. Quantitative DNA fiber mapping

    DOEpatents

    Gray, Joe W.; Weier, Heinz-Ulrich G.

    1998-01-01

    The present invention relates generally to the DNA mapping and sequencing technologies. In particular, the present invention provides enhanced methods and compositions for the physical mapping and positional cloning of genomic DNA. The present invention also provides a useful analytical technique to directly map cloned DNA sequences onto individual stretched DNA molecules.

  10. Determination of apurinic/apyrimidinic lesions in DNA with high-performance liquid chromatography and tandem mass spectrometry.

    PubMed

    Roberts, Kenneth P; Sobrino, Justin A; Payton, Julie; Mason, Lavinnia B; Turesky, Robert J

    2006-02-01

    A new method has been developed to accurately measure apurinic and apyrimidinic (AP) DNA damage sites, which are lesions in DNA formed by loss of a nucleobase from oxidative stress or carcinogen adducts. If AP sites are left unrepaired (or if improperly repaired), these sites can lead to DNA mutations that may ultimately result in the formation of cancer. Hence, detection of AP sites may provide a useful indicator of exposure and susceptibility to chemical carcinogens and oxidative stress. AP detection is currently accomplished by immunodetection methods using an aldehyde reactive probe [Nakamura, J., Walker, V. E., Upton, P. B., Chiang, S.-Y., Kow, Y. W., and Swenberg, J. A. (1998) Cancer Res. 58, 222-225; Atamna, H., Cheung, I., and Ames, B. N. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 686-691]; however, these approaches lack the specificity required for unequivocal identification of the AP site. Therefore, we have developed an accurate method based on mass spectrometry detection of AP sites from AP DNA that have been prelabeled with O-4-nitrobenzylhydroxylamine (NBHA). Once labeled and once the excess labeling agent has been removed, enzymatic digestion of DNA to monomeric subunits can be accomplished, followed by isolation and detection with high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Optimization and validation of the experimental procedures and detection limits have been established using a model DNA oligomer (11-mer) containing uracil. Enzymatic removal of uracil with uracil glycosylase generates well-defined AP sites in both single- and double-stranded DNA. The addition of NBHA labels the AP site in the oligomer, creating a labeled 11-mer. HPLC-ESI-MS/MS in the negative ionization mode was used to monitor and confirm binding of NBHA to the AP oligomer. The NBHA-tagged oligomer underwent endo- and exonuclease digestion to the 5'-deoxyribose monophosphate (5'-dRp) level, thereby releasing

  11. [Exercise training in hypoxia prevents hypoxia induced mitochondrial DNA oxidative damage in skeletal muscle].

    PubMed

    Bo, Hai; Li, Ling; Duan, Fu-Qiang; Zhu, Jiang

    2014-10-25

    This study was undertaken to investigate the effect of exercise training on mitochondrial DNA (mtDNA) oxidative damage and 8-oxoguanine DNA glycosylase-1 (OGG1) expression in skeletal muscle of rats under continuous exposure to hypoxia. Male Sprague-Dawley rats were randomly divided into 4 groups (n = 8): normoxia control group (NC), normoxia training group (NT), hypoxia control group (HC), and hypoxia training group (HT). The hypoxia-treated animals were housed in normobaric hypoxic tent containing 11.3% oxygen for consecutive 4 weeks. The exercise-trained animals were exercised on a motor-driven rodent treadmill at a speed of 15 m/min, 5% grade for 60 min/day, 5 days per week for 4 weeks. The results showed that, compared with NC group, hypoxia attenuated complex I, II, IV and ATP synthase activities of the electron transport chain, and the level of mitochondrial membrane potential in HC group (P < 0.05 or P < 0.01). Moreover, hypoxia decreased mitochondrial OGG1, MnSOD, and GPx activities (P < 0.05 or P < 0.01), whereas elevated reactive oxygen species (ROS) generation and the level of 8-oxo-deoxyguanosine (8-oxodG) in mtDNA (P < 0.01). Furthermore, hypoxia attenuated muscle and mitochondrial [NAD⁺]/ [NADH] ratio, and SIRT3 protein expression (P < 0.05 or P < 0.01). Compared with HC group, exercise training in hypoxia elevated complex I, II, IV and ATP synthase activities, and the level of mitochondrial membrane potential in HT group (P < 0.05 or P < 0.01). Moreover, exercise training in hypoxia increased MnSOD and GPx activities and mitochondrial OGG1 level (P < 0.01), whereas decreased ROS generation and the level of 8-oxodG in mtDNA (P < 0.01). Furthermore, exercise training in hypoxia increased muscle and mitochondrial [NAD⁺]/[NADH] ratio, as well as SIRT3 protein expression (P < 0.05 or P < 0.01). These findings suggest that exercise training in hypoxia can decrease hypoxia-induced mtDNA oxidative damage in the skeletal muscle through up

  12. The role of polycyclic aromatic hydrocarbon-DNA adducts in inducing mutations in mouse skin

    PubMed Central

    Chakravarti, Dhrubajyoti; Venugopal, Divya; Mailander, Paula C.; Meza, Jane L.; Higginbotham, Sheila; Cavalieri, Ercole L.; Rogan, Eleanor G.

    2008-01-01

    Polycyclic aromatic hydrocarbons (PAH) form stable and depurinating DNA adducts in mouse skin to induce preneoplastic mutations. Some mutations transform cells, which then clonally expand to establish tumors. Strong clues about the mutagenic mechanism can be obtained if the PAH-DNA adducts can be correlated with both preneoplastic and tumor mutations. To this end, we studied mutagenesis in PAH-treated early preneoplastic skin (1 day after exposure) and in the induced papillomas in SENCAR mice. Papillomas were studied by PCR amplification of the H-ras gene and sequencing. For benzo[a]pyrene (BP), BP-7,8-dihydrodiol (BPDHD), 7,12-dimethylbenz[a]anthracene (DMBA) and dibenzo[a,l]pyrene (DB[a,l]P), the codon 13 (GGC to GTC) and codon 61 (CAA to CTA) mutations in papillomas corresponded to the relative levels of Gua and Ade-depurinating adducts, despite BP and BPDHD forming significant amounts of stable DNA adducts. Such a relationship was expected for DMBA and DB[a,l]P, as they formed primarily depurinating adducts. These results suggest that depurinating adducts play a major role in forming the tumorigenic mutations. To validate this correlation, preneoplastic skin mutations were studied by cloning H-ras PCR products and sequencing individual clones. DMBA- and DB[a,l]P-treated skin showed primarily A.T to G.C mutations, which correlated with the high ratio of the Ade/Gua-depurinating adducts. Incubation of skin DNA with T.G-DNA glycosylase eliminated most of these A.T to G.C mutations, indicating that they existed as G.T heteroduplexes, as would be expected if they were formed by errors in the repair of abasic sites generated by the depurinating adducts. BP and its metabolites induced mainly G.C to T.A mutations in preneoplastic skin. However, PCR over unrepaired anti-BPDE-N2dG adducts can generate similar mutations as artifacts of the study protocol, making it difficult to establish an adduct-mutation correlation for determining which BP-DNA adducts induce the early

  13. Pretreatment of primary rat cutaneous epidermal keratinocyte culture with a low concentration of MNNG: Effect on DNA cross-linking measured in situ after challenge with bis-2-chloroethyl sulfide

    SciTech Connect

    Sorsher, D.H.; Conolly, R.B. )

    1989-01-01

    Bis-2-chloroethyl sulfide- (BCES-) induced DNA cross-links in confluent, primary cultures of newborn rat cutaneous epidermal keratinocytes were detected using an assay that includes in situ unwinding of the DNA followed by separation of single-stranded DNA and double-stranded DNA (DSDNA) with hydroxylapatite. DNA cross-links in BCES-challenged cultures were inferred form increases in the percentage of DNA the remained double-stranded, compared with control cultures, after a 60-min alkaline unwinding incubation. The amount of DNA cross-linking after 5 or 10 {mu}M BCES was increased when keratinocytes were first pretreated with 0.05 {mu}M MNNG for 1 h at 8 a.m., 2 p.m., and 8 p.m. for two consecutive days and challenged with BCES the following morning. This increase was statistically significant. For example, after 5{mu}M BCES challenge, cultures not pretreated with MNNG had 114.14% control DSDNA, whereas MNNG pretreated cultures had 122.78% control DSDNA. The level of BCES-induced cross-linking was maximal immediately after 30-min challenge and decreased during postchallenge incubation. At 24 and 48 h post 5, 10, or 20 {mu}M BCES challenge, the level of DSDNA was actually depressed below unchallenged levels. This postchallenge decreased in the level of DSDNA, indicative of SSB in DNA, suggests repair activity by glycosylases and endonucleases. However completion of repair (i.e., a return to control levels of DSDNA) was not seen in these experiments. The activity that resulted in decreases in the level of DSDNA during postchallenge incubation response was unaffected by MNNG pretreatment.

  14. Identification of Escherichia coli ygaQ and rpmG as novel mitomycin C resistance factors implicated in DNA repair.

    PubMed

    Bolt, Edward L; Jenkins, Tabitha; Russo, Valeria Moreira; Ahmed, Sharlene; Cavey, James; Cass, Simon D

    2016-01-01

    Using the ASKA (A Complete Set of Escherichia coli K-12 ORF Archive) library for genome-wide screening of E. coli proteins we identified that expression of ygaQ and rpmG promotes mitomycin C resistance (MMC(R)). YgaQ mediated MMC(R) was independent of homologous recombination involving RecA or RuvABC, but required UvrD. YgaQ is an uncharacterized protein homologous with α-amylases that we identified to have nuclease activity directed to ssDNA of 5' flaps. Nuclease activity was inactivated by mutation of two amino acid motifs, which also abolished MMC(R). RpmG is frequently annotated as a bacterial ribosomal protein, although forms an operon with MutM glycosylase and a putative deubiquitinating (DUB) enzyme, YicR. RpmG associated MMC(R) was dependent on MutM. MMC(R) from RpmG resembles DNA repair phenotypes reported for 'idiosyncratic ribosomal proteins' in eukaryotes. PMID:26704888

  15. Non-canonical uracil processing in DNA gives rise to double-strand breaks and deletions: relevance to class switch recombination.

    PubMed

    Bregenhorn, Stephanie; Kallenberger, Lia; Artola-Borán, Mariela; Peña-Diaz, Javier; Jiricny, Josef

    2016-04-01

    During class switch recombination (CSR), antigen-stimulated B-cells rearrange their immunoglobulin constant heavy chain (CH) loci to generate antibodies with different effector functions. CSR is initiated by activation-induced deaminase (AID), which converts cytosines in switch (S) regions, repetitive sequences flanking the CH loci, to uracils. Although U/G mispairs arising in this way are generally efficiently repaired to C/Gs by uracil DNA glycosylase (UNG)-initiated base excision repair (BER), uracil processing in S-regions of activated B-cells occasionally gives rise to double strand breaks (DSBs), which trigger CSR. Surprisingly, genetic experiments revealed that CSR is dependent not only on AID and UNG, but also on mismatch repair (MMR). To elucidate the role of MMR in CSR, we studied the processing of uracil-containing DNA substrates in extracts of MMR-proficient and -deficient human cells, as well as in a system reconstituted from recombinant BER and MMR proteins. Here, we show that the interplay of these repair systems gives rise to DSBs in vitro and to genomic deletions and mutations in vivo, particularly in an S-region sequence. Our findings further suggest that MMR affects pathway choice in DSB repair. Given its amenability to manipulation, our system represents a powerful tool for the molecular dissection of CSR. PMID:26743004

  16. Identification of Escherichia coli ygaQ and rpmG as novel mitomycin C resistance factors implicated in DNA repair

    PubMed Central

    Bolt, Edward L.; Jenkins, Tabitha; Russo, Valeria Moreira; Ahmed, Sharlene; Cavey, James; Cass, Simon D.

    2015-01-01

    Using the ASKA (A Complete Set of Escherichia coli K-12 ORF Archive) library for genome-wide screening of E. coli proteins we identified that expression of ygaQ and rpmG promotes mitomycin C resistance (MMCR). YgaQ mediated MMCR was independent of homologous recombination involving RecA or RuvABC, but required UvrD. YgaQ is an uncharacterized protein homologous with α-amylases that we identified to have nuclease activity directed to ssDNA of 5′ flaps. Nuclease activity was inactivated by mutation of two amino acid motifs, which also abolished MMCR. RpmG is frequently annotated as a bacterial ribosomal protein, although forms an operon with MutM glycosylase and a putative deubiquitinating (DUB) enzyme, YicR. RpmG associated MMCR was dependent on MutM. MMCR from RpmG resembles DNA repair phenotypes reported for ‘idiosyncratic ribosomal proteins’ in eukaryotes. PMID:26704888

  17. Non-canonical uracil processing in DNA gives rise to double-strand breaks and deletions: relevance to class switch recombination

    PubMed Central

    Bregenhorn, Stephanie; Kallenberger, Lia; Artola-Borán, Mariela; Peña-Diaz, Javier; Jiricny, Josef

    2016-01-01

    During class switch recombination (CSR), antigen-stimulated B-cells rearrange their immunoglobulin constant heavy chain (CH) loci to generate antibodies with different effector functions. CSR is initiated by activation-induced deaminase (AID), which converts cytosines in switch (S) regions, repetitive sequences flanking the CH loci, to uracils. Although U/G mispairs arising in this way are generally efficiently repaired to C/Gs by uracil DNA glycosylase (UNG)-initiated base excision repair (BER), uracil processing in S-regions of activated B-cells occasionally gives rise to double strand breaks (DSBs), which trigger CSR. Surprisingly, genetic experiments revealed that CSR is dependent not only on AID and UNG, but also on mismatch repair (MMR). To elucidate the role of MMR in CSR, we studied the processing of uracil-containing DNA substrates in extracts of MMR-proficient and –deficient human cells, as well as in a system reconstituted from recombinant BER and MMR proteins. Here, we show that the interplay of these repair systems gives rise to DSBs in vitro and to genomic deletions and mutations in vivo, particularly in an S-region sequence. Our findings further suggest that MMR affects pathway choice in DSB repair. Given its amenability to manipulation, our system represents a powerful tool for the molecular dissection of CSR. PMID:26743004

  18. Activity-based assay for ricin-like toxins

    DOEpatents

    Keener, William K.; Ward, Thomas E.

    2007-02-06

    A method of detecting N-glycosylase activity in a sample involves incubating an oligodeoxyribonucleotide substrate containing a deoxyadenosine or deoxyuridine residue with the sample to be tested such that the N-glycosylase, if present, hydrolyzes the deoxyadenosine or deoxyuridine residue to result in an N-glycosylase product having an abasic site. A primer is annealed to the N-glycosylase product, and the primer is extended with a DNA polymerase, such as Taq DNA polymerase, that pauses at abasic sites. The resulting extension products are melted from the N-glycosylase product, allowed to form hairpins due to self-complementarity, and further extended in the presence of labeled precursors to result in labeled products. Extension products synthesized from undigested substrate as template do not result in labeled products. Thus, detection of labeled products results in detection of N-glycosylase activity. Oligodeoxyribonucleotide substrates, primer, and positive controls and a kit for N-glycosylase assay are also disclosed.

  19. RNA-binding protein regulates plant DNA methylation by controlling mRNA processing at the intronic heterochromatin-containing gene IBM1

    PubMed Central

    Wang, Xingang; Duan, Cheng-Guo; Tang, Kai; Wang, Bangshing; Zhang, Huiming; Lei, Mingguang; Lu, Kun; Mangrauthia, Satendra K.; Wang, Pengcheng; Zhao, Yang; Zhu, Jian-Kang

    2013-01-01

    DNA methylation-dependent heterochromatin formation is a conserved mechanism of epigenetic silencing of transposons and other repeat elements in many higher eukaryotes. Genes adjacent to repetitive elements are often also subjected to this epigenetic silencing. Consequently, plants have evolved antisilencing mechanisms such as active DNA demethylation mediated by the REPRESSOR OF SILENCING 1 (ROS1) family of 5-methylcytosine DNA glycosylases to protect these genes from silencing. Some transposons and other repeat elements have found residence in the introns of genes. It is unclear how these intronic repeat elements-containing genes are regulated. We report here the identification of ANTI-SILENCING 1 (ASI1), a bromo-adjacent homology domain and RNA recognition motif-containing protein, from a forward genetic screen for cellular antisilencing factors in Arabidopsis thaliana. ASI1 is required to prevent promoter DNA hypermethylation and transcriptional silencing of some transgenes. Genome-wide DNA methylation analysis reveals that ASI1 has a similar role to that of the histone H3K9 demethylase INCREASE IN BONSAI METHYLATION 1 (IBM1) in preventing CHG methylation in the bodies of thousands of genes. We found that ASI1 is an RNA-binding protein and ensures the proper expression of IBM1 full-length transcript by associating with an intronic heterochromatic repeat element of IBM1. Through mRNA sequencing, we identified many genes containing intronic transposon elements that require ASI1 for proper expression. Our results suggest that ASI1 associates with intronic heterochromatin and binds the gene transcripts to promote their 3′ distal polyadenylation. The study thus reveals a unique mechanism by which higher eukaryotes deal with the collateral effect of silencing intronic repeat elements. PMID:24003136

  20. An electrochemiluminescence biosensor for 8-oxo-7,8-dihydro-2'-deoxyguanosine quantification and DNA repair enzyme activity analysis using a novel bifunctional probe.

    PubMed

    Wu, Yiping; Yang, Xiqiang; Zhang, Bintian; Guo, Liang-Hong

    2015-07-15

    A new electrochemiluminescence (ECL) sensor was developed for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) quantification and Escherichia coli formamidopyrimidine-DNA glycosylase (FPG) activity assay. The sensor employed a novel spermine conjugated ruthenium tris-(bipyridine) derivative (spermine-Ru) which binds specifically with 8-oxodGuo through a one-step reaction and also acts as an ECL signal reporter. In the sensor, an 8-oxodGuo-containing ds-DNA film was first immobilized on a gold electrode by self-assembly. The DNA film was then incubated with spermine-Ru under oxidative condition for 8-oxodGuo labeling. The ECL intensity was found to correlate with the amount of 8-oxodGuo on the surface and the detection limit was estimated to be about 1 lesion in 500 DNA bases. Addition of FPG resulted in some loss of the signal due to the excision of 8-oxodGuo by the enzyme. An inverse relationship between ECL intensity and FPG concentration was observed in a range from 0 to 4.0U/µL, demonstrating that this sensor could be used for FPG activity assay. A number of metal ions were screened by the sensor for their inhibition effect on FPG activity. Among them, Hg(2+) and methyl Hg(II) shown very potent inhibition, with IC50 values of 4.04µM and 4.34nM respectively. The result may suggest that interference on the DNA repair system could be another mechanism for the high toxicity of MeHg. PMID:25747509

  1. LCAT DNA shearing.

    PubMed

    Okabe, Yuka; Lee, Abraham P

    2014-04-01

    We present a novel method to fragment DNA by using lateral cavity acoustic transducers (LCATs). DNA solution is placed within a microfluidic device containing LCATs. The LCATs cause microstreaming, which fragments DNA within the solution without any need for purification or downstream processing. The LCAT-based DNA fragmentation method offers an easy-to-use, low-cost, low-energy way to fragment DNA that is amenable to integration on microfluidic platforms to further automate DNA processing. Furthermore, the LCAT microdevice requires less than 10 µL of sample, and no external equipment is needed besides a piezoelectric transducer. PMID:23850863

  2. Structural Organization of DNA.

    ERIC Educational Resources Information Center

    Banfalvi, Gaspar

    1986-01-01

    Explains the structural organization of DNA by providing information on the primary, secondary, tertiary, and higher organization levels of the molecule. Also includes illustrations and descriptions of sign-inversion and rotating models for supercoiling of DNA. (ML)

  3. Modeling DNA Replication.

    ERIC Educational Resources Information Center

    Bennett, Joan

    1998-01-01

    Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

  4. HPV DNA test

    MedlinePlus

    The HPV DNA test is used to check for high-risk HPV infection in women. HPV infection around the genitals is ... warts spread when you have sex. The HPV-DNA test is generally not recommended for detecting low- ...

  5. DNA tagged microparticles

    DOEpatents

    Farquar, George Roy; Leif, Roald N; Wheeler, Elizabeth

    2015-05-05

    A simulant that includes a carrier and DNA encapsulated in the carrier. Also a method of making a simulant including the steps of providing a carrier and encapsulating DNA in the carrier to produce the simulant.

  6. Is DNA a language?

    PubMed

    Tsonis, A A; Elsner, J B; Tsonis, P A

    1997-01-01

    DNA sequences usually involve local construction rules that affect different scales. As such their "dictionary" may not follow Zipf's law (a power law) which is followed in every natural language. Indeed, analysis of many DNA sequences suggests that no linguistics connections to DNA exist and that even though it has structure DNA is not a language. Computer simulations and a biological approach to this problem further support these results. PMID:9039397

  7. DNAzymes in DNA Nanomachines and DNA Analysis

    NASA Astrophysics Data System (ADS)

    He, Yu; Tian, Ye; Chen, Yi; Mao, Chengde

    This chapter discusses our efforts in using DNAzymes in DNA nano-machines and DNA analysis systems. 10-23 DNAzymes can cleave specific phos-phodiester bonds in RNA. We use them to construct an autonomous DNA-RNA chimera nanomotor, which constantly extracts chemical energy from RNA substrates and transduces the energy into a mechanical motion: cycles of contraction and extension. The motor's motion can be reversibly turned on and off by a DNA analogue (brake) of the RNA substrate. Addition and removal of the brake stops and restarts, respectively, the motor's motion. Furthermore, when the RNA substrates are preorganized into a one-dimensional track, a DNAzyme can continuously move along the track so long as there are substrates available ahead. Based on a similar mechanism, a novel DNA detection system has been developed. A target DNA activates a DNAzyme to cleave RNA-containing molecular beacons (MB), which generates an enhanced fluorescence signal. A following work integrates two steps of signal amplifications: a rolling-circle amplification (RCA) to synthesize multiple copies of DNAzymes, and the DNAzymes catalyze a chemical reaction to generate a colorimetric signal. This method allows detection of DNA analytes whose concentration is as low as 1 pM.

  8. The Many Sides of DNA.

    ERIC Educational Resources Information Center

    Flannery, Maura C.

    1997-01-01

    Explores the meaning of DNA. Discusses histories of DNA, literature on DNA, the contributions of Max Delbruck and Barbara McClintock, life, views of control, current research, and the language of DNA. Contains 24 references. (JRH)

  9. DNA Sequencing apparatus

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1992-01-01

    An automated DNA sequencing apparatus having a reactor for providing at least two series of DNA products formed from a single primer and a DNA strand, each DNA product of a series differing in molecular weight and having a chain terminating agent at one end; separating means for separating the DNA products to form a series bands, the intensity of substantially all nearby bands in a different series being different, band reading means for determining the position an This invention was made with government support including a grant from the U.S. Public Health Service, contract number AI-06045. The U.S. government has certain rights in the invention.

  10. DNA in Nanoscale Electronics

    NASA Astrophysics Data System (ADS)

    Slinker, Jason

    2012-10-01

    DNA, the quintessential molecule of life, possesses a number of attractive properties for use in nanoscale circuits. Charge transport (CT) through DNA itself is of both fundamental and practical interest. Fundamentally, DNA has a unique configuration of π-stacked bases in a well ordered, double helical structure. Given its unparalleled importance to life processes and its arrangement of conjugated subunits, DNA has been a compelling target of conductivity studies. In addition, further understanding of DNA CT will elucidate the biological implications of this process and advance its use in sensing technologies. We have investigated the fundamentals of DNA CT by measuring the electrochemistry of DNA monolayers under biologically-relevant conditions. We have uncovered both fundamental kinetic parameters to distinguish between competing models of operation as well as the practical implications of DNA CT for sensing. Furthermore, we are leveraging our studies of DNA conductivity for the manufacture of nanoscale circuits. We are investigating the electrical properties and self-assembly of DNA nanowires containing artificial base pair surrogates, which can be prepared through low cost and high throughput automated DNA synthesis. This unique and economically viable approach will establish a new paradigm for the scalable manufacture of nanoscale semiconductor devices.

  11. DNA structure and function.

    PubMed

    Travers, Andrew; Muskhelishvili, Georgi

    2015-06-01

    The proposal of a double-helical structure for DNA over 60 years ago provided an eminently satisfying explanation for the heritability of genetic information. But why is DNA, and not RNA, now the dominant biological information store? We argue that, in addition to its coding function, the ability of DNA, unlike RNA, to adopt a B-DNA structure confers advantages both for information accessibility and for packaging. The information encoded by DNA is both digital - the precise base specifying, for example, amino acid sequences - and analogue. The latter determines the sequence-dependent physicochemical properties of DNA, for example, its stiffness and susceptibility to strand separation. Most importantly, DNA chirality enables the formation of supercoiling under torsional stress. We review recent evidence suggesting that DNA supercoiling, particularly that generated by DNA translocases, is a major driver of gene regulation and patterns of chromosomal gene organization, and in its guise as a promoter of DNA packaging enables DNA to act as an energy store to facilitate the passage of translocating enzymes such as RNA polymerase. PMID:25903461

  12. Human DNA repair genes.

    PubMed

    Wood, R D; Mitchell, M; Sgouros, J; Lindahl, T

    2001-02-16

    Cellular DNA is subjected to continual attack, both by reactive species inside cells and by environmental agents. Toxic and mutagenic consequences are minimized by distinct pathways of repair, and 130 known human DNA repair genes are described here. Notable features presently include four enzymes that can remove uracil from DNA, seven recombination genes related to RAD51, and many recently discovered DNA polymerases that bypass damage, but only one system to remove the main DNA lesions induced by ultraviolet light. More human DNA repair genes will be found by comparison with model organisms and as common folds in three-dimensional protein structures are determined. Modulation of DNA repair should lead to clinical applications including improvement of radiotherapy and treatment with anticancer drugs and an advanced understanding of the cellular aging process. PMID:11181991

  13. DNA polymerases β and λ do not directly affect Ig variable region somatic hypermutation although their absence reduces the frequency of mutations

    PubMed Central

    Schrader, Carol E.; Linehan, Erin K.; Ucher, Anna J.; Bertocci, Barbara; Stavnezer, Janet

    2014-01-01

    During somatic hypermutation (SHM) of antibody variable (V) region genes, activation-induced cytidine deaminase (AID) converts dC to dU, and dUs can either be excised by uracil DNA glycosylase (UNG), by mismatch repair, or replicated over. If UNG excises the dU, the abasic site could be cleaved by AP-endonuclease (APE), introducing the single-strand DNA breaks (SSBs) required for generating mutations at A:T bp, which are known to depend upon mismatch repair and DNA Pol η. DNA Pol β or λ could instead repair the lesion correctly. To assess the involvement of Pols β and λ in SHM of antibody genes, we analyzed mutations in the VDJh4 3′ flanking region in Peyer’s patch germinal center (GC) B cells from polβ−/−polλ−/−, polλ−/−, and polβ−/− mice. We find that deficiency of either or both polymerases results in a modest but significant decrease in V region SHM, with Pol β having a greater effect, but there is no effect on mutation specificity, suggesting they have no direct role in SHM. Instead, the effect on SHM appears to be due to a role for these enzymes in GC B cell proliferation or viability. The results suggest that the BER pathway is not important during V region SHM for generating mutations at A:T bp. Furthermore, this implies that most of the SSBs required for Pol η to enter and create A:T mutations are likely generated during replication instead. These results contrast with the inhibitory effect of Pol β on mutations at the Ig Sμ locus, Sμ DSBs and class switch recombination (CSR) reported previously. We show here that B cells deficient in Pol λ or both Pol β and λ proliferate normally in culture and undergo slightly elevated CSR, as shown previously for Pol β-deficient B cells. PMID:24084171

  14. Apurinic/apyrimidinic endonucleases of Mycobacterium tuberculosis protect against DNA damage but are dispensable for the growth of the pathogen in guinea pigs.

    PubMed

    Puri, Rupangi Verma; Reddy, P Vineel; Tyagi, Anil K

    2014-01-01

    In host cells, Mycobacterium tuberculosis encounters an array of reactive molecules capable of damaging its genome. Non-bulky DNA lesions are the most common damages produced on the exposure of the pathogen to reactive species and base excision repair (BER) pathway is involved in the repair of such damage. During BER, apurinic/apyrimidinic (AP) endonuclease enzymes repair the abasic sites that are generated after spontaneous DNA base loss or by the action of DNA glycosylases, which if left unrepaired lead to inhibition of replication and transcription. However, the role of AP endonucleases in imparting protection against DNA damage and in the growth and pathogenesis of M.tuberculosis has not yet been elucidated. To demonstrate the biological significance of these enzymes in M.tuberculosis, it would be desirable to disrupt the relevant genes and evaluate the resulting mutants for their ability to grow in the host and cause disease. In this study, we have generated M.tuberculosis mutants of the base excision repair (BER) system, disrupted in either one (MtbΔend or MtbΔxthA) or both the AP endonucleases (MtbΔendΔxthA). We demonstrate that these genes are crucial for bacteria to withstand alkylation and oxidative stress in vitro. In addition, the mutant disrupted in both the AP endonucleases (MtbΔendΔxthA) exhibited a significant reduction in its ability to survive inside human macrophages. However, infection of guinea pigs with either MtbΔend or MtbΔxthA or MtbΔendΔxthA resulted in the similar bacillary load and pathological damage in the organs as observed in the case of infection with wild-type M.tuberculosis. The implications of these observations are discussed. PMID:24800740

  15. Exposure to runoff from coal-tar-sealed pavement induces genotoxicity and impairment of DNA repair capacity in the RTL-W1 fish liver cell line.

    PubMed

    Kienzler, Aude; Mahler, Barbara J; Van Metre, Peter C; Schweigert, Nathalie; Devaux, Alain; Bony, Sylvie

    2015-07-01

    Coal-tar-based (CTB) sealcoat, frequently applied to parking lots and driveways in North America, contains elevated concentrations of polycyclic aromatic hydrocarbons (PAHs) and related compounds. The RTL-W1 fish liver cell line was used to investigate two endpoints (genotoxicity and DNA-repair-capacity impairment) associated with exposure to runoff from asphalt pavement with CTB sealcoat or with an asphalt-based sealcoat hypothesized to contain about 7% CTB sealcoat (AS-blend). Genotoxic potential was assessed by the Formamido pyrimidine glycosylase (Fpg)-modified comet assay for 1:10 and 1:100 dilutions of runoff samples collected from 5 h to 36 d following sealcoat application. DNA-repair capacity was assessed by the base excision repair comet assay for 1:10 dilution of samples collected 26 h and 36 d following application. Both assays were run with and without co-exposure to ultraviolet-A radiation (UVA). With co-exposure to UVA, genotoxic effects were significant for both dilutions of CTB runoff for three of four sample times, and for some samples of AS-blend runoff. Base excision repair was significantly impaired for CTB runoff both with and without UVA exposure, and for AS-blend runoff only in the absence of UVA. This study is the first to investigate the effects of exposure to the complex mixture of chemicals in coal tar on DNA repair capacity. The results indicate that co-exposure to runoff from CT-sealcoated pavement and UVA as much as a month after sealcoat application has the potential to cause genotoxicity and impair DNA repair capacity. PMID:25795989

  16. Exposure to runoff from coal-tar-sealed pavement induces genotoxicity and impairment of DNA repair capacity in the RTL-W1 fish liver cell line

    USGS Publications Warehouse

    Kienzler, Aude; Mahler, Barbara J.; Van Metre, Peter C.; Schweigert, Nathalie; Devaux, Alain; Bony, Sylvie

    2015-01-01

    Coal-tar-based (CTB) sealcoat, frequently applied to parking lots and driveways in North America, contains elevated concentrations of polycyclic aromatic hydrocarbons (PAHs) and related compounds. The RTL-W1 fish liver cell line was used to investigate two endpoints (genotoxicity and DNA-repair-capacity impairment) associated with exposure to runoff from asphalt pavement with CTB sealcoat or with an asphalt-based sealcoat hypothesized to contain about 7% CTB sealcoat (AS-blend). Genotoxic potential was assessed by the Formamido pyrimidine glycosylase (Fpg)-modified comet assay for 1:10 and 1:100 dilutions of runoff samples collected from 5 h to 36 d following sealcoat application. DNA-repair capacity was assessed by the base excision repair comet assay for 1:10 dilution of samples collected 26 h and 36 d following application. Both assays were run with and without co-exposure to ultraviolet-A radiation (UVA). With co-exposure to UVA, genotoxic effects were significant for both dilutions of CTB runoff for three of four sample times, and for some samples of AS-blend runoff. Base excision repair was significantly impaired for CTB runoff both with and without UVA exposure, and for AS-blend runoff only in the absence of UVA. This study is the first to investigate the effects of exposure to the complex mixture of chemicals in coal tar on DNA repair capacity. The results indicate that co-exposure to runoff from CT-sealcoated pavement and UVA as much as a month after sealcoat application has the potential to cause genotoxicity and impair DNA repair capacity.

  17. DNA methylation in plants.

    PubMed

    Vanyushin, B F

    2006-01-01

    DNA in plants is highly methylated, containing 5-methylcytosine (m5C) and N6-methyladenine (m6A); m5C is located mainly in symmetrical CG and CNG sequences but it may occur also in other non-symmetrical contexts. m6A but not m5C was found in plant mitochondrial DNA. DNA methylation in plants is species-, tissue-, organelle- and age-specific. It is controlled by phytohormones and changes on seed germination, flowering and under the influence of various pathogens (viral, bacterial, fungal). DNA methylation controls plant growth and development, with particular involvement in regulation of gene expression and DNA replication. DNA replication is accompanied by the appearance of under-methylated, newly formed DNA strands including Okazaki fragments; asymmetry of strand DNA methylation disappears until the end of the cell cycle. A model for regulation of DNA replication by methylation is suggested. Cytosine DNA methylation in plants is more rich and diverse compared with animals. It is carried out by the families of specific enzymes that belong to at least three classes of DNA methyltransferases. Open reading frames (ORF) for adenine DNA methyltransferases are found in plant and animal genomes, and a first eukaryotic (plant) adenine DNA methyltransferase (wadmtase) is described; the enzyme seems to be involved in regulation of the mitochondria replication. Like in animals, DNA methylation in plants is closely associated with histone modifications and it affects binding of specific proteins to DNA and formation of respective transcription complexes in chromatin. The same gene (DRM2) in Arabidopsis thaliana is methylated both at cytosine and adenine residues; thus, at least two different, and probably interdependent, systems of DNA modification are present in plants. Plants seem to have a restriction-modification (R-M) system. RNA-directed DNA methylation has been observed in plants; it involves de novo methylation of almost all cytosine residues in a region of siRNA-DNA

  18. Forensic DNA analysis.

    PubMed

    McDonald, Jessica; Lehman, Donald C

    2012-01-01

    Before the routine use of DNA profiling, blood typing was an important forensic tool. However, blood typing was not very discriminating. For example, roughly 30% of the United States population has type A-positive blood. Therefore, if A-positive blood were found at a crime scene, it could have come from 30% of the population. DNA profiling has a much better ability for discrimination. Forensic laboratories no longer routinely determine blood type. If blood is found at a crime scene, DNA profiling is performed. From Jeffrey's discovery of DNA fingerprinting to the development of PCR of STRs to the formation of DNA databases, our knowledge of DNA and DNA profiling have expanded greatly. Also, the applications for which we use DNA profiling have increased. DNA profiling is not just used for criminal case work, but it has expanded to encompass paternity testing, disaster victim identification, monitoring bone marrow transplants, detecting fetal cells in a mother's blood, tracing human history, and a multitude of other areas. The future of DNA profiling looks expansive with the development of newer instrumentation and techniques. PMID:22693781

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

    DOEpatents

    McCutchen-Maloney, Sandra L.

    2002-01-01

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

  20. DNA profiles from fingermarks.

    PubMed

    Templeton, Jennifer E L; Linacre, Adrian

    2014-11-01

    Criminal investigations would be considerably improved if DNA profiles could be routinely generated from single fingermarks. Here we report a direct DNA profiling method that was able to generate interpretable profiles from 71% of 170 fingermarks. The data are based on fingermarks from all 5 digits of 34 individuals. DNA was obtained from the fingermarks using a swab moistened with Triton-X, and the fibers were added directly to one of two commercial DNA profiling kits. All profiles were obtained without increasing the number of amplification cycles; therefore, our method is ideally suited for adoption by the forensic science community. We indicate the use of the technique in a criminal case in which a DNA profile was generated from a fingermark on tape that was wrapped around a drug seizure. Our direct DNA profiling approach is rapid and able to generate profiles from touched items when current forensic practices have little chance of success. PMID:25391915

  1. Electrocatalysis in DNA Sensors.

    PubMed

    Furst, Ariel; Hill, Michael G; Barton, Jacqueline K

    2014-12-14

    Electrocatalysis is often thought of solely in the inorganic realm, most often applied to energy conversion in fuel cells. However, the ever-growing field of bioelectrocatalysis has made great strides in advancing technology for both biofuel cells as well as biological detection platforms. Within the context of bioelectrocatalytic detection systems, DNA-based platforms are especially prevalent. One subset of these platforms, the one we have developed, takes advantage of the inherent charge transport properties of DNA. Electrocatalysis coupled with DNA-mediated charge transport has enabled specific and sensitive detection of lesions, mismatches and DNA-binding proteins. Even greater signal amplification from these platforms is now being achieved through the incorporation of a secondary electrode to the platform both for patterning DNA arrays and for detection. Here, we describe the evolution of this new DNA sensor technology. PMID:25435647

  2. DNA Media Storage

    PubMed Central

    Bogard, Christy M.; Rouchka, Eric C.

    2010-01-01

    In 1994, University of Southern California computer scientist Dr. Leonard Adelman solved the Hamiltonian Path Problem using DNA as a computational mechanism. He proved the principle that DNA computing could be used to solve computationally complex problems. Because of the limitations in discovery time, resource requirements, and sequence mismatches, DNA computing has not yet become a commonly accepted practice. However, advancements are continually being discovered that are evolving the field of DNA Computing. Practical applications of DNA are not restricted to computation alone. This research presents a novel approach in which DNA could be used as a means of storing files. Through the use of Multiple Sequence Alignment combined with intelligent heuristics, the most probabilistic file contents can be determined with minimal errors. PMID:20622994

  3. DNA Media Storage.

    PubMed

    Bogard, Christy M; Rouchka, Eric C

    2007-09-01

    In 1994, University of Southern California computer scientist Dr. Leonard Adelman solved the Hamiltonian Path Problem using DNA as a computational mechanism. He proved the principle that DNA computing could be used to solve computationally complex problems. Because of the limitations in discovery time, resource requirements, and sequence mismatches, DNA computing has not yet become a commonly accepted practice. However, advancements are continually being discovered that are evolving the field of DNA Computing. Practical applications of DNA are not restricted to computation alone. This research presents a novel approach in which DNA could be used as a means of storing files. Through the use of Multiple Sequence Alignment combined with intelligent heuristics, the most probabilistic file contents can be determined with minimal errors. PMID:20622994

  4. Ribonucleotides in Bacterial DNA

    PubMed Central

    Schroeder, Jeremy W.; Randall, Justin R.; Matthews, Lindsay A.; Simmons, Lyle A.

    2014-01-01

    In all living cells, DNA is the storage medium for genetic information. Being quite stable, DNA is well-suited for its role in storage and propagation of information, but RNA is also covalently included in DNA through various mechanisms. Recent studies also demonstrate useful aspects of including ribonucleotides in the genome during repair. Therefore, our understanding of the consequences of RNA inclusion into bacterial genomic DNA is just beginning, but with its high frequency of occurrence the consequences and potential benefits are likely to be numerous and diverse. In this review, we discuss the processes that cause ribonucleotide inclusion in genomic DNA, the pathways important for ribonucleotide removal and the consequences that arise should ribonucleotides remain nested in genomic DNA. PMID:25387798

  5. Electrocatalysis in DNA Sensors

    PubMed Central

    Furst, Ariel; Hill, Michael G.; Barton, Jacqueline K.

    2014-01-01

    Electrocatalysis is often thought of solely in the inorganic realm, most often applied to energy conversion in fuel cells. However, the ever-growing field of bioelectrocatalysis has made great strides in advancing technology for both biofuel cells as well as biological detection platforms. Within the context of bioelectrocatalytic detection systems, DNA-based platforms are especially prevalent. One subset of these platforms, the one we have developed, takes advantage of the inherent charge transport properties of DNA. Electrocatalysis coupled with DNA-mediated charge transport has enabled specific and sensitive detection of lesions, mismatches and DNA-binding proteins. Even greater signal amplification from these platforms is now being achieved through the incorporation of a secondary electrode to the platform both for patterning DNA arrays and for detection. Here, we describe the evolution of this new DNA sensor technology. PMID:25435647

  6. DNA-based machines.

    PubMed

    Wang, Fuan; Willner, Bilha; Willner, Itamar

    2014-01-01

    The base sequence in nucleic acids encodes substantial structural and functional information into the biopolymer. This encoded information provides the basis for the tailoring and assembly of DNA machines. A DNA machine is defined as a molecular device that exhibits the following fundamental features. (1) It performs a fuel-driven mechanical process that mimics macroscopic machines. (2) The mechanical process requires an energy input, "fuel." (3) The mechanical operation is accompanied by an energy consumption process that leads to "waste products." (4) The cyclic operation of the DNA devices, involves the use of "fuel" and "anti-fuel" ingredients. A variety of DNA-based machines are described, including the construction of "tweezers," "walkers," "robots," "cranes," "transporters," "springs," "gears," and interlocked cyclic DNA structures acting as reconfigurable catenanes, rotaxanes, and rotors. Different "fuels", such as nucleic acid strands, pH (H⁺/OH⁻), metal ions, and light, are used to trigger the mechanical functions of the DNA devices. The operation of the devices in solution and on surfaces is described, and a variety of optical, electrical, and photoelectrochemical methods to follow the operations of the DNA machines are presented. We further address the possible applications of DNA machines and the future perspectives of molecular DNA devices. These include the application of DNA machines as functional structures for the construction of logic gates and computing, for the programmed organization of metallic nanoparticle structures and the control of plasmonic properties, and for controlling chemical transformations by DNA machines. We further discuss the future applications of DNA machines for intracellular sensing, controlling intracellular metabolic pathways, and the use of the functional nanostructures for drug delivery and medical applications. PMID:24647836

  7. DNA ELECTROPHORESIS AT SURFACES

    SciTech Connect

    RAFAILOVICH, MIRIAM; SOKOLOV, JONATHAN; GERSAPPE, DILIP

    2003-09-01

    During this year we performed two major projects: I. We developed a detailed theoretical model which complements our experiments on surface DNA electrophoresis. We found that it was possible to enhance the separation of DNA chains by imposing a chemical nanoscale pattern on the surface. This approach utilized the surface interaction effect of the DNA chains with the substrate and is a refinement to our previous method in which DNA chains were separated on homogeneous flat surfaces. By introducing the nano-patterns on the surface, the conformational changes of DNA chains of different lengths can be amplified, which results in the different friction strengths with the substrate surface. Our results also show that, when compared to the DNA electrophoresis performed on homogeneous flat surfaces, nanopatterned surfaces offer a larger window in choosing different surface interactions to achieve separation. II. In collaboration with a large international manufacturer of skin care products we also embarked on a project involving photo toxicity of titanium dioxide nanoparticles, which are a key ingredient in sunscreen and cosmetic lotions. The results clearly implicated the nanoparticles in catalyzing damage to chromosomal DNA. We then used this knowledge to develop a polymer/anti-oxidant coating which prevented the photocatalytic reaction on DNA while still retaining the UV absorptive properties of the nanoparticles. The standard gel electrophoresis was not sufficient in determining the extent of the DNA damage. The conclusions of this study were based predominantly on analysis obtained with the surface electrophoresis method.

  8. Tiny telomere DNA

    PubMed Central

    Ren, Jinsong; Qu, Xiaogang; Trent, John O.; Chaires, Jonathan B.

    2002-01-01

    We describe the design, synthesis and biophysical characterization of a novel DNA construct in which a folded quadruplex structure is joined to a standard double helix. Circular dichroism, gel electrophoresis, three-dimensional UV melting and differential scanning calorimetry were all used to characterize the structure. Rigorous molecular dynamics simulations were used to build a plausible atomic-level structural model of the DNA construct. This novel DNA construct provides a model for the duplex–quadruplex junction region at the end of chromosomal DNA and offers a system for the study of structure-selective ligand binding. PMID:12034817

  9. DNA Damage Response

    PubMed Central

    Giglia-Mari, Giuseppina; Zotter, Angelika; Vermeulen, Wim

    2011-01-01

    Structural changes to DNA severely affect its functions, such as replication and transcription, and play a major role in age-related diseases and cancer. A complicated and entangled network of DNA damage response (DDR) mechanisms, including multiple DNA repair pathways, damage tolerance processes, and cell-cycle checkpoints safeguard genomic integrity. Like transcription and replication, DDR is a chromatin-associated process that is generally tightly controlled in time and space. As DNA damage can occur at any time on any genomic location, a specialized spatio-temporal orchestration of this defense apparatus is required. PMID:20980439

  10. High Potency of a Novel Resveratrol Derivative, 3,3',4,4'-Tetrahydroxy-trans-stilbene, against Ovarian Cancer Is Associated with an Oxidative Stress-Mediated Imbalance between DNA Damage Accumulation and Repair.

    PubMed

    Mikuła-Pietrasik, Justyna; Sosińska, Patrycja; Murias, Marek; Wierzchowski, Marcin; Brewińska-Olchowik, Marta; Piwocka, Katarzyna; Szpurek, Dariusz; Książek, Krzysztof

    2015-01-01

    We explored the effect of a new resveratrol (RVT) derivative, 3,3',4,4'-tetrahydroxy-trans-stilbene (3,3',4,4'-THS), on viability, apoptosis, proliferation, and senescence of three representative lines of ovarian cancer cells, that is, A2780, OVCAR-3, and SKOV-3, in vitro. In addition, the mechanistic aspects of 3,3',4,4'-THS activity, including cell redox homeostasis (the production of reactive oxygen species, activity of enzymatic antioxidants, and magnitude of DNA damage accumulation and repair), and the activity of caspases (3, 8, and 9) and p38 MAPK were examined. The study showed that 3,3',4,4'-THS affects cancer cell viability much more efficiently than its parent drug. This effect coincided with increased generation of reactive oxygen species, downregulated activity of superoxide dismutase and catalase, and excessive accumulation of 8-hydroxy-2'-deoxyguanosine and its insufficient repair due to decreased expression of DNA glycosylase I. Cytotoxicity elicited by 3,3',4,4'-THS was related to increased incidence of apoptosis, which was mediated by caspases 3 and 9. Moreover, 3,3',4,4'-THS inhibited cancer cell proliferation and accelerated senescence, which was accompanied by the activation of p38 MAPK. Collectively, our findings indicate that 3,3',4,4'-THS may constitute a valuable tool in the fight against ovarian malignancy and that the anticancer capabilities of this stilbene proceed in an oxidative stress-dependent mechanism. PMID:26229578

  11. Replicative DNA polymerases.

    PubMed

    Johansson, Erik; Dixon, Nicholas

    2013-06-01

    In 1959, Arthur Kornberg was awarded the Nobel Prize for his work on the principles by which DNA is duplicated by DNA polymerases. Since then, it has been confirmed in all branches of life that replicative DNA polymerases require a single-stranded template to build a complementary strand, but they cannot start a new DNA strand de novo. Thus, they also depend on a primase, which generally assembles a short RNA primer to provide a 3'-OH that can be extended by the replicative DNA polymerase. The general principles that (1) a helicase unwinds the double-stranded DNA, (2) single-stranded DNA-binding proteins stabilize the single-stranded DNA, (3) a primase builds a short RNA primer, and (4) a clamp loader loads a clamp to (5) facilitate the loading and processivity of the replicative polymerase, are well conserved among all species. Replication of the genome is remarkably robust and is performed with high fidelity even in extreme environments. Work over the last decade or so has confirmed (6) that a common two-metal ion-promoted mechanism exists for the nucleotidyltransferase reaction that builds DNA strands, and (7) that the replicative DNA polymerases always act as a key component of larger multiprotein assemblies, termed replisomes. Furthermore (8), the integrity of replisomes is maintained by multiple protein-protein and protein-DNA interactions, many of which are inherently weak. This enables large conformational changes to occur without dissociation of replisome components, and also means that in general replisomes cannot be isolated intact. PMID:23732474

  12. Many Ways to Loop DNA

    PubMed Central

    Griffith, Jack D.

    2013-01-01

    In the 1960s, I developed methods for directly visualizing DNA and DNA-protein complexes using an electron microscope. This made it possible to examine the shape of DNA and to visualize proteins as they fold and loop DNA. Early applications included the first visualization of true nucleosomes and linkers and the demonstration that repeating tracts of adenines can cause a curvature in DNA. The binding of DNA repair proteins, including p53 and BRCA2, has been visualized at three- and four-way junctions in DNA. The trombone model of DNA replication was directly verified, and the looping of DNA at telomeres was discovered. PMID:24005675

  13. Nanoparticle bridge DNA biosensor

    NASA Astrophysics Data System (ADS)

    Huang, Hong-Wen

    A new DNA sensing method is demonstrated in which DNA hybridization events lead to the formation of nanoparticle satellites that bridge two electrodes and are detected electrically. The hybridization events are exclusively carried out only on specific locations, the surfaces of C-ssDNA modified 50 nm GNPs. The uniqueness of this work is that only a small number of T-ccDNA molecules (<10) is required to form the nanoparticle satellites, allowing ultra-sensitive DNA sensing. The principle of this new DNA sensing technique has been demonstrated using target DNA and three-base-pair-mismatched DNA in 20nM concentrations. Three single-stranded DNA (ssDNA) system is used in our experiment which includes Capture-ssDNA (C-ssDNA), Target-ssDNA (T-ssDNA) and Probe-ssDNA (P-ssDNA). Both C-ssDNA and P-ssDNA are modified by a thiol group and can hybridize with different portions of T-ssDNA. T-ssDNA requires no modification in three ssDNA system, which is beneficial in many applications. C-ssDNA modified 50nm gold nanoparticle (C-50au) and P-ssDNA modified 30nm gold nanoparticle (P-30au) are prepared through the reaction of thiol-gold chemical bonding between thiolated ssDNA and gold nanoparticle (GNP) (C-ssDNA with 50nm GNP, P-ssDNA with 30nm GNP). We controllably place the C-50au only on the SiO2 band surface (˜ 90nm width) between two gold electrodes (source and drain electrodes) by forming positively- and negatively-charged self-assembled monolayers (SAMs) on SiO2 and gold surface, respectively. DNA modified GNP is negatively charged due to ionization of phosphate group on DNA back bone. C-50au therefore is negatively charged and can only be attracted toward SiO2 area (repelled by negatively charged gold electrode surface). The amine group of positively-charged SAMs on SiO2 surface is then passivated by converting to non-polar methyl functional group after C-50au placement. P-30au is first hybridized with T-ssDNA in the solution phase (T-P- 30au formed) and is introduced

  14. Curating DNA specimens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA data are used in a variety of ethnobiological disciplines including archaeology, conservation, ecology, medicinal plants and natural products research, taxonomy and systematics, crop evolution and domestication, and genetic diversity. It frequently is convenient to store and share DNA among coop...

  15. Routine DNA testing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Routine DNA testing. It’s done once you’ve Marker-Assisted Breeding Pipelined promising Qantitative Trait Loci within your own breeding program and thereby established the performance-predictive power of each DNA test for your germplasm under your conditions. By then you are ready to screen your par...

  16. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  17. MICROWAVE RESONANCES IN DNA

    EPA Science Inventory

    This report describes spectroscopic studies of DNA which were undertaken to better understand a physical basis for microwave absorption by this molecule. hree types of studies are described. ) The low frequency scattered light spectrum of DNA was studied by two methods. irst, Ram...

  18. Replicating repetitive DNA.

    PubMed

    Tognetti, Silvia; Speck, Christian

    2016-05-27

    The function and regulation of repetitive DNA, the 'dark matter' of the genome, is still only rudimentarily understood. Now a study investigating DNA replication of repetitive centromeric chromosome segments has started to expose a fascinating replication program that involves suppression of ATR signalling, in particular during replication stress. PMID:27230530

  19. Hydrogels: DNA bulks up

    NASA Astrophysics Data System (ADS)

    Labean, Thom

    2006-10-01

    Since the 1940s DNA has been known as the genetic material connected to heredity, and from the early 1980s it has also been considered as a potential structural material for nanoscale construction. Now, a hydrogel made entirely of DNA brings this molecule into the realm of bulk materials.

  20. Translesion DNA synthesis

    PubMed Central

    Vaisman, Alexandra; McDonald, John P.; Woodgate, Roger

    2014-01-01

    All living organisms are continually exposed to agents that damage their DNA, which threatens the integrity of their genome. As a consequence, cells are equipped with a plethora of DNA repair enzymes to remove the damaged DNA. Unfortunately, situations nevertheless arise where lesions persist, and these lesions block the progression of the cell’s replicase. Under these situations, cells are forced to choose between recombination-mediated “damage avoidance” pathways, or use a specialized DNA polymerase (pol) to traverse the blocking lesion. The latter process is referred to as Translesion DNA Synthesis (TLS). As inferred by its name, TLS not only results in bases being (mis)incorporated opposite DNA lesions, but also downstream of the replicase-blocking lesion, so as to ensure continued genome duplication and cell survival. Escherichia coli and Salmonella typhimurium possess five DNA polymerases, and while all have been shown to facilitate TLS under certain experimental conditions, it is clear that the LexA-regulated and damage-inducible pols II, IV and V perform the vast majority of TLS under physiological conditions. Pol V can traverse a wide range of DNA lesions and performs the bulk of mutagenic TLS, whereas pol II and pol IV appear to be more specialized TLS polymerases. PMID:26442823

  1. Characterization of muntjac DNA

    SciTech Connect

    Davis, R.C.

    1981-05-27

    Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange.

  2. DNA as information.

    PubMed

    Wills, Peter R

    2016-03-13

    This article reviews contributions to this theme issue covering the topic 'DNA as information' in relation to the structure of DNA, the measure of its information content, the role and meaning of information in biology and the origin of genetic coding as a transition from uninformed to meaningful computational processes in physical systems. PMID:26857666

  3. DNA-cell conjugates

    DOEpatents

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

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

    PubMed Central

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

    2015-01-01

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

  5. Curcumin protects against cytotoxic and inflammatory effects of quartz particles but causes oxidative DNA damage in a rat lung epithelial cell line.

    PubMed

    Li, Hui; van Berlo, Damien; Shi, Tingming; Speit, Günter; Knaapen, Ad M; Borm, Paul J A; Albrecht, Catrin; Schins, Roel P F

    2008-02-15

    Chronic inhalation of high concentrations of respirable quartz particles has been implicated in various lung diseases including lung fibrosis and cancer. Generation of reactive oxygen species (ROS) and oxidative stress is considered a major mechanism of quartz toxicity. Curcumin, a yellow pigment from Curcuma longa, has been considered as nutraceutical because of its strong anti-inflammatory, antitumour and antioxidant properties. The aim of our present study was to investigate whether curcumin can protect lung epithelial cells from the cytotoxic, genotoxic and inflammatory effects associated with quartz (DQ12) exposure. Electron paramagnetic resonance (EPR) measurements using the spin-trap DMPO demonstrated that curcumin reduces hydrogen peroxide-dependent hydroxyl-radical formation by quartz. Curcumin was also found to reduce quartz-induced cytotoxicity and cyclooxygenase 2 (COX-2) mRNA expression in RLE-6TN rat lung epithelial cells (RLE). Curcumin also inhibited the release of macrophage inflammatory protein-2 (MIP-2) from RLE cells as observed upon treatment with interleukin-1 beta (IL-1beta) and tumour necrosis factor-alpha (TNFalpha). However, curcumin failed to protect the RLE cells from oxidative DNA damage induced by quartz, as shown by formamidopyrimidine glycosylase (FPG)-modified comet assay and by immunocytochemistry for 8-hydroxydeoxyguanosine. In contrast, curcumin was found to be a strong inducer of oxidative DNA damage itself at non-cytotoxic and anti-inflammatory concentrations. In line with this, curcumin also enhanced the mRNA expression of the oxidative stress response gene heme oxygenase-1 (ho-1). Curcumin also caused oxidative DNA damage in NR8383 rat alveolar macrophages and A549 human lung epithelial cells. Taken together, these observations indicate that one should be cautious in considering the potential use of curcumin in the prevention or treatment of lung diseases associated with quartz exposure. PMID:18001810

  6. An oxidative DNA "damage" and repair mechanism localized in the VEGF promoter is important for hypoxia-induced VEGF mRNA expression.

    PubMed

    Pastukh, Viktor; Roberts, Justin T; Clark, David W; Bardwell, Gina C; Patel, Mita; Al-Mehdi, Abu-Bakr; Borchert, Glen M; Gillespie, Mark N

    2015-12-01

    In hypoxia, mitochondria-generated reactive oxygen species not only stimulate accumulation of the transcriptional regulator of hypoxic gene expression, hypoxia inducible factor-1 (Hif-1), but also cause oxidative base modifications in hypoxic response elements (HREs) of hypoxia-inducible genes. When the hypoxia-induced base modifications are suppressed, Hif-1 fails to associate with the HRE of the VEGF promoter, and VEGF mRNA accumulation is blunted. The mechanism linking base modifications to transcription is unknown. Here we determined whether recruitment of base excision DNA repair (BER) enzymes in response to hypoxia-induced promoter modifications was required for transcription complex assembly and VEGF mRNA expression. Using chromatin immunoprecipitation analyses in pulmonary artery endothelial cells, we found that hypoxia-mediated formation of the base oxidation product 8-oxoguanine (8-oxoG) in VEGF HREs was temporally associated with binding of Hif-1α and the BER enzymes 8-oxoguanine glycosylase 1 (Ogg1) and redox effector factor-1 (Ref-1)/apurinic/apyrimidinic endonuclease 1 (Ape1) and introduction of DNA strand breaks. Hif-1α colocalized with HRE sequences harboring Ref-1/Ape1, but not Ogg1. Inhibition of BER by small interfering RNA-mediated reduction in Ogg1 augmented hypoxia-induced 8-oxoG accumulation and attenuated Hif-1α and Ref-1/Ape1 binding to VEGF HRE sequences and blunted VEGF mRNA expression. Chromatin immunoprecipitation-sequence analysis of 8-oxoG distribution in hypoxic pulmonary artery endothelial cells showed that most of the oxidized base was localized to promoters with virtually no overlap between normoxic and hypoxic data sets. Transcription of genes whose promoters lost 8-oxoG during hypoxia was reduced, while those gaining 8-oxoG was elevated. Collectively, these findings suggest that the BER pathway links hypoxia-induced introduction of oxidative DNA modifications in promoters of hypoxia-inducible genes to transcriptional

  7. Curcumin protects against cytotoxic and inflammatory effects of quartz particles but causes oxidative DNA damage in a rat lung epithelial cell line

    SciTech Connect

    Li Hui; Berlo, Damien van; Shi Tingming; Speit, Guenter; Knaapen, Ad M.; Borm, Paul J.A.; Albrecht, Catrin; Schins, Roel P.F.

    2008-02-15

    Chronic inhalation of high concentrations of respirable quartz particles has been implicated in various lung diseases including lung fibrosis and cancer. Generation of reactive oxygen species (ROS) and oxidative stress is considered a major mechanism of quartz toxicity. Curcumin, a yellow pigment from Curcuma longa, has been considered as nutraceutical because of its strong anti-inflammatory, antitumour and antioxidant properties. The aim of our present study was to investigate whether curcumin can protect lung epithelial cells from the cytotoxic, genotoxic and inflammatory effects associated with quartz (DQ12) exposure. Electron paramagnetic resonance (EPR) measurements using the spin-trap DMPO demonstrated that curcumin reduces hydrogen peroxide-dependent hydroxyl-radical formation by quartz. Curcumin was also found to reduce quartz-induced cytotoxicity and cyclooxygenase 2 (COX-2) mRNA expression in RLE-6TN rat lung epithelial cells (RLE). Curcumin also inhibited the release of macrophage inflammatory protein-2 (MIP-2) from RLE cells as observed upon treatment with interleukin-1 beta (IL-1{beta}) and tumour necrosis factor-alpha (TNF{alpha}). However, curcumin failed to protect the RLE cells from oxidative DNA damage induced by quartz, as shown by formamidopyrimidine glycosylase (FPG)-modified comet assay and by immunocytochemistry for 8-hydroxydeoxyguanosine. In contrast, curcumin was found to be a strong inducer of oxidative DNA damage itself at non-cytotoxic and anti-inflammatory concentrations. In line with this, curcumin also enhanced the mRNA expression of the oxidative stress response gene heme oxygenase-1 (ho-1). Curcumin also caused oxidative DNA damage in NR8383 rat alveolar macrophages and A549 human lung epithelial cells. Taken together, these observations indicate that one should be cautious in considering the potential use of curcumin in the prevention or treatment of lung diseases associated with quartz exposure.

  8. Premeltons in DNA.

    PubMed

    Sobell, Henry M

    2016-03-01

    Premeltons are examples of emergent-structures (i.e., structural-solitons) that arise spontaneously in DNA due to the presence of nonlinear-excitations in its structure. They are of two kinds: B-B (or A-A) premeltons form at specific DNA-regions to nucleate site-specific DNA melting. These are stationary and, being globally-nontopological, undergo breather-motions that allow drugs and dyes to intercalate into DNA. B-A (or A-B) premeltons, on the other hand, are mobile, and being globally-topological, act as phase-boundaries transforming B- into A-DNA during the structural phase-transition. They are not expected to undergo breather motions. A key feature of both types of premeltons is the presence of an intermediate structural-form in their central regions (proposed as being a transition-state intermediate in DNA-melting and in the B- to A-transition), which differs from either A- or B-DNA. Called beta-DNA, this is both metastable and hyperflexible--and contains an alternating sugar-puckering pattern along the polymer backbone combined with the partial unstacking (in its lower energy-forms) of every-other base-pair. Beta-DNA is connected to either B- or to A-DNA on either side by boundaries possessing a gradation of nonlinear structural-change, these being called the kink and the antikink regions. The presence of premeltons in DNA leads to a unifying theory to understand much of DNA physical chemistry and molecular biology. In particular, premeltons are predicted to define the 5' and 3' ends of genes in naked-DNA and DNA in active-chromatin, this having important implications for understanding physical aspects of the initiation, elongation and termination of RNA-synthesis during transcription. For these and other reasons, the model will be of broader interest to the general-audience working in these areas. The model explains a wide variety of data, and carries with it a number of experimental predictions--all readily testable--as will be described in this review

  9. Advances in DNA photonics

    NASA Astrophysics Data System (ADS)

    Heckman, Emily M.; Aga, Roberto S.; Fehrman Cory, Emily M.; Ouchen, Fahima; Lesko, Alyssa; Telek, Brian; Lombardi, Jack; Bartsch, Carrie M.; Grote, James G.

    2012-10-01

    In this paper we present our current research in exploring a DNA biopolymer for photonics applications. A new processing technique has been adopted that employs a modified soxhlet-dialysis (SD) rinsing technique to completely remove excess ionic contaminants from the DNA biopolymer, resulting in a material with greater mechanical stability and enhanced performance reproducibility. This newly processed material has been shown to be an excellent material for cladding layers in poled polymer electro-optic (EO) waveguide modulator applications. Thin film poling results are reported for materials using the DNA biopolymer as a cladding layer, as are results for beam steering devices also using the DNA biopolymer. Finally, progress on fabrication of a Mach Zehnder EO modulator with DNA biopolymer claddings using nanoimprint lithography techniques is reported.

  10. Archaeal DNA replication.

    PubMed

    Kelman, Lori M; Kelman, Zvi

    2014-01-01

    DNA replication is essential for all life forms. Although the process is fundamentally conserved in the three domains of life, bioinformatic, biochemical, structural, and genetic studies have demonstrated that the process and the proteins involved in archaeal DNA replication are more similar to those in eukaryal DNA replication than in bacterial DNA replication, but have some archaeal-specific features. The archaeal replication system, however, is not monolithic, and there are some differences in the replication process between different species. In this review, the current knowledge of the mechanisms governing DNA replication in Archaea is summarized. The general features of the replication process as well as some of the differences are discussed. PMID:25421597

  11. DNA Align Editor: DNA Alignment Editor Tool

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The SNPAlignEditor is a DNA sequence alignment editor that runs on Windows platforms. The purpose of the program is to provide an intuitive, user-friendly tool for manual editing of multiple sequence alignments by providing functions for input, editing, and output of nucleotide sequence alignments....

  12. Studying DNA in the Classroom.

    ERIC Educational Resources Information Center

    Zarins, Silja

    1993-01-01

    Outlines a workshop for teachers that illustrates a method of extracting DNA and provides instructions on how to do some simple work with DNA without sophisticated and expensive equipment. Provides details on viscosity studies and breaking DNA molecules. (DDR)

  13. Simple & Safe Genomic DNA Isolation.

    ERIC Educational Resources Information Center

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A procedure for purifying DNA using either bacteria or rat liver is presented. Directions for doing a qualitative DNA assay using diphenylamine and a quantitative DNA assay using spectroscopy are included. (KR)

  14. Quantitive DNA Fiber Mapping

    SciTech Connect

    Lu, Chun-Mei; Wang, Mei; Greulich-Bode, Karin M.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-01-28

    Several hybridization-based methods used to delineate single copy or repeated DNA sequences in larger genomic intervals take advantage of the increased resolution and sensitivity of free chromatin, i.e., chromatin released from interphase cell nuclei. Quantitative DNA fiber mapping (QDFM) differs from the majority of these methods in that it applies FISH to purified, clonal DNA molecules which have been bound with at least one end to a solid substrate. The DNA molecules are then stretched by the action of a receding meniscus at the water-air interface resulting in DNA molecules stretched homogeneously to about 2.3 kb/{micro}m. When non-isotopically, multicolor-labeled probes are hybridized to these stretched DNA fibers, their respective binding sites are visualized in the fluorescence microscope, their relative distance can be measured and converted into kilobase pairs (kb). The QDFM technique has found useful applications ranging from the detection and delineation of deletions or overlap between linked clones to the construction of high-resolution physical maps to studies of stalled DNA replication and transcription.

  15. Reversible DNA compaction.

    PubMed

    González-Pérez, Alfredo

    2014-01-01

    In this review we summarize and discuss the different methods we can use to achieve reversible DNA compaction in vitro. Reversible DNA compaction is a natural process that occurs in living cells and viruses. As a result these process long sequences of DNA can be concentrated in a small volume (compacted) to be decompacted only when the information carried by the DNA is needed. In the current work we review the main artificial compacting agents looking at their suitability for decompaction. The different approaches used for decompaction are strongly influenced by the nature of the compacting agent that determines the mechanism of compaction. We focus our discussion on two main artificial compacting agents: multivalent cations and cationic surfactants that are the best known compacting agents. The reversibility of the process can be achieved by adding chemicals like divalent cations, alcohols, anionic surfactants, cyclodextrins or by changing the chemical nature of the compacting agents via pH modifications, light induced conformation changes or by redox-reactions. We stress the relevance of electrostatic interactions and self-assembly as a main approach in order to tune up the DNA conformation in order to create an on-off switch allowing a transition between coil and compact states. The recent advances to control DNA conformation in vitro, by means of molecular self-assembly, result in a better understanding of the fundamental aspects involved in the DNA behavior in vivo and serve of invaluable inspiration for the development of potential biomedical applications. PMID:24444152

  16. Tracking Mitochondrial DNA In Situ.

    PubMed

    Ligasová, Anna; Koberna, Karel

    2016-01-01

    The methods of the detection of (1) non-labeled and (2) BrdU-labeled mitochondrial DNA (mtDNA) are described. They are based on the production of singlet oxygen by monovalent copper ions and the subsequent induction of DNA gaps. The ends of interrupted DNA serve as origins for the labeling of mtDNA by DNA polymerase I or they are utilized by exonuclease that degrades DNA strands, unmasking BrdU in BrdU-labeled DNA. Both methods are sensitive approaches without the need of additional enhancement of the signal or the use of highly sensitive optical systems. PMID:26530676

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

    DOEpatents

    McCutchen-Maloney, Sandra L.

    2002-01-01

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

  18. DNA-PK assay

    DOEpatents

    Anderson, Carl W.; Connelly, Margery A.

    2004-10-12

    The present invention provides a method for detecting DNA-activated protein kinase (DNA-PK) activity in a biological sample. The method includes contacting a biological sample with a detectably-labeled phosphate donor and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. A compostion and a kit for the detection of DNA-PK activity are also provided. Methods for detecting DNA, protein phosphatases and substances that alter the activity of DNA-PK are also provided. The present invention also provides a method of monitoring protein kinase and DNA-PK activity in living cells. -A composition and a kit for monitoring protein kinase activity in vitro and a composition and a kit for monitoring DNA-PK activities in living cells are also provided. A method for identifying agents that alter protein kinase activity in vitro and a method for identifying agents that alter DNA-PK activity in living cells are also provided.

  19. DNA Mismatch Repair

    PubMed Central

    MARINUS, M. G.

    2014-01-01

    DNA mismatch repair functions to correct replication errors in newly synthesized DNA and to prevent recombination between related, but not identical (homeologous), DNA sequences. The mechanism of mismatch repair is best understood in Escherichia coli and is the main focus of this review. The early genetic studies of mismatch repair are described as a basis for the subsequent biochemical characterization of the system. The effects of mismatch repair on homologous and homeologous recombination are described. The relationship of mismatch repair to cell toxicity induced by various drugs is included. The VSP (Very Short Patch) repair system is described in detail. PMID:26442827

  20. Focus: DNA probes

    SciTech Connect

    Not Available

    1986-11-01

    Progress in the development of DNA probes for the identification and quantitation of specific genetic sequences in biological samples is reviewed. Current research efforts in the development of DNA probes for the diagnosis of a wide variety of bacterial, viral, and other infectious diseases, such as herpes simplex and cytomegalovirus, and inherited genetic diseases such as cystic fibrosis and sickle cell anemia are discussed. Progress in development of DNA probe assays for cancer diagnosis, detection of Salmonella food poisoning, tissue typing (detection of histocompatibility antigens), mutagen screening, and animal diseases, among other applications is included.

  1. Interaction of DNA and DNA-anti-DNA complexes to fibronectin

    SciTech Connect

    Gupta, R.C.; Simpson, W.A.; Raghow, R.; Hasty, K.

    1986-03-01

    Fibronectin (Fn) is