Transient and Switchable (Triethylsilyl)ethynyl Protection of DNA against Cleavage by Restriction Endonucleases

Czech Academy of Sciences Publication Activity Database

Kielkowski, Pavel; Macíčková-Cahová, Hana; Pohl, Radek; Hocek, Michal

2011-01-01

Roč. 50, č. 37 (2011), s. 8727-8730 ISSN 1433-7851 R&D Projects: GA ČR GA203/09/0317 Institutional research plan: CEZ:AV0Z40550506 Keywords : alkynes * DNA * protecting groups * nucleotides * restriction endonucleases Subject RIV: CC - Organic Chemistry Impact factor: 13.455, year: 2011

Cleavage of DNA containing 5-fluorocytosine or 5-fluorouracil by type II restriction endonucleases

Czech Academy of Sciences Publication Activity Database

Olszewska, Agata; Daďová, Jitka; Mačková, Michaela; Hocek, Michal

2015-01-01

Roč. 23, č. 21 (2015), s. 6885-6890 ISSN 0968-0896 R&D Projects: GA ČR GA14-04289S Institutional support: RVO:61388963 Keywords : modified nucleotides * DNA * restriction endonucleases * DNA polymerase * pyrimidine nucleosides Subject RIV: CC - Organic Chemistry Impact factor: 2.923, year: 2015

Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

Energy Technology Data Exchange (ETDEWEB)

Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.; Lashkov, A. A., E-mail: alashkov83@gmail.com [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

2012-05-15

Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized by interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.

Purification and characterization of VDE, a site-specific endonuclease from the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Gimble, F S; Thorner, J

1993-10-15

The 119-kDa primary translation product of the VMA1 gene of Saccharomyces cerevisiae undergoes a self-catalyzed rearrangement ("protein splicing") that excises an internal 50-kDa segment of the polypeptide and joins the amino-terminal and carboxyl-terminal segments to generate the 69-kDa subunit of the vacuolar membrane-associated H(+)-ATPase. We have shown previously that the internal segment is a site-specific endonuclease (Gimble, F. S., and Thorner, J. (1992) Nature 357, 301-306). Here we describe methods for the high level expression and purification to near homogeneity of both the authentic VMA1-derived endonuclease (or VDE) from yeast (yield 18%) and a recombinant form of VDE made in bacteria (yield 29%). Detailed characterization of these preparations demonstrated that the yeast-derived and bacterially produced enzymes were indistinguishable, as judged by: (a) behavior during purification; (b) apparent native molecular mass (50 kDa); (c) immunological reactivity; and (d) catalytic properties (specific activity; cleavage site recognition; and optima for pH, temperature, divalent cation and ionic strength). The minimal site required for VDE cleavage was delimited to a 30-base pair sequence within its specific substrate (the VMA1 delta vde allele).

Purification, crystallization, X-ray diffraction analysis and phasing of an engineered single-chain PvuII restriction endonuclease

International Nuclear Information System (INIS)

Meramveliotaki, Chrysi; Kotsifaki, Dina; Androulaki, Maria; Hountas, Athanasios; Eliopoulos, Elias; Kokkinidis, Michael

2007-01-01

PvuII is the first type II restriction endonuclease to be converted from its wild-type homodimeric form into an enzymatically active single-chain variant. The enzyme was crystallized and phasing was successfully performed by molecular replacement. The restriction endonuclease PvuII from Proteus vulgaris has been converted from its wild-type homodimeric form into the enzymatically active single-chain variant scPvuII by tandemly joining the two subunits through the peptide linker Gly-Ser-Gly-Gly. scPvuII, which is suitable for the development of programmed restriction endonucleases for highly specific DNA cleavage, was purified and crystallized. The crystals diffract to a resolution of 2.35 Å and belong to space group P4 2 , with unit-cell parameters a = b = 101.92, c = 100.28 Å and two molecules per asymmetric unit. Phasing was successfully performed by molecular replacement

Quantification of DNA cleavage specificity in Hi-C experiments.

Science.gov (United States)

Meluzzi, Dario; Arya, Gaurav

2016-01-08

Hi-C experiments produce large numbers of DNA sequence read pairs that are typically analyzed to deduce genomewide interactions between arbitrary loci. A key step in these experiments is the cleavage of cross-linked chromatin with a restriction endonuclease. Although this cleavage should happen specifically at the enzyme's recognition sequence, an unknown proportion of cleavage events may involve other sequences, owing to the enzyme's star activity or to random DNA breakage. A quantitative estimation of these non-specific cleavages may enable simulating realistic Hi-C read pairs for validation of downstream analyses, monitoring the reproducibility of experimental conditions and investigating biophysical properties that correlate with DNA cleavage patterns. Here we describe a computational method for analyzing Hi-C read pairs to estimate the fractions of cleavages at different possible targets. The method relies on expressing an observed local target distribution downstream of aligned reads as a linear combination of known conditional local target distributions. We validated this method using Hi-C read pairs obtained by computer simulation. Application of the method to experimental Hi-C datasets from murine cells revealed interesting similarities and differences in patterns of cleavage across the various experiments considered. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Yeast redoxyendonuclease, a DNA repair enzyme similar to Escherichia coli endonuclease III

International Nuclear Information System (INIS)

Gossett, J.; Lee, K.; Cunningham, R.P.; Doetsch, P.W.

1988-01-01

A DNA repair endonuclease (redoxyendonuclease) was isolated from bakers' yeast (Saccharomyces cerevisiae). The enzyme has been purified by a series of column chromatography steps and cleaves OsO 4 -damaged, double-stranded DNA at sites of thymine glycol and heavily UV-irradiated DNA at sites of cytosine, thymine, and guanine photoproducts. The base specificity and mechanism of phosphodiester bond cleavage for the yeast redoxyendonuclease appear to be identical with those of Escherichia coli endonuclease III when thymine glycol containing, end-labeled DNA fragments of defined sequence are employed as substrates. Yeast redoxyendonuclease has an apparent molecular size of 38,000-42,000 daltons and is active in the absence of divalent metal cations. The identification of such an enzyme in yeast may be of value in the elucidation of the biochemical basis for radiation sensitivity in certain yeast mutants

Human RECQL5beta stimulates flap endonuclease 1

DEFF Research Database (Denmark)

Speina, Elzbieta; Dawut, Lale; Hedayati, Mohammad

2010-01-01

devoid of RECQL1 and RECQL5 display increased chromosomal instability. Here, we report the physical and functional interaction of the large isomer of RECQL5, RECQL5beta, with the human flap endonuclease 1, FEN1, which plays a critical role in DNA replication, recombination and repair. RECQL5beta...... dramatically stimulates the rate of FEN1 cleavage of flap DNA substrates. Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests...

Selective inhibition by harmane of the apurinic apyrimidinic endonuclease activity of phage T4-induced UV endonuclease.

Science.gov (United States)

Warner, H R; Persson, M L; Bensen, R J; Mosbaugh, D W; Linn, S

1981-11-25

1-Methyl-9H-pyrido-[3,4-b]indole (harmane) inhibits the apurinic/apyrimidinic (AP) endonuclease activity of the UV endonuclease induced by phage T4, whereas it stimulates the pyrimidine dimer-DNA glycosylase activity of that enzyme. E. coli endonuclease IV, E. coli endonuclease VI (the AP endonuclease activity associated with E. coli exonuclease III), and E. coli uracil-DNA glycosylase were not inhibited by harmane. Human fibroblast AP endonucleases I and II also were only slightly inhibited. Therefore, harmane is neither a general inhibitor of AP endonucleases, nor a general inhibitor of Class I AP endonucleases which incise DNA on the 3'-side of AP sites. However, E. coli endonuclease III and its associated dihydroxythymine-DNA glycosylase activity were both inhibited by harmane. This observation suggests that harmane may inhibit only AP endonucleases which have associated glycosylase activities.

Ultrafast spectroscopy on DNA-cleavage by endonuclease in molecular crowding.

Science.gov (United States)

Singh, Priya; Choudhury, Susobhan; Dutta, Shreyasi; Adhikari, Aniruddha; Bhattacharya, Siddhartha; Pal, Debasish; Pal, Samir Kumar

2017-10-01

The jam-packed intracellular environments differ the activity of a biological macromolecule from that in laboratory environments (in vitro) through a number of mechanisms called molecular crowding related to structure, function and dynamics of the macromolecule. Here, we have explored the structure, function and dynamics of a model enzyme protein DNase I in molecular crowing of polyethylene glycol (PEG; MW 3350). We have used steady state and picosecond resolved dynamics of a well-known intercalator ethidium bromide (EB) in a 20-mer double-stranded DNA (dsDNA) to monitor the DNA-cleavage by the enzyme in absence and presence PEG. We have also labelled the enzyme by a well-known fluorescent probe 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS) to study the molecular mechanism of the protein-DNA association through exited state relaxation of the probe in absence (dictated by polarity) and presence of EB in the DNA (dictated by Förster resonance energy transfer (FRET)). The overall and local structures of the protein in presence of PEG have been followed by circular dichroism and time resolved polarization gated spectroscopy respectively. The enhanced dynamical flexibility of protein in presence of PEG as revealed from excited state lifetime and polarization gated anisotropy of ANS has been correlated with the stronger DNA-binding for the higher nuclease activity. We have also used conventional experimental strategy of agarose gel electrophoresis to monitor DNA-cleavage and found consistent results of enhanced nuclease activities both on synthetic 20-mer oligonucleotide and long genomic DNA from calf thymus. Copyright © 2017 Elsevier B.V. All rights reserved.

Divalent metal ion differentially regulates the sequential nicking reactions of the GIY-YIG homing endonuclease I-BmoI.

Directory of Open Access Journals (Sweden)

Benjamin P Kleinstiver

Full Text Available Homing endonucleases are site-specific DNA endonucleases that function as mobile genetic elements by introducing double-strand breaks or nicks at defined locations. Of the major families of homing endonucleases, the modular GIY-YIG endonucleases are least understood in terms of mechanism. The GIY-YIG homing endonuclease I-BmoI generates a double-strand break by sequential nicking reactions during which the single active site of the GIY-YIG nuclease domain must undergo a substantial reorganization. Here, we show that divalent metal ion plays a significant role in regulating the two independent nicking reactions by I-BmoI. Rate constant determination for each nicking reaction revealed that limiting divalent metal ion has a greater impact on the second strand than the first strand nicking reaction. We also show that substrate mutations within the I-BmoI cleavage site can modulate the first strand nicking reaction over a 314-fold range. Additionally, in-gel DNA footprinting with mutant substrates and modeling of an I-BmoI-substrate complex suggest that amino acid contacts to a critical GC-2 base pair are required to induce a bottom-strand distortion that likely directs conformational changes for reaction progress. Collectively, our data implies mechanistic roles for divalent metal ion and substrate bases, suggesting that divalent metal ion facilitates the re-positioning of the GIY-YIG nuclease domain between sequential nicking reactions.

Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis.

Science.gov (United States)

Abeldenov, Sailau; Talhaoui, Ibtissam; Zharkov, Dmitry O; Ishchenko, Alexander A; Ramanculov, Erlan; Saparbaev, Murat; Khassenov, Bekbolat

2015-09-01

Apurinic/apyrimidinic (AP) endonucleases are key enzymes involved in the repair of abasic sites and DNA strand breaks. Pathogenic bacteria Mycobacterium tuberculosis contains two AP endonucleases: MtbXthA and MtbNfo members of the exonuclease III and endonuclease IV families, which are exemplified by Escherichia coli Xth and Nfo, respectively. It has been shown that both MtbXthA and MtbNfo contain AP endonuclease and 3'→5' exonuclease activities. However, it remains unclear whether these enzymes hold 3'-repair phosphodiesterase and nucleotide incision repair (NIR) activities. Here, we report that both mycobacterial enzymes have 3'-repair phosphodiesterase and 3'-phosphatase, and MtbNfo contains in addition a very weak NIR activity. Interestingly, depending on pH, both enzymes require different concentrations of divalent cations: 0.5mM MnCl2 at pH 7.6 and 10 mM at pH 6.5. MtbXthA requires a low ionic strength and 37 °C, while MtbNfo requires high ionic strength (200 mM KCl) and has a temperature optimum at 60 °C. Point mutation analysis showed that D180 and N182 in MtbXthA and H206 and E129 in MtbNfo are critical for enzymes activities. The steady-state kinetic parameters indicate that MtbXthA removes 3'-blocking sugar-phosphate and 3'-phosphate moieties at DNA strand breaks with an extremely high efficiency (kcat/KM=440 and 1280 μM(-1)∙min(-1), respectively), while MtbNfo exhibits much lower 3'-repair activities (kcat/KM=0.26 and 0.65 μM(-1)∙min(-1), respectively). Surprisingly, both MtbXthA and MtbNfo exhibited very weak AP site cleavage activities, with kinetic parameters 100- and 300-fold lower, respectively, as compared with the results reported previously. Expression of MtbXthA and MtbNfo reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to methylmethanesulfonate and H2O2 to various degrees. Taken together, these data establish the DNA substrate specificity of M. tuberculosis AP endonucleases and suggest their possible role

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.

Science.gov (United States)

Sternberg, Samuel H; Redding, Sy; Jinek, Martin; Greene, Eric C; Doudna, Jennifer A

2014-03-06

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

Science.gov (United States)

Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

2014-03-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

Restriction enzyme cleavage of ultraviolet-damaged Simian virus 40 and pBR322 DNA

International Nuclear Information System (INIS)

Cleaver, J.E.

1983-01-01

Cleavage of specific DNA sequences by the restriction enzymes EcoRI, HindIII and TaqI was prevented when the DNA was irradiated with ultraviolet light. Most of the effects were attributed to cyclobutane pyrimidine dimers in the recognition sequences; the effectiveness of irradiation was directly proportional to the number of potential dimer sites in the DNA. Combining EcoRI with dimer-specific endonuclease digestion revealed that pyrimidine dimers blocked cleavage within one base-pair on the strand opposite to the dimer but did not block cleavage three to four base-pairs away on the same strand. These are the probable limits for the range of influence of pyrimidine dimers along the DNA, at least for this enzyme. The effect of irradiation on cleavage by TaqI seemed far greater than expected for the cyclobutane dimer yield, possibly because of effects from photoproducts flanking the tetranucleotide recognition sequence and the effect of non-cyclobutane (6-4)pyrimidine photoproducts involving adjacent T and C bases. (author)

Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage.

Science.gov (United States)

Brok-Volchanskaya, Vera S; Kadyrov, Farid A; Sivogrivov, Dmitry E; Kolosov, Peter M; Sokolov, Andrey S; Shlyapnikov, Michael G; Kryukov, Valentine M; Granovsky, Igor E

2008-04-01

Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3' 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TpsiC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages.

Restriction endonuclease analysis of chloroplast DNA in interspecies somatic Hybrids of Petunia.

Science.gov (United States)

Kumar, A; Cocking, E C; Bovenberg, W A; Kool, A J

1982-12-01

Restriction endonuclease cleavage pattern analysis of chloroplast DNA (cpDNA) of three different interspecific somatic hybrid plants revealed that the cytoplasms of the hybrids contained only cpDNA of P. parodii. The somatic hybrid plants analysed were those between P. parodii (wild type) + P. hybrida (wild type); P. parodii (wild type)+P. inflata (cytoplasmic albino mutant); P. parodii (wild type) + P. parviflora (nuclear albino mutant). The presence of only P. parodii chloroplasts in the somatic hybrid of P. parodii + P. inflata is possibly due to the stringent selection used for somatic hybrid production. However, in the case of the two other somatic hybrids P. parodii + P. hybrida and P. parodii + P. parviflora it was not possible to determine whether the presence of only P. parodii chloroplasts in these somatic hybrid plants was due to the nature of the selection schemes used or simply occurred by chance. The relevance of such somatic hybrid material for the study of genomic-cytoplasmic interaction is discussed, as well as the use of restriction endonuclease fragment patterns for the analysis of taxonomic and evolutionary inter-relationships in the genus Petunia.

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

KAUST Repository

Rashid, Fahad

2017-02-23

Human flap endonuclease 1 (FEN1) and related structure-specific 5\\'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5\\'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually \\'locks\\' protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.

Polymerase-endonuclease amplification reaction (PEAR for large-scale enzymatic production of antisense oligonucleotides.

Directory of Open Access Journals (Sweden)

Xiaolong Wang

Full Text Available Antisense oligonucleotides targeting microRNAs or their mRNA targets prove to be powerful tools for molecular biology research and may eventually emerge as new therapeutic agents. Synthetic oligonucleotides are often contaminated with highly homologous failure sequences. Synthesis of a certain oligonucleotide is difficult to scale up because it requires expensive equipment, hazardous chemicals and a tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR, for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves over 100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation. PEAR has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs.

The elastic network model reveals a consistent picture on intrinsic functional dynamics of type II restriction endonucleases

International Nuclear Information System (INIS)

Uyar, A; Kurkcuoglu, O; Doruker, P; Nilsson, L

2011-01-01

The vibrational dynamics of various type II restriction endonucleases, in complex with cognate/non-cognate DNA and in the apo form, are investigated with the elastic network model in order to reveal common functional mechanisms in this enzyme family. Scissor-like and tong-like motions observed in the slowest modes of all enzymes and their complexes point to common DNA recognition and cleavage mechanisms. Normal mode analysis further points out that the scissor-like motion has an important role in differentiating between cognate and non-cognate sequences at the recognition site, thus implying its catalytic relevance. Flexible regions observed around the DNA-binding site of the enzyme usually concentrate on the highly conserved β-strands, especially after DNA binding. These β-strands may have a structurally stabilizing role in functional dynamics for target site recognition and cleavage. In addition, hot spot residues based on high-frequency modes reveal possible communication pathways between the two distant cleavage sites in the enzyme family. Some of these hot spots also exist on the shortest path between the catalytic sites and are highly conserved

Endonuclease IV Is the major apurinic/apyrimidinic endonuclease in Mycobacterium tuberculosis and is important for protection against oxidative damage.

Directory of Open Access Journals (Sweden)

Rupangi Verma Puri

Full Text Available During the establishment of an infection, bacterial pathogens encounter oxidative stress resulting in the production of DNA lesions. Majority of these lesions are repaired by base excision repair (BER pathway. Amongst these, abasic sites are the most frequent lesions in DNA. Class II apurinic/apyrimidinic (AP endonucleases play a major role in BER of damaged DNA comprising of abasic sites. Mycobacterium tuberculosis, a deadly pathogen, resides in the human macrophages and is continually subjected to oxidative assaults. We have characterized for the first time two AP endonucleases namely Endonuclease IV (End and Exonuclease III (XthA that perform distinct functions in M.tuberculosis. We demonstrate that M.tuberculosis End is a typical AP endonuclease while XthA is predominantly a 3'→5' exonuclease. The AP endonuclease activity of End and XthA was stimulated by Mg(2+ and Ca(2+ and displayed a preferential recognition for abasic site paired opposite to a cytosine residue in DNA. Moreover, End exhibited metal ion independent 3'→5' exonuclease activity while in the case of XthA this activity was metal ion dependent. We demonstrate that End is not only a more efficient AP endonuclease than XthA but it also represents the major AP endonuclease activity in M.tuberculosis and plays a crucial role in defense against oxidative stress.

Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations

Science.gov (United States)

Zuo, Zhicheng; Liu, Jin

2016-11-01

The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. II. Characterization Of the binding and cleavage abilities by site-directed mutagenesis.

OpenAIRE

Komori, K; Ichiyanagi, K; Morikawa, K; Ishino, Y

1999-01-01

PI- Pfu I and PI- Pfu II from Pyrococcus furiosus are homing endonucleases, as shown in the accompanying paper. These two endonucleases are produced by protein splicing from the precursor protein including ribonucleotide reductase (RNR). We show here that both enzymes specifically interact with their substrate DNA and distort the DNA strands by 73 degrees and 67 degrees, respectively. They have two copies of the amino acid sequence motif LAGLIDADG, which is present in the majority of homing e...

Modulation of the Pyrococcus abyssi NucS endonuclease activity by replication clamp at functional and structural levels.

Science.gov (United States)

Creze, Christophe; Ligabue, Alessio; Laurent, Sébastien; Lestini, Roxane; Laptenok, Sergey P; Khun, Joelle; Vos, Marten H; Czjzek, Mirjam; Myllykallio, Hannu; Flament, Didier

2012-05-04

Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5' and 3' flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction.

Modulation of the Pyrococcus abyssi NucS Endonuclease Activity by Replication Clamp at Functional and Structural Levels*

Science.gov (United States)

Creze, Christophe; Ligabue, Alessio; Laurent, Sébastien; Lestini, Roxane; Laptenok, Sergey P.; Khun, Joelle; Vos, Marten H.; Czjzek, Mirjam; Myllykallio, Hannu; Flament, Didier

2012-01-01

Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5′ and 3′ flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction. PMID:22431731

Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage

DEFF Research Database (Denmark)

Stella, Stefano; Alcón, Pablo; Montoya, Guillermo

2017-01-01

involved in DNA unwinding to form a CRISPR RNA (crRNA)-DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner...... and the crRNA-DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1....

Programmable RNA recognition and cleavage by CRISPR/Cas9.

Science.gov (United States)

O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

2014-12-11

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

Science.gov (United States)

Hilbert, Brendan J.; Hayes, Janelle A.; Stone, Nicholas P.; Xu, Rui-Gang

2017-01-01

Abstract Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA. PMID:28082398

Identification and characterization of inhibitors of human apurinic/apyrimidinic endonuclease APE1.

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Anton Simeonov

2009-06-01

Full Text Available APE1 is the major nuclease for excising abasic (AP sites and particular 3'-obstructive termini from DNA, and is an integral participant in the base excision repair (BER pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5-fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC(1280, a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays -- a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen -- and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals.

Endonuclease activities in extracts of Micrococcus luteus that act on. gamma. -irradiated DNA

Energy Technology Data Exchange (ETDEWEB)

Schoen-Bopp, A; Schaefer, G; Hagen, U [Kernforschungszentrum Karlsruhe (Germany, F.R.). Inst. fuer Strahlenbiologie

1977-03-01

Several protein fractions containing endonuclease activity against ..gamma..-irradiated DNA (..gamma..-endonuclease) were isolated from M.luteus. The crude extract was eluted on a phosphocellulose column and chromatographed on TEAE cellulose and subsequently on hydroxypatite. Five peaks of ..gamma..-endonuclease were obtained from each preparation. Repeated experiments showed comparable chromatographic behaviour of the fractions. There was no detectable activity of uv-endonuclease in the fractions with ..gamma..-endonuclease but a small contamination of endonuclease against unirradiated DNA and against DNA with apurinic sites. The ..gamma..-endonuclease was stimulated by, but was not dependent on, magnesium. Several tests for endonuclease activity have been used: the analysis of strand breaks in calf-thymus DNA or in PM2 DNA, and the determination of end-groups formed by endonuclease, either 3'OH end-groups or phosphomonoester end groups. From the results obtained it can be assumed that the strand breaks induced by the ..gamma..-endonuclease carry 3'OH and 5' phosphate end groups.

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a).

Science.gov (United States)

Singh, Digvijay; Mallon, John; Poddar, Anustup; Wang, Yanbo; Tippana, Ramreddy; Yang, Olivia; Bailey, Scott; Ha, Taekjip

2018-05-22

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9. They both bind any DNA in search of protospacer-adjacent motif (PAM) sequences, verify the target sequence directionally from the PAM-proximal end, and rapidly reject any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ∼16 bp for cleavage, Cpf1 requires an ∼17-bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions, and 5' guanine in guide-RNA differentially affected different Cpf1 orthologs. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

SaCas9 Requires 5'-NNGRRT-3' PAM for Sufficient Cleavage and Possesses Higher Cleavage Activity than SpCas9 or FnCpf1 in Human Cells.

Science.gov (United States)

Xie, Haihua; Tang, Lianchao; He, Xiubin; Liu, Xiexie; Zhou, Chenchen; Liu, Junjie; Ge, Xianglian; Li, Jin; Liu, Changbao; Zhao, Junzhao; Qu, Jia; Song, Zongming; Gu, Feng

2018-04-01

CRISPR/Cas9-mediated gene therapy holds great promise for the treatment of human diseases. The protospacer adjacent motif (PAM), the sequence adjacent to the target sequence, is an essential targeting component for the design of CRISPR/Cas9-mediated gene editing. However, currently, very few studies have attempted to directly study the PAM sequence in human cells. To address this issue, the authors develop a dual fluorescence reporter system that could be harnessed for identifying functional PAMs for genome editing endonuclease, including Cas9. With this system, the authors investigate the effects of different PAM sequences for SaCas9, which is small and has the advantage of allowing in vivo genome editing, and found only 5'-NNGRRT-3' PAM could induced sufficient target cleavage with multi-sites. The authors also found SaCas9 possesses higher activity than SpCas9 or FnCpf1 via plasmids (episomal) and chromosomes with integrated eGFP-based comparison. Taken together, the authors show that a dual fluorescence reporter system is a means to identifying a functional PAM and quantitatively comparing the efficiency of different genome editing endonucleases with the similar or identical target sequence in human cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multiplex, rapid and sensitive isothermal detection of nucleic-acid sequence by endonuclease restriction-mediated real-time multiple cross displacement amplification

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Yi eWang

2016-05-01

Full Text Available We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA, which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5’ end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labelled at the 5’ end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5’ end short sequences and their complementary sequences, which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 minutes, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism.

Multiplex, Rapid, and Sensitive Isothermal Detection of Nucleic-Acid Sequence by Endonuclease Restriction-Mediated Real-Time Multiple Cross Displacement Amplification.

Science.gov (United States)

Wang, Yi; Wang, Yan; Zhang, Lu; Liu, Dongxin; Luo, Lijuan; Li, Hua; Cao, Xiaolong; Liu, Kai; Xu, Jianguo; Ye, Changyun

2016-01-01

We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA), which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5' end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labeled at the 5' end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 min, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here, we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism.

Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway

KAUST Repository

Zaher, Manal S.; Rashid, Fahad; Song, Bo; Joudeh, Luay I; Sobhy, Mohamed Abdelmaboud; Tehseen, Muhammad; Hingorani, Manju M; Hamdan, Samir

2018-01-01

RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5'-single-stranded flap is cleaved by structure-specific 5'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway.

Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway

KAUST Repository

Zaher, Manal S.

2018-01-27

RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5\\'-single-stranded flap is cleaved by structure-specific 5\\'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway.

Crystal structure of the apurinic/apyrimidinic endonuclease IV from Mycobacterium tuberculosis.

Science.gov (United States)

Zhang, Wei; Xu, Yueyang; Yan, Mengrong; Li, Shanshan; Wang, Huiying; Yang, Haitao; Zhou, Weihong; Rao, Zihe

2018-03-25

Endonuclease IV is a typical endonuclease of the apurinic-apyrimidinic (AP) or abasic endonuclease superfamily. It repairs damaged DNA through base excision repair by cleaving the DNA backbone immediately 5' of an AP site. In Mycobacterium tuberculosis, endonuclease IV is the major AP endonuclease. This enzyme is absent from mammalian cells, making it an attractive target for anti-tuberculosis drug development. In this study, the structure of the recombinant endonuclease IV from M. tuberculosis (MtbEndo IV) was determined at a high resolution of 1.18 Å. MtbEndo IV was found to have a classical α8β8-fold TIM barrel with loops on its surface connecting the α-helices and β-strands that constitute a groove for DNA binding. Three zinc ions were identified at the active site. A comparison between the structures of MtbEndo IV and Escherichia coli End IV suggested that Gln32 of MtbEndo IV may plays a role in regulating substrate binding. Copyright © 2018. Published by Elsevier Inc.

Endonuclease IV of Escherichia coli is induced by paraquat

Energy Technology Data Exchange (ETDEWEB)

Chan, E.; Weiss, B.

1987-05-01

The addition of paraquat (methyl viologen) to a growing culture of Escherichia coli K-12 led within 1 hr to a 10- to 20-fold increase in the level of endonuclease IV, a DNase for apurinic/apyrimidinic sites. The induction was blocked by chloramphenicol. Increases of 3-fold or more were also seen with plumbagin, menadione, and phenazine methosulfate. H/sub 2/O/sub 2/ produced no more than a 2-fold increase in endonuclease IV activity. The following agents had no significant effect: streptonigrin, nitrofurantoin, tert-butyl hydroperoxide, ..gamma.. rays, 260-nm UV radiation, methyl methanesulfonate, mitomycin C, and ascorbate. Paraquat, plumbagin, menadione, and phenazine methosulfate are known to generate superoxide radical anions via redox cycling in vivo. A mutant lacking superoxide dismutase was unusually sensitive to induction by paraquat. In addition, endonuclease IV could be induced by merely growing the mutant in pure O/sub 2/. The levels of endonuclease IV in uninduced or paraquat-treated cells were unaffected by mutations of oxyR, a H/sub 2/O/sub 2/-inducible gene that governs an oxidative-stress regulon. The results indicate that endonuclease IV is an inducible DNA-repair enzyme and that its induction can be mediated via the production of superoxide radicals.

Endonuclease IV of Escherichia coli is induced by paraquat

International Nuclear Information System (INIS)

Chan, E.; Weiss, B.

1987-01-01

The addition of paraquat (methyl viologen) to a growing culture of Escherichia coli K-12 led within 1 hr to a 10- to 20-fold increase in the level of endonuclease IV, a DNase for apurinic/apyrimidinic sites. The induction was blocked by chloramphenicol. Increases of 3-fold or more were also seen with plumbagin, menadione, and phenazine methosulfate. H 2 O 2 produced no more than a 2-fold increase in endonuclease IV activity. The following agents had no significant effect: streptonigrin, nitrofurantoin, tert-butyl hydroperoxide, γ rays, 260-nm UV radiation, methyl methanesulfonate, mitomycin C, and ascorbate. Paraquat, plumbagin, menadione, and phenazine methosulfate are known to generate superoxide radical anions via redox cycling in vivo. A mutant lacking superoxide dismutase was unusually sensitive to induction by paraquat. In addition, endonuclease IV could be induced by merely growing the mutant in pure O 2 . The levels of endonuclease IV in uninduced or paraquat-treated cells were unaffected by mutations of oxyR, a H 2 O 2 -inducible gene that governs an oxidative-stress regulon. The results indicate that endonuclease IV is an inducible DNA-repair enzyme and that its induction can be mediated via the production of superoxide radicals

Isolation and properties of the acid site-specific endonuclease from mature eggs of the sea urchin Strongylocentrotus intermedius

International Nuclear Information System (INIS)

Sibirtsev, Yu.T.; Konechnyi, A.A.; Rasskazov, V.A.

1986-01-01

An acid site-specific endonuclease has been detected in mature sea urchin eggs and cells of embryos at early stages of differentiation. Fractionation with ammonium sulfate, followed by chromatography on columns with DEAE, phosphocellulose, and hydroxyapatite resulted in an 18,000-fold purification. The molecular weight of the enzyme was determined at ∼ 29,000, the optimum pH 5.5. The activity of the enzyme does not depend on divalent metal ions, EDTA, ATP, and tRNA, but it is modulated to a substantial degree by NaCl. The maximum rate of cleavage of the DNA supercoil (form I) is observed at 100 mM NaCl. Increasing the NaCl concentration to 350 mM only slightly lowers the rate of cleavage of form I, yielding form II, but entirely suppresses the accumulation of form III. Restriction analysis of the products of enzymatic hydrolysis of Co1E1 and pBR322 DNA showed that at the early stages of hydrolysis the enzyme exhibits pronounced specificity for definite sites, the number of which is 12 for Co1 E1 DNA and 8 sites for pBR322 DNA

On the torbernite from Kakinoki Mine, Hyogo Prefecture

International Nuclear Information System (INIS)

Kiji, Michio

1982-01-01

Upon visiting Kakinoki Mine, Hyogo Prefecture, in August, 1980, the author collected the green-colored mineral in square plate form adhering on the surfaces of waste ore. It was discovered that this was torbernite, the first uranium-containing mineral discovered in the area. Its deposit classified as hydatogenetic deposit, since the Kakinoki Mine being an abandoned copper mine. Torbernite exists only in blackish pelite (waste ore), and adheres on the surface and the cleavage, either as small platy crystals or their aggregate. In the blackish pelite, numerous quartz veins were observed, where torbernite occasionally appeared. The torbernite adhering on the surface and cleavage was grass-green and translucent, and the one in the quartz veins was emerald-green and transparent or translucent. The detection of torbernite in the waste ore suggests the potentiality of finding uranium in the Kakinoki Mine. (J.P.N.)

Identification of a mismatch-specific endonuclease in hyperthermophilic Archaea.

Science.gov (United States)

Ishino, Sonoko; Nishi, Yuki; Oda, Soichiro; Uemori, Takashi; Sagara, Takehiro; Takatsu, Nariaki; Yamagami, Takeshi; Shirai, Tsuyoshi; Ishino, Yoshizumi

2016-04-20

The common mismatch repair system processed by MutS and MutL and their homologs was identified in Bacteria and Eukarya. However, no evidence of a functional MutS/L homolog has been reported for archaeal organisms, and it is not known whether the mismatch repair system is conserved in Archaea. Here, we describe an endonuclease that cleaves double-stranded DNA containing a mismatched base pair, from the hyperthermophilic archaeon Pyrococcus furiosus The corresponding gene revealed that the activity originates from PF0012, and we named this enzyme Endonuclease MS (EndoMS) as the mismatch-specific Endonuclease. The sequence similarity suggested that EndoMS is the ortholog of NucS isolated from Pyrococcus abyssi, published previously. Biochemical characterizations of the EndoMS homolog from Thermococcus kodakarensis clearly showed that EndoMS specifically cleaves both strands of double-stranded DNA into 5'-protruding forms, with the mismatched base pair in the central position. EndoMS cleaves G/T, G/G, T/T, T/C and A/G mismatches, with a more preference for G/T, G/G and T/T, but has very little or no effect on C/C, A/C and A/A mismatches. The discovery of this endonuclease suggests the existence of a novel mismatch repair process, initiated by the double-strand break generated by the EndoMS endonuclease, in Archaea and some Bacteria. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Karyopherin-Mediated Nuclear Import of the Homing Endonuclease VMA1-Derived Endonuclease Is Required for Self-Propagation of the Coding Region

OpenAIRE

Nagai, Yuri; Nogami, Satoru; Kumagai-Sano, Fumi; Ohya, Yoshikazu

2003-01-01

VMA1-derived endonuclease (VDE), a site-specific endonuclease in Saccharomyces cerevisiae, enters the nucleus to generate a double-strand break in the VDE-negative allelic locus, mediating the self-propagating gene conversion called homing. Although VDE is excluded from the nucleus in mitotic cells, it relocalizes at premeiosis, becoming localized in both the nucleus and the cytoplasm in meiosis. The nuclear localization of VDE is induced by inactivation of TOR kinases, which constitute centr...

Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1.

Science.gov (United States)

Gloor, Jason W; Balakrishnan, Lata; Campbell, Judith L; Bambara, Robert A

2012-08-01

In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼ 30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation.

Endonuclease α from Saccharomyces cerevisiae shows increased activity on ultraviolet irradiated native DNA

International Nuclear Information System (INIS)

Bryant, D.W.; Haynes, R.H.

1978-01-01

Endonuclease α isolated from the nucleus of the yeast Saccharomyces cerevisiae is a DNA endonuclease which has been shown to act preferentially on denatured T7 DNA. The purified enzyme is more active with UV-irradiated native T7 DNA than with unirradiated substrate. The relation between damage, measured by pyrimidine dimer concentration, and excess endonuclease activity is most readily explained by local denaturation caused by the presence of pyrimidine dimers. When three radiation sensitive mutants of yeast were tested for the level of endonuclease α present, none were found lacking the enzyme. However, nuclei of strain rad 1-1, a mutant that may be defective in heteroduplex repair as well as excision repair, were found to contain reduced levels of the endonuclease. (orig./AJ) [de

Total sequence decomposition distinguishes functional modules, "molegos" in apurinic/apyrimidinic endonucleases

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Braun Werner

2002-11-01

Full Text Available Abstract Background Total sequence decomposition, using the web-based MASIA tool, identifies areas of conservation in aligned protein sequences. By structurally annotating these motifs, the sequence can be parsed into individual building blocks, molecular legos ("molegos", that can eventually be related to function. Here, the approach is applied to the apurinic/apyrimidinic endonuclease (APE DNA repair proteins, essential enzymes that have been highly conserved throughout evolution. The APEs, DNase-1 and inositol 5'-polyphosphate phosphatases (IPP form a superfamily that catalyze metal ion based phosphorolysis, but recognize different substrates. Results MASIA decomposition of APE yielded 12 sequence motifs, 10 of which are also structurally conserved within the family and are designated as molegos. The 12 motifs include all the residues known to be essential for DNA cleavage by APE. Five of these molegos are sequentially and structurally conserved in DNase-1 and the IPP family. Correcting the sequence alignment to match the residues at the ends of two of the molegos that are absolutely conserved in each of the three families greatly improved the local structural alignment of APEs, DNase-1 and synaptojanin. Comparing substrate/product binding of molegos common to DNase-1 showed that those distinctive for APEs are not directly involved in cleavage, but establish protein-DNA interactions 3' to the abasic site. These additional bonds enhance both specific binding to damaged DNA and the processivity of APE1. Conclusion A modular approach can improve structurally predictive alignments of homologous proteins with low sequence identity and reveal residues peripheral to the traditional "active site" that control the specificity of enzymatic activity.

Bcl2-independent chromatin cleavage is a very early event during induction of apoptosis in mouse thymocytes after treatment with either dexamethasone or ionizing radiation.

Science.gov (United States)

Hahn, Peter J; Lai, Zhi-Wei; Nevaldine, Barbara; Schiff, Ninel; Fiore, Nancy C; Silverstone, Allen E

2003-11-01

We have quantified the emergence of early chromatin breaks during the signal transduction phase of apoptosis in mouse thymocytes after treatment with either ionizing radiation or dexamethasone. Dexamethasone at 1 microM can induce significant levels of DNA breaks (equivalent to the amount induced directly by 7.5 Gy ionizing radiation) within 0.5 h of treatment. The execution phase of apoptosis was not observed until 4-6 h after the same treatment. The presence of the Bcl2 transgene under the control of the p56lck promoter almost completely inhibited apoptosis up to 24 h after treatment, but it had virtually no effect on the early chromatin cleavage occurring in the first 6 h. Ionizing radiation induced chromatin cleavage both directly by damaging DNA and indirectly with kinetics similar to the induction of chromatin cleavage by dexamethasone. The presence of the Bcl2 transgene had no effect on the direct or indirect radiation-induced cleavage in the first 6 h, but after the first 6 h, the Bcl2 gene inhibited further radiation-induced chromatin cleavage. These results suggest that endonucleases are activated within minutes of treatment with either dexamethasone or ionizing radiation as part of the very early signal transduction phase of apoptosis, and prior to the irreversible commitment to cell death.

Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP Assay for the Detection of Bacterial Meningitis Pathogens

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Owen Higgins

2018-02-01

Full Text Available Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae. Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae, N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology.

Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens

Science.gov (United States)

Clancy, Eoin; Cormican, Martin; Boo, Teck Wee; Cunney, Robert

2018-01-01

Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae. Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae, N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology. PMID:29425124

Predictors of hepatitis B cure using gene therapy to deliver DNA cleavage enzymes: a mathematical modeling approach.

Directory of Open Access Journals (Sweden)

Joshua T Schiffer

Full Text Available Most chronic viral infections are managed with small molecule therapies that inhibit replication but are not curative because non-replicating viral forms can persist despite decades of suppressive treatment. There are therefore numerous strategies in development to eradicate all non-replicating viruses from the body. We are currently engineering DNA cleavage enzymes that specifically target hepatitis B virus covalently closed circular DNA (HBV cccDNA, the episomal form of the virus that persists despite potent antiviral therapies. DNA cleavage enzymes, including homing endonucleases or meganucleases, zinc-finger nucleases (ZFNs, TAL effector nucleases (TALENs, and CRISPR-associated system 9 (Cas9 proteins, can disrupt specific regions of viral DNA. Because DNA repair is error prone, the virus can be neutralized after repeated cleavage events when a target sequence becomes mutated. DNA cleavage enzymes will be delivered as genes within viral vectors that enter hepatocytes. Here we develop mathematical models that describe the delivery and intracellular activity of DNA cleavage enzymes. Model simulations predict that high vector to target cell ratio, limited removal of delivery vectors by humoral immunity, and avid binding between enzyme and its DNA target will promote the highest level of cccDNA disruption. Development of de novo resistance to cleavage enzymes may occur if DNA cleavage and error prone repair does not render the viral episome replication incompetent: our model predicts that concurrent delivery of multiple enzymes which target different vital cccDNA regions, or sequential delivery of different enzymes, are both potentially useful strategies for avoiding multi-enzyme resistance. The underlying dynamics of cccDNA persistence are unlikely to impact the probability of cure provided that antiviral therapy is given concurrently during eradication trials. We conclude by describing experiments that can be used to validate the model, which

Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1

KAUST Repository

Sobhy, M.; Joudeh, L.; Huang, X.; Takahashi, Masateru; Hamdan, S.

2013-01-01

Human flap endonuclease 1 (FEN1), one of the structure-specific 5' nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5' nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5' end junctions. Using single-molecule Förster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5' nuclease mechanisms. This is achieved by coordinating threading of the 5' flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5' nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection ofDNA junctions from nonspecific bending and cleavage. 2013 The Authors.

Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1

KAUST Repository

Sobhy, M.

2013-06-06

Human flap endonuclease 1 (FEN1), one of the structure-specific 5\\' nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5\\' nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5\\' end junctions. Using single-molecule Förster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5\\' nuclease mechanisms. This is achieved by coordinating threading of the 5\\' flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5\\' nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection ofDNA junctions from nonspecific bending and cleavage. 2013 The Authors.

Binding of T4 endonuclease V to deoxyribonucleic acid irradiated with ultraviolet light

International Nuclear Information System (INIS)

Seawell, P.C.; Simon, T.J.; Ganesan, A.K.

1980-01-01

Endonuclease V of bacteriophage T4 binds to uv-irradiated deoxyribonucleic acid (DNA) but not to unirradiated DNA. We have developed an assay to detect this binding, based on the retention of enzyme - DNA complexes on nitrocellulose filters. The amount of complex retained, ascertained by using radioactive DNA, is a measure of T4 endonuclease V activity. From our data we conclude that (1) T4 endonuclease V binds to uv-irradiated DNA but not to DNA that has been previously incised by the endonuclease, (2) equilibrium between the free and complexed form of the enzyme is attained under our reaction conditions, (3) dissociation of enzyme - DNA complexes is retarded by sodium cyanide, and (4) retention of enzyme - DNA complexes on nitrocellulose filters is enhanced by high concentrations of saline-citrate

Simple and sensitive fluorescence assay of restriction endonuclease on graphene oxide

International Nuclear Information System (INIS)

Gang, Jong Back

2015-01-01

Restriction endonucleases hydrolyze internal phosphodiester bonds at specific sites in a DNA sequence. These enzymes are essential in a variety of fields, such as biotechnology and clinical diagnostics. It is of great importance and necessity for the scientific and biomedical use of enzymes to measure endonuclease activity. In this study, graphene oxide (GO) has been used as a platform to measure enzyme activity with high sensitivity. To increase the detection sensitivity of Hinf I, the endonuclease-digested reaction was treated with exonuclease III (Exo III) and a fluorescence assay was conducted to measure the emission. Results showed that Exo III treatment enhanced 2.7-fold signal-to-background ratio for the detection of Hinf I compared with that done without Exo III in the presence of GO

Properties of an endonuclease activity in Micrococcus luteus acting on γ-irradiated DNA and on apurinic DNA

International Nuclear Information System (INIS)

Schaefer, G.; Haas, P.; Coquerelle, Th.; Hagen, U.

1980-01-01

A protein fraction from Micrococcus luteus with endonuclease activity against γ-irradiated DNA was isolated and characterized. An additional activity on apurinic sites could not be separated, either by sucrose gradient sedimentation or by gel filtration through Sephadex G 100. From gel filtration, a molecular weight of about 25 000 was calculated for both endonuclease activities. The endonuclease activity against γ-irradiated DNA was stimulated five-fold with 5 mM Mg ++ , whereas that against apurinic sites was less dependent on the Mg ++ concentration. 100 mM KCl inhibited the γ-ray endonuclease, but not the apurinic endonuclease activity. In γ-irradiated DNA the protein recognized 1.65 endonuclease sensitive sites per radiation-induced single-strand break, among which are 0.45 alkali labile lesions in the nucleotide strand. The was evaluated resulting in a Ksub(m)-value of 73 nM. (author)

Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.

Science.gov (United States)

Anders, Carolin; Niewoehner, Ole; Duerst, Alessia; Jinek, Martin

2014-09-25

The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.

Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9.

Science.gov (United States)

Anders, Carolin; Bargsten, Katja; Jinek, Martin

2016-03-17

The RNA-guided endonuclease Cas9 from Streptococcus pyogenes (SpCas9) forms the core of a powerful genome editing technology. DNA cleavage by SpCas9 is dependent on the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) in the target DNA, restricting the choice of targetable sequences. To address this limitation, artificial SpCas9 variants with altered PAM specificities have recently been developed. Here we report crystal structures of the VQR, EQR, and VRER SpCas9 variants bound to target DNAs containing their preferred PAM sequences. The structures reveal that the non-canonical PAMs are recognized by an induced fit mechanism. Besides mediating sequence-specific base recognition, the amino acid substitutions introduced in the SpCas9 variants facilitate conformational remodeling of the PAM region of the bound DNA. Guided by the structural data, we engineered a SpCas9 variant that specifically recognizes NAAG PAMs. Taken together, these studies inform further development of Cas9-based genome editing tools. Copyright © 2016 Elsevier Inc. All rights reserved.

Evolutionary maintenance of selfish homing endonuclease genes in the absence of horizontal transfer.

Science.gov (United States)

Yahara, Koji; Fukuyo, Masaki; Sasaki, Akira; Kobayashi, Ichizo

2009-11-03

Homing endonuclease genes are "selfish" mobile genetic elements whose endonuclease promotes the spread of its own gene by creating a break at a specific target site and using the host machinery to repair the break by copying and inserting the gene at this site. Horizontal transfer across the boundary of a species or population within which mating takes place has been thought to be necessary for their evolutionary persistence. This is based on the assumption that they will become fixed in a host population, where opportunities of homing will disappear, and become susceptible to degeneration. To test this hypothesis, we modeled behavior of a homing endonuclease gene that moves during meiosis through double-strand break repair. We mathematically explored conditions for persistence of the homing endonuclease gene and elucidated their parameter dependence as phase diagrams. We found that, if the cost of the pseudogene is lower than that of the homing endonuclease gene, the 2 forms can persist in a population through autonomous periodic oscillation. If the cost of the pseudogene is higher, 2 types of dynamics appear that enable evolutionary persistence: bistability dependent on initial frequency or fixation irrespective of initial frequency. The prediction of long persistence in the absence of horizontal transfer was confirmed by stochastic simulations in finite populations. The average time to extinction of the endonuclease gene was found to be thousands of meiotic generations or more based on realistic parameter values. These results provide a solid theoretical basis for an understanding of these and other extremely selfish elements.

Substrate specificity of Micrococcus luteus uv endonuclease and its overlap with DNA photolyase activity

International Nuclear Information System (INIS)

Patrick, M.H.

1975-01-01

The action of an endonuclease from Micrococcus luteus that operates on uv damage in DNA overlaps with that of DNA photolyase from yeast: homo- and heterocyclobutane dipyrimidines in DNA are substrates for both enzymes, but pyrimidine adducts or the spore photoproduct in DNA are not. As expected from this overlap, the action of the two enzymes is mutually interfering: single-strand nicks introduced by the endonuclease effectively preclude photoreactivation; conversely, formation of a photolyase-cyclobutane dipyrimidine complex can prevent nicking by the endonuclease

Properties of an endonuclease activity in Micrococcus luteus acting on. gamma. -irradiated DNA and on apurinic DNA

Energy Technology Data Exchange (ETDEWEB)

Schaefer, G; Haas, P; Coquerelle, Th; Hagen, U [Kernforschungszentrum Karlsruhe G.m.b.H. (Germany, F.R.). Inst. fuer Genetik und fuer Toxikologie von Spaltstoffen

1980-01-01

A protein fraction from Micrococcus luteus with endonuclease activity against ..gamma..-irradiated DNA was isolated and characterized. An additional activity on apurinic sites could not be separated, either by sucrose gradient sedimentation or by gel filtration through Sephadex G 100. From gel filtration, a molecular weight of about 25 000 was calculated for both endonuclease activities. The endonuclease activity against ..gamma..-irradiated DNA was stimulated five-fold with 5 mM Mg/sup + +/, whereas that against apurinic sites was less dependent on the Mg/sup + +/ concentration. 100 mM KCl inhibited the ..gamma..-ray endonuclease, but not the apurinic endonuclease activity. In ..gamma..-irradiated DNA the protein recognized 1.65 endonuclease sensitive sites per radiation-induced single-strand break, among which are 0.45 alkali labile lesions in the nucleotide strand. The was evaluated resulting in a Ksub(m)-value of 73 nM.

Identification of human flap endonuclease 1 (FEN1) inhibitors using a machine learning based consensus virtual screening.

Science.gov (United States)

Deshmukh, Amit Laxmikant; Chandra, Sharat; Singh, Deependra Kumar; Siddiqi, Mohammad Imran; Banerjee, Dibyendu

2017-07-25

Human Flap endonuclease1 (FEN1) is an enzyme that is indispensable for DNA replication and repair processes and inhibition of its Flap cleavage activity results in increased cellular sensitivity to DNA damaging agents (cisplatin, temozolomide, MMS, etc.), with the potential to improve cancer prognosis. Reports of the high expression levels of FEN1 in several cancer cells support the idea that FEN1 inhibitors may target cancer cells with minimum side effects to normal cells. In this study, we used large publicly available, high-throughput screening data of small molecule compounds targeted against FEN1. Two machine learning algorithms, Support Vector Machine (SVM) and Random Forest (RF), were utilized to generate four classification models from huge PubChem bioassay data containing probable FEN1 inhibitors and non-inhibitors. We also investigated the influence of randomly selected Zinc-database compounds as negative data on the outcome of classification modelling. The results show that the SVM model with inactive compounds was superior to RF with Matthews's correlation coefficient (MCC) of 0.67 for the test set. A Maybridge database containing approximately 53 000 compounds was screened and top ranking 5 compounds were selected for enzyme and cell-based in vitro screening. The compound JFD00950 was identified as a novel FEN1 inhibitor with in vitro inhibition of flap cleavage activity as well as cytotoxic activity against a colon cancer cell line, DLD-1.

Processive nicking activity of T4 endonuclease V on UV-irradiated chromatin

International Nuclear Information System (INIS)

Gruskin, E.A.; Lloyd, R.S.

1986-01-01

T4 endonuclease V initiates the excision repair of pyrimidine dimers in UV-irradiated T4 infected E. coli cells. The pyrimidine dimer specific nicking activity of T4 endonuclease V functions by a processive scanning on UV-irradiated DNA. Previously it has been demonstrated that introduction of endonuclease V into repair-deficient human cells causes a restoration of UV survival in these cells. This demonstrates that endonuclease V is competent to incise mammalian DNA at the site of pyrimidine dimers. In order to assess the ability of endonuclease V to act processively on DNA associated as chromatin, minichromosomes were prepared for use as a substrate. Form I DNA was reconstituted with H3, H4 +/- H1 histones by sequential dialysis steps from 2.0 M NaCl to 50 mM NaCl. Time course reactions were performed with minichromosomes containing 10 and 25 dimers per molecule. In each case the rate of disappearance of form I DNA which was associated as chromatin was decreased relative to that of naked form I DNA. Concurrent with that observation, the rate and extent of appearance of form III DNA was increased with the DNA in minichromosomes relative to naked DNA. This is diagnostic of an enhancement of processivity. The inclusion of H1 in the minichromosomes resulted in a slight additional increase in processivity relative to minichromosomes consisting only of H3 and H4

Computational Characterization of Small Molecules Binding to the Human XPF Active Site and Virtual Screening to Identify Potential New DNA Repair Inhibitors Targeting the ERCC1-XPF Endonuclease

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Francesco Gentile

2018-04-01

Full Text Available The DNA excision repair protein ERCC-1-DNA repair endonuclease XPF (ERCC1-XPF is a heterodimeric endonuclease essential for the nucleotide excision repair (NER DNA repair pathway. Although its activity is required to maintain genome integrity in healthy cells, ERCC1-XPF can counteract the effect of DNA-damaging therapies such as platinum-based chemotherapy in cancer cells. Therefore, a promising approach to enhance the effect of these therapies is to combine their use with small molecules, which can inhibit the repair mechanisms in cancer cells. Currently, there are no structures available for the catalytic site of the human ERCC1-XPF, which performs the metal-mediated cleavage of a DNA damaged strand at 5′. We adopted a homology modeling strategy to build a structural model of the human XPF nuclease domain which contained the active site and to extract dominant conformations of the domain using molecular dynamics simulations followed by clustering of the trajectory. We investigated the binding modes of known small molecule inhibitors targeting the active site to build a pharmacophore model. We then performed a virtual screening of the ZINC Is Not Commercial 15 (ZINC15 database to identify new ERCC1-XPF endonuclease inhibitors. Our work provides structural insights regarding the binding mode of small molecules targeting the ERCC1-XPF active site that can be used to rationally optimize such compounds. We also propose a set of new potential DNA repair inhibitors to be considered for combination cancer therapy strategies.

The metabolic enhancer piracetam attenuates mitochondrion-specific endonuclease G translocation and oxidative DNA fragmentation.

Science.gov (United States)

Gupta, Sonam; Verma, Dinesh Kumar; Biswas, Joyshree; Rama Raju, K Siva; Joshi, Neeraj; Wahajuddin; Singh, Sarika

2014-08-01

This study was performed to investigate the involvement of mitochondrion-specific endonuclease G in piracetam (P)-induced protective mechanisms. Studies have shown the antiapoptotic effects of piracetam but the mechanism of action of piracetam is still an enigma. To assess the involvement of endonuclease G in piracetam-induced protective effects, astrocyte glial cells were treated with lipopolysaccharide (LPS) and piracetam. LPS treatment caused significantly decreased viability, mitochondrial activity, oxidative stress, chromatin condensation, and DNA fragmentation, which were attenuated by piracetam cotreatment. Cotreatment of astrocytes with piracetam showed its significantly time-dependent absorption as observed with high-performance liquid chromatography. Astrocytes treated with piracetam alone showed enhanced mitochondrial membrane potential (MMP) in comparison to control astrocytes. However, in LPS-treated cells no significant alteration in MMP was observed in comparison to control cells. Protein and mRNA levels of the terminal executor of the caspase-mediated pathway, caspase-3, were not altered significantly in LPS or LPS + piracetam-treated astrocytes, whereas endonuclease G was significantly translocated to the nucleus in LPS-treated astrocytes. Piracetam cotreatment attenuated the LPS-induced endonuclease G translocation. In conclusion this study indicates that LPS treatment of astrocytes caused decreased viability, oxidative stress, mitochondrial dysfunction, chromatin condensation, DNA damage, and translocation of endonuclease G to the nucleus, which was inhibited by piracetam cotreatment, confirming that the mitochondrion-specific endonuclease G is one of the factors involved in piracetam-induced protective mechanisms. Copyright © 2014 Elsevier Inc. All rights reserved.

A functional endonuclease Q exists in the bacterial domain: identification and characterization of endonuclease Q from Bacillus pumilus.

Science.gov (United States)

Shiraishi, Miyako; Ishino, Sonoko; Cann, Isaac; Ishino, Yoshizumi

2017-05-01

DNA base deamination occurs spontaneously under physiological conditions and is promoted by high temperature. Therefore, hyperthermophiles are expected to have efficient repair systems of the deaminated bases in their genomes. Endonuclease Q (EndoQ) was originally identified from the hyperthermophlic archaeon, Pyrococcus furiosus, as a hypoxanthine-specific endonuclease recently. Further biochemical analyses revealed that EndoQ also recognizes uracil, xanthine, and the AP site in DNA, and is probably involved in a specific repair process for damaged bases. Initial phylogenetic analysis showed that an EndoQ homolog is found only in the Thermococcales and some of the methanogens in Archaea, and is not present in most members of the domains Bacteria and Eukarya. A better understanding of the distribution of the EndoQ-mediated repair system is, therefore, of evolutionary interest. We showed here that an EndoQ-like polypeptide from Bacillus pumilus, belonging to the bacterial domain, is functional and has similar properties with the archaeal EndoQs.

Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

KAUST Repository

Aouida, Mustapha

2015-07-30

The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

KAUST Repository

Aouida, Mustapha; Eid, Ayman; Ali, Zahir; Cradick, Thomas; Lee, Ciaran; Deshmukh, Harshavardhan; Atef, Ahmed; Abu Samra, Dina Bashir Kamil; Gadhoum, Samah Zeineb; Merzaban, Jasmeen; Bao, Gang; Mahfouz, Magdy M.

2015-01-01

The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

A new restriction endonuclease from Citrobacter freundii

OpenAIRE

Janulaitis, A.A.; Stakenas, P.S.; Lebedenko, E.N.; Berlin, Yu.A.

1982-01-01

CfrI, a new restriction endonuclease of unique substrate specificity, has been isolated from a Citrobacter freundii strain. The enzyme recognizes a degenerated sequence PyGGCCPu in double-strand DNA and cleaves it between Py and G residues to yield 5′ -protruding tetranucleotide ends GGCC.

A new restriction endonuclease from Citrobacter freundii

Science.gov (United States)

Janulaitis, A.A.; Stakenas, P.S.; Lebedenko, E.N.; Berlin, Yu.A.

1982-01-01

CfrI, a new restriction endonuclease of unique substrate specificity, has been isolated from a Citrobacter freundii strain. The enzyme recognizes a degenerated sequence PyGGCCPu in double-strand DNA and cleaves it between Py and G residues to yield 5′ -protruding tetranucleotide ends GGCC. Images PMID:6294607

Visualizing phosphodiester-bond hydrolysis by an endonuclease

DEFF Research Database (Denmark)

Molina, Rafael; Stella, Stefano; Redondo, Pilar

2015-01-01

The enzymatic hydrolysis of DNA phosphodiester bonds has been widely studied, but the chemical reaction has not yet been observed. Here we follow the generation of a DNA double-strand break (DSB) by the Desulfurococcus mobilis homing endonuclease I-DmoI, trapping sequential stages of a two-metal-...

Excision repair of gamma-ray-induced alkali-stable DNA lesions with the help of γ-endonuclease from Micrococcus luteus

International Nuclear Information System (INIS)

Tomilin, N.V.; Barenfeld, L.S.

1979-01-01

γ-endonuclease Y, an enzyme that hydrolyses phosphodiester bonds at alkali-stable lesions in γ-irradiated (N 2 , tris buffer) DNA, has been partially purified from Micrococcus luteus. The enzyme has a molecular weight of about 19 000, induces single-strand breaks with 3'OH-5'PO 4 termini and contains endonuclease activity towards DNA treated with 7-bromomethylbenz(a)anthracene. γ-endonuclease Y induces breaks in OsO 4 -treated poly(dA-dT) and apparently is specific towards γ-ray-induced base lesions of the t' type. The complete excision repair of γ-endonuclease Y substrate sites has been performed in vitro by γ-endonuclease Y, DNA polymerase and ligase. (author)

Excision repair of gamma-ray-induced alkali-stable DNA lesions with the help of. gamma. -endonuclease from Micrococcus luteus

Energy Technology Data Exchange (ETDEWEB)

Tomilin, N V; Barenfeld, L S [AN SSSR, Leningrad. Inst. Tsitologii

1979-03-01

..gamma..-endonuclease Y, an enzyme that hydrolyses phosphodiester bonds at alkali-stable lesions in ..gamma..-irradiated (N/sub 2/, tris buffer) DNA, has been partially purified from Micrococcus luteus. The enzyme has a molecular weight of about 19 000, induces single-strand breaks with 3'OH-5'PO/sub 4/ termini and contains endonuclease activity towards DNA treated with 7-bromomethylbenz(a)anthracene. ..gamma..-endonuclease Y induces breaks in OsO/sub 4/-treated poly(dA-dT) and apparently is specific towards ..gamma..-ray-induced base lesions of the t' type. The complete excision repair of ..gamma..-endonuclease Y substrate sites has been performed in vitro by ..gamma..-endonuclease Y, DNA polymerase and ligase.

DENV gene of bacteriophage T4 codes for both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities

International Nuclear Information System (INIS)

McMillan, S.; Edenberg, H.J.; Radany, E.H.; Friedberg, R.C.; Friedberg, E.C.

1981-01-01

Recent studies have shown that purified preparations of phage T4 UV DNA-incising activity (T4 UV endonuclease or endonuclease V of phase T4) contain a pyrimidine dimer-DNA glycosylase activity that catalyzes hydrolysis of the 5' glycosyl bond of dimerized pyrimidines in UV-irradiated DNA. Such enzyme preparations have also been shown to catalyze the hydrolysis of phosphodiester bonds in UV-irradiated DNA at a neutral pH, presumably reflecting the action of an apurinic/apyrimidinic endonuclease at the apyrimidinic sites created by the pyrimidine dimer-DNA glycosylase. In this study we found that preparations of T4 UV DNA-incising activity contained apurinic/apyrimidinic endonuclease activity that nicked depurinated form I simian virus 40 DNA. Apurinic/apyrimidinic endonuclease activity was also found in extracts of Escherichia coli infected with T4 denV + phage. Extracts of cells infected with T4 denV mutants contained significantly lower levels of apurinic/apyrimidinic endonuclease activity; these levels were no greater than the levels present in extracts of uninfected cells. Furthermore, the addition of DNA containing UV-irradiated DNA and T4 enzyme resulted in competition for pyrimidine dimer-DNA glycosylase activity against the UV-irradiated DNA. On the basis of these results, we concluded that apurinic/apyrimidinic endonuclease activity is encoded by the denV gene of phage T4, the same gene that codes for pyrimidine dimer-DNA glycosylase activity

Cloning and analysis of a bifunctional methyltransferase/restriction endonuclease TspGWI, the prototype of a Thermus sp. enzyme family

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Zylicz-Stachula Agnieszka

2009-05-01

Full Text Available Abstract Background Restriction-modification systems are a diverse class of enzymes. They are classified into four major types: I, II, III and IV. We have previously proposed the existence of a Thermus sp. enzyme family, which belongs to type II restriction endonucleases (REases, however, it features also some characteristics of types I and III. Members include related thermophilic endonucleases: TspGWI, TaqII, TspDTI, and Tth111II. Results Here we describe cloning, mutagenesis and analysis of the prototype TspGWI enzyme that recognises the 5'-ACGGA-3' site and cleaves 11/9 nt downstream. We cloned, expressed, and mutagenised the tspgwi gene and investigated the properties of its product, the bifunctional TspGWI restriction/modification enzyme. Since TspGWI does not cleave DNA completely, a cloning method was devised, based on amino acid sequencing of internal proteolytic fragments. The deduced amino acid sequence of the enzyme shares significant sequence similarity with another representative of the Thermus sp. family – TaqII. Interestingly, these enzymes recognise similar, yet different sequences in the DNA. Both enzymes cleave DNA at the same distance, but differ in their ability to cleave single sites and in the requirement of S-adenosylmethionine as an allosteric activator for cleavage. Both the restriction endonuclease (REase and methyltransferase (MTase activities of wild type (wt TspGWI (either recombinant or isolated from Thermus sp. are dependent on the presence of divalent cations. Conclusion TspGWI is a bifunctional protein comprising a tandem arrangement of Type I-like domains; particularly noticeable is the central HsdM-like module comprising a helical domain and a highly conserved S-adenosylmethionine-binding/catalytic MTase domain, containing DPAVGTG and NPPY motifs. TspGWI also possesses an N-terminal PD-(D/EXK nuclease domain related to the corresponding domains in HsdR subunits, but lacks the ATP-dependent translocase module

A new endonuclease recognizing the deoxynucleotide sequence CCNNGG from the cyanobacterium Synechocystis 6701.

Science.gov (United States)

Calléja, F; Tandeau de Marsac, N; Coursin, T; van Ormondt, H; de Waard, A

1985-09-25

A new sequence-specific endonuclease from the cyanobacterium Synechocystis species PCC 6701 has been purified and characterized. This enzyme, SecI, is unique in recognizing the nucleotide sequence: 5' -CCNNGG-3' 3' -GGNNCC-5' and cleaves it at the position indicated by the symbol. Two other restriction endonucleases, SecII and SecIII, found in this organism are isoschizomers of MspI and MstII, respectively.

PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance.

Science.gov (United States)

van Oers, Johanna M M; Roa, Sergio; Werling, Uwe; Liu, Yiyong; Genschel, Jochen; Hou, Harry; Sellers, Rani S; Modrich, Paul; Scharff, Matthew D; Edelmann, Winfried

2010-07-27

The DNA mismatch repair protein PMS2 was recently found to encode a novel endonuclease activity. To determine the biological functions of this activity in mammals, we generated endonuclease-deficient Pms2E702K knock-in mice. Pms2EK/EK mice displayed increased genomic mutation rates and a strong cancer predisposition. In addition, class switch recombination, but not somatic hypermutation, was impaired in Pms2EK/EK B cells, indicating a specific role in Ig diversity. In contrast to Pms2-/- mice, Pms2EK/EK male mice were fertile, indicating that this activity is dispensable in spermatogenesis. Therefore, the PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance and tumor suppression.

Adenosine triphosphate stimulates Aquifex aeolicus MutL endonuclease activity.

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Jerome Mauris

2009-09-01

Full Text Available Human PMS2 (hPMS2 homologues act to nick 5' and 3' to misincorporated nucleotides during mismatch repair in organisms that lack MutH. Mn(++ was previously found to stimulate the endonuclease activity of these homologues. ATP was required for the nicking activity of hPMS2 and yPMS1, but was reported to inhibit bacterial MutL proteins from Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X(2E(X(4E motif present in the C-terminus of PMS2 homologues as important for endonuclease activity.We examined the effect ATP had on the Mn(++ induced nicking of supercoiled pBR322 by full-length and mutant A. aeolicus MutL (Aae MutL proteins. Assays were single time point, enzyme titration experiments or reaction time courses. The maximum velocity for MutL nicking was determined to be 1.6+/-0.08x10(-5 s(-1 and 4.2+/-0.3x10(-5 s(-1 in the absence and presence of ATP, respectively. AMPPNP stimulated the nicking activity to a similar extent as ATP. A truncated Aae MutL protein composed of only the C-terminal 123 amino acid residues was found to nick supercoiled DNA. Furthermore, mutations in the conserved C-terminal DQHA(X(2E(X(4E and CPHGRP motifs were shown to abolish Aae MutL endonuclease activity.ATP stimulated the Mn(++ induced endonuclease activity of Aae MutL. Experiments utilizing AMPPNP implied that the stimulation did not require ATP hydrolysis. A mutation in the DQHA(X(2E(X(4E motif of Aae MutL further supported the role of this region in endonclease activity. For the first time, to our knowledge, we demonstrate that changing the histidine residue in the conserved CPHGRP motif abolishes endonucleolytic activity of a hPMS2 homologue. Finally, the C-terminal 123 amino acid residues of Aae MutL were sufficient to display Mn(++ induced nicking activity.

Evolution of divergent DNA recognition specificities in VDE homing endonucleases from two yeast species.

Science.gov (United States)

Posey, Karen L; Koufopanou, Vassiliki; Burt, Austin; Gimble, Frederick S

2004-01-01

Homing endonuclease genes (HEGs) are mobile DNA elements that are thought to confer no benefit to their host. They encode site-specific DNA endonucleases that perpetuate the element within a species population by homing and disseminate it between species by horizontal transfer. Several yeast species contain the VMA1 HEG that encodes the intein-associated VMA1-derived endonuclease (VDE). The evolutionary state of VDEs from 12 species was assessed by assaying their endonuclease activities. Only two enzymes are active, PI-ZbaI from Zygosaccharomyces bailii and PI-ScaI from Saccharomyces cariocanus. PI-ZbaI cleaves the Z.bailii recognition sequence significantly faster than the Saccharomyces cerevisiae site, which differs at six nucleotide positions. A mutational analysis indicates that PI-ZbaI cleaves the S.cerevisiae substrate poorly due to the absence of a contact that is analogous to one made in PI-SceI between Gln-55 and nucleotides +9/+10. PI-ZbaI cleaves the Z.bailii substrate primarily due to a single base-pair substitution (A/T+5 --> T/A+5). Structural modeling of the PI-ZbaI/DNA complex suggests that Arg-331, which is absent in PI-SceI, contacts T/A+5, and the reduced activity observed in a PI-ZbaI R331A mutant provides evidence for this interaction. These data illustrate that homing endonucleases evolve altered specificity as they adapt to recognize alternative target sites.

Evolution of divergent DNA recognition specificities in VDE homing endonucleases from two yeast species

Science.gov (United States)

Posey, Karen L.; Koufopanou, Vassiliki; Burt, Austin; Gimble, Frederick S.

2004-01-01

Homing endonuclease genes (HEGs) are mobile DNA elements that are thought to confer no benefit to their host. They encode site-specific DNA endonucleases that perpetuate the element within a species population by homing and disseminate it between species by horizontal transfer. Several yeast species contain the VMA1 HEG that encodes the intein-associated VMA1-derived endonuclease (VDE). The evolutionary state of VDEs from 12 species was assessed by assaying their endonuclease activities. Only two enzymes are active, PI-ZbaI from Zygosaccharomyces bailii and PI-ScaI from Saccharomyces cariocanus. PI-ZbaI cleaves the Z.bailii recognition sequence significantly faster than the Saccharomyces cerevisiae site, which differs at six nucleotide positions. A mutational analysis indicates that PI-ZbaI cleaves the S.cerevisiae substrate poorly due to the absence of a contact that is analogous to one made in PI-SceI between Gln-55 and nucleotides +9/+10. PI-ZbaI cleaves the Z.bailii substrate primarily due to a single base-pair substitution (A/T+5 → T/A+5). Structural modeling of the PI-ZbaI/DNA complex suggests that Arg-331, which is absent in PI-SceI, contacts T/A+5, and the reduced activity observed in a PI-ZbaI R331A mutant provides evidence for this interaction. These data illustrate that homing endonucleases evolve altered specificity as they adapt to recognize alternative target sites. PMID:15280510

Purification, crystallization and preliminary crystallographic analysis of a thermostable endonuclease IV from Thermotoga maritima

International Nuclear Information System (INIS)

Hughes, Ronny C.; Tomanicek, Stephen J.; Ng, Joseph D.; Coates, Leighton

2009-01-01

The overexpression, purification and crystallization of endonuclease IV from T. maritima are reported. The crystals belonged to the hexagonal space group P6 1 and diffracted to 2.36 Å resolution. The DNA-repair enzyme endonuclease IV from the thermophilic bacterium Thermotoga maritima MSB8 (reference sequence NC-000853) has been expressed in Escherichia coli and crystallized for X-ray analysis. T. maritima endonuclease IV is a 287-amino-acid protein with 32% sequence identity to E. coli endonuclease IV. The protein was purified to homogeneity and was crystallized using the sitting-drop vapor-diffusion method. The protein crystallized in space group P6 1 , with one biological molecule in the asymmetric unit, corresponding to a Matthews coefficient of 2.39 Å 3 Da −1 and 47% solvent content. The unit-cell parameters of the crystals were a = b = 123.2, c = 35.6 Å. Microseeding and further optimization yielded crystals with an X-ray diffraction limit of 2.36 Å. A single 70° data set was collected and processed, resulting in an overall R merge and a completeness of 9.5% and 99.3%, respectively

Expression analysis of a ''Cucurbita'' cDNA encoding endonuclease

International Nuclear Information System (INIS)

Szopa, J.

1995-01-01

The nuclear matrices of plant cell nuclei display intrinsic nuclease activity which consists in nicking supercoiled DNA. A cDNA encoding a 32 kDa endonuclease has been cloned and sequenced. The nucleotide and deduced amino-acid sequences show high homology to known 14-3-3-protein sequences from other sources. The amino-acid sequence shows agreement with consensus sequences for potential phosphorylation by protein kinase A and C and for calcium, lipid and membrane-binding sites. The nucleotide-binding site is also present within the conserved part of the sequence. By Northern blot analysis, the differential expression of the corresponding mRNA was detected; it was the strongest in sink tissues. The endonuclease activity found on DNA-polyacrylamide gel electrophoresis coincided with mRNA content and was the highest in tuber. (author). 22 refs, 6 figs

Conserved structural chemistry for incision activity in structurally non-homologous apurinic/apyrimidinic endonuclease APE1 and endonuclease IV DNA repair enzymes.

Energy Technology Data Exchange (ETDEWEB)

Tsutakawa, Susan E.; Shin, David S.; Mol, Clifford D.; Izum, Tadahide; Arvai, Andrew S.; Mantha, Anil K.; Szczesny, Bartosz; Ivanov, Ivaylo N.; Hosfield, David J.; Maiti, Buddhadev; Pique, Mike E.; Frankel, Kenneth A.; Hitomi, Kenichi; Cunningham, Richard P.; Mitra, Sankar; Tainer, John A.

2013-03-22

Non-coding apurinic/apyrimidinic (AP) sites in DNA form spontaneously and as DNA base excision repair intermediates are the most common toxic and mutagenic in vivo DNA lesion. For repair, AP sites must be processed by 5' AP endonucleases in initial stages of base repair. Human APE1 and bacterial Nfo represent the two conserved 5' AP endonuclease families in the biosphere; they both recognize AP sites and incise the phosphodiester backbone 5' to the lesion, yet they lack similar structures and metal ion requirements. Here, we determined and analyzed crystal structures of a 2.4 ? resolution APE1-DNA product complex with Mg(2+) and a 0.92 Nfo with three metal ions. Structural and biochemical comparisons of these two evolutionarily distinct enzymes characterize key APE1 catalytic residues that are potentially functionally similar to Nfo active site components, as further tested and supported by computational analyses. We observe a magnesium-water cluster in the APE1 active site, with only Glu-96 forming the direct protein coordination to the Mg(2+). Despite differences in structure and metal requirements of APE1 and Nfo, comparison of their active site structures surprisingly reveals strong geometric conservation of the catalytic reaction, with APE1 catalytic side chains positioned analogously to Nfo metal positions, suggesting surprising functional equivalence between Nfo metal ions and APE1 residues. The finding that APE1 residues are positioned to substitute for Nfo metal ions is supported by the impact of mutations on activity. Collectively, the results illuminate the activities of residues, metal ions, and active site features for abasic site endonucleases.

Murine leukemia virus pol gene products: analysis with antisera generated against reverse transcriptase and endonuclease fusion proteins expressed in Escherichia coli

International Nuclear Information System (INIS)

Hu, S.C.; Court, D.L.; Zweig, M.; Levin, J.G.

1986-01-01

The organization of the murine leukemia virus (MuLV) pol gene was investigated by expressing molecular clones containing AKR MuLV reverse transcriptase or endonuclease or both gene segments in Escherichia coli and generating specific antisera against the expressed bacterial proteins. Reaction of these antisera with detergent-disrupted virus precipitated and 80-kilodalton (kDa) protein, the MuLV reverse transcriptase, and a 46-kDa protein which we believe is the viral endonuclease. A third (50-kDa) protein, related to reverse transcriptase, was also precipitated. Bacterial extracts of clones expressing reverse transcriptase and endonuclease sequences competed with the viral 80- and 46-kDa proteins, respectively. These results demonstrate that the antisera are specific for viral reverse transcriptase and endonuclease. Immunoprecipitation of AKR MuLV with antisera prepared against a bacterial protein containing only endonuclease sequences led to the observation that reverse transcriptase and endonuclease can be associated as a complex involving a disulfide bond(s)

Physical association of pyrimidine dimer DNA glycosylase and apurinic/apyrimidinic DNA endonuclease essential for repair of ultraviolet-damaged DNA

International Nuclear Information System (INIS)

Nakabeppu, Y.; Sekiguchi, M.

1981-01-01

T4 endonuclease, which is involved in repair of uv-damaged DNA, has been purified to apparent physical homogeneity. Incubation of uv-irradiated poly(dA).poly(dT) with the purified enzyme preparations resulted in production of alkali-labile apyrimidinic sites, followed by formation of nicks in the polymer. By performing a limited reaction with T4 endonuclease V at pH 8.5, irradiated polymer was converted to an intermediate form that carried a large number of alkali-labile sites but only a few nicks. The intermediate was used as substrate for the assay of apurinic/apyrimidinic DNA endonuclease activity. The two activities, a pyrimidine dimer DNA glycosylase and an apurinic/apyrimidinic DNA endonuclease, were copurified and found in enzyme preparations that contained only a 16,000-dalton polypeptide. These results strongly suggested that a DNA glycosylase specific for pyrimidine dimers and an apurinic/apyrimidinic DNA endonuclease reside in a single polypeptide chain coded by the denV gene of bacteriophage T4

Biochemical properties and base excision repair complex formation of apurinic/apyrimidinic endonuclease from Pyrococcus furiosus

OpenAIRE

Kiyonari, Shinichi; Tahara, Saki; Shirai, Tsuyoshi; Iwai, Shigenori; Ishino, Sonoko; Ishino, Yoshizumi

2009-01-01

Apurinic/apyrimidinic (AP) sites are the most frequently found mutagenic lesions in DNA, and they arise mainly from spontaneous base loss or modified base removal by damage-specific DNA glycosylases. AP sites are cleaved by AP endonucleases, and the resultant gaps in the DNA are repaired by DNA polymerase/DNA ligase reactions. We identified the gene product that is responsible for the AP endonuclease activity in the hyperthermophilic euryarchaeon, Pyrococcus furiosus. Furthermore, we detected...

DNA scanning mechanism of T4 endonuclease V. Effect of NaCl concentration on processive nicking activity

International Nuclear Information System (INIS)

Gruskin, E.A.; Lloyd, R.S.

1986-01-01

T4 endonuclease V is a pyrimidine dimer-specific endonuclease which generates incisions in DNA at the sites of pyrimidine dimers by a processive reaction mechanism. A model is presented in which the degree of processivity is directly related to the efficacy of the one-dimensional diffusion of endonuclease V on DNA by which the enzyme locates pyrimidine dimers. The modulation of the processive nicking activity of T4 endonuclease V on superhelical covalently closed circular DNA (form I) which contains pyrimidine dimers has been investigated as a function of the ionic strength of the reaction. Agarose gel electrophoresis was used to separate the three topological forms of the DNA which were generated in time course reactions of endonuclease V with dimer-containing form I DNA in the absence of NaCl, and in 25, 50, and 100 mM NaCl. The degree of processivity was evaluated in terms of the mass fraction of form III (linear) DNA which was produced as a function of the fraction of form I DNA remaining. Processivity is maximal in the absence of NaCl and decreases as the NaCl concentration is increased. At 100 mM NaCl, processivity is abolished and endonuclease V generates incisions in DNA at the site of dimers by a distributive reaction mechanism. The change from the distributive to a processive reaction mechanism occurs at NaCl concentrations slightly below 50 mM. The high degree of processivity which is observed in the absence of NaCl is reversible to the distributive mechanism, as demonstrated by experiments in which the NaCl concentration was increased during the time course reaction. In addition, unirradiated DNA inhibited the incision of irradiated DNA only at NaCl concentrations at which processivity was observed

Type III restriction endonucleases are heterotrimeric: comprising one helicase–nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

Science.gov (United States)

Butterer, Annika; Pernstich, Christian; Smith, Rachel M.; Sobott, Frank; Szczelkun, Mark D.; Tóth, Júlia

2014-01-01

Fundamental aspects of the biochemistry of Type III restriction endonucleases remain unresolved despite being characterized by numerous research groups in the past decades. One such feature is the subunit stoichiometry of these hetero-oligomeric enzyme complexes, which has important implications for the reaction mechanism. In this study, we present a series of results obtained by native mass spectrometry and size exclusion chromatography with multi-angle light scattering consistent with a 1:2 ratio of Res to Mod subunits in the EcoP15I, EcoPI and PstII complexes as the main holoenzyme species and a 1:1 stoichiometry of specific DNA (sDNA) binding by EcoP15I and EcoPI. Our data are also consistent with a model where ATP hydrolysis activated by recognition site binding leads to release of the enzyme from the site, dissociation from the substrate via a free DNA end and cleavage of the DNA. These results are discussed critically in the light of the published literature, aiming to resolve controversies and discuss consequences in terms of the reaction mechanism. PMID:24510100

Karyopherin-mediated nuclear import of the homing endonuclease VMA1-derived endonuclease is required for self-propagation of the coding region.

Science.gov (United States)

Nagai, Yuri; Nogami, Satoru; Kumagai-Sano, Fumi; Ohya, Yoshikazu

2003-03-01

VMA1-derived endonuclease (VDE), a site-specific endonuclease in Saccharomyces cerevisiae, enters the nucleus to generate a double-strand break in the VDE-negative allelic locus, mediating the self-propagating gene conversion called homing. Although VDE is excluded from the nucleus in mitotic cells, it relocalizes at premeiosis, becoming localized in both the nucleus and the cytoplasm in meiosis. The nuclear localization of VDE is induced by inactivation of TOR kinases, which constitute central regulators of cell differentiation in S. cerevisiae, and by nutrient depletion. A functional genomic approach revealed that at least two karyopherins, Srp1p and Kap142p, are required for the nuclear localization pattern. Genetic and physical interactions between Srp1p and VDE imply direct involvement of karyopherin-mediated nuclear transport in this process. Inactivation of TOR signaling or acquisition of an extra nuclear localization signal in the VDE coding region leads to artificial nuclear localization of VDE and thereby induces homing even during mitosis. These results serve as evidence that VDE utilizes the host systems of nutrient signal transduction and nucleocytoplasmic transport to ensure the propagation of its coding region.

[Restriction endonuclease digest - melting curve analysis: a new SNP genotyping and its application in traditional Chinese medicine authentication].

Science.gov (United States)

Jiang, Chao; Huang, Lu-Qi; Yuan, Yuan; Chen, Min; Hou, Jing-Yi; Wu, Zhi-Gang; Lin, Shu-Fang

2014-04-01

Single nucleotide polymorphisms (SNP) is an important molecular marker in traditional Chinese medicine research, and it is widely used in TCM authentication. The present study created a new genotyping method by combining restriction endonuclease digesting with melting curve analysis, which is a stable, rapid and easy doing SNP genotyping method. The new method analyzed SNP genotyping of two chloroplast SNP which was located in or out of the endonuclease recognition site, the results showed that when attaching a 14 bp GC-clamp (cggcgggagggcgg) to 5' end of the primer and selecting suited endonuclease to digest the amplification products, the melting curve of Lonicera japonica and Atractylodes macrocephala were all of double peaks and the adulterants Shan-yin-hua and A. lancea were of single peaks. The results indicated that the method had good stability and reproducibility for identifying authentic medicines from its adulterants. It is a potential SNP genotyping method and named restriction endonuclease digest - melting curve analysis.

The mitochondrial LSU rRNA group II intron of Ustilago maydis encodes an active homing endonuclease likely involved in intron mobility.

Directory of Open Access Journals (Sweden)

Anja Pfeifer

Full Text Available BACKGROUND: The a2 mating type locus gene lga2 is critical for uniparental mitochondrial DNA inheritance during sexual development of Ustilago maydis. Specifically, the absence of lga2 results in biparental inheritance, along with efficient transfer of intronic regions in the large subunit rRNA gene between parental molecules. However, the underlying role of the predicted LAGLIDADG homing endonuclease gene I-UmaI located within the group II intron LRII1 has remained unresolved. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the enzymatic activity of I-UmaI in vitro based on expression of a tagged full-length and a naturally occurring mutant derivative, which harbors only the N-terminal LAGLIDADG domain. This confirmed Mg²⁺-dependent endonuclease activity and cleavage at the LRII1 insertion site to generate four base pair extensions with 3' overhangs. Specifically, I-UmaI recognizes an asymmetric DNA sequence with a minimum length of 14 base pairs (5'-GACGGGAAGACCCT-3' and tolerates subtle base pair substitutions within the homing site. Enzymatic analysis of the mutant variant indicated a correlation between the activity in vitro and intron homing. Bioinformatic analyses revealed that putatively functional or former functional I-UmaI homologs are confined to a few members within the Ustilaginales and Agaricales, including the phylogenetically distant species Lentinula edodes, and are linked to group II introns inserted into homologous positions in the LSU rDNA. CONCLUSIONS/SIGNIFICANCE: The present data provide strong evidence that intron homing efficiently operates under conditions of biparental inheritance in U. maydis. Conversely, uniparental inheritance may be critical to restrict the transmission of mobile introns. Bioinformatic analyses suggest that I-UmaI-associated introns have been acquired independently in distant taxa and are more widespread than anticipated from available genomic data.

Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus.

Science.gov (United States)

Yang, Ying-Jie; Wang, Ye; Li, Zhi-Feng; Gong, Ya; Zhang, Peng; Hu, Wen-Chao; Sheng, Duo-Hong; Li, Yue-Zhong

2017-08-16

The CRISPR/Cas9 system is a powerful tool for genome editing, in which the sgRNA binds and guides the Cas9 protein for the sequence-specific cleavage. The protocol is employable in different organisms, but is often limited by cell damage due to the endonuclease activity of the introduced Cas9 and the potential off-target DNA cleavage from incorrect guide by the 20 nt spacer. In this study, after resolving some critical limits, we have established an efficient CRISPR/Cas9 system for the deletion of large genome fragments related to the biosynthesis of secondary metabolites in Myxococcus xanthus cells. We revealed that the high expression of a codon-optimized cas9 gene in M. xanthus was cytotoxic, and developed a temporally high expression strategy to reduce the cell damage from high expressions of Cas9. We optimized the deletion protocol by using the tRNA-sgRNA-tRNA chimeric structure to ensure correct sgRNA sequence. We found that, in addition to the position-dependent nucleotide preference, the free energy of a 20 nt spacer was a key factor for the deletion efficiency. By using the developed protocol, we achieved the CRISPR/Cas9-induced deletion of large biosynthetic gene clusters for secondary metabolites in M. xanthus DK1622 and its epothilone-producing mutant. The findings and the proposals described in this paper were suggested to be workable in other organisms, for example, other Gram negative bacteria with high GC content.

Home and away- the evolutionary dynamics of homing endonucleases

Directory of Open Access Journals (Sweden)

Barzel Adi

2011-11-01

Full Text Available Abstract Background Homing endonucleases (HEases are a large and diverse group of site-specific DNAases. They reside within self-splicing introns and inteins, and promote their horizontal dissemination. In recent years, HEases have been the focus of extensive research due to their promising potential use in gene targeting procedures for the treatment of genetic diseases and for the genetic engineering of crop, animal models and cell lines. Results Using mathematical analysis and computational modeling, we present here a novel account for the evolution and population dynamics of HEase genes (HEGs. We describe HEGs as paradoxical selfish elements whose long-term persistence in a single population relies on low transmission rates and a positive correlation between transmission efficiency and toxicity. Conclusion Plausible conditions allow HEGs to sustain at high frequency through long evolutionary periods, with the endonuclease frequency being either at equilibrium or periodically oscillating. The predictions of our model may prove important not only for evolutionary theory but also for gene therapy and bio-engineering applications of HEases.

Photogrammetry in mining - possibilities, state of art, perspectives

Energy Technology Data Exchange (ETDEWEB)

Sitek, Z.; Mierzwa, W.

1987-01-01

Presents a systematic review of the application of aerial and terrestrial photogrammetry in mining. Photogrammetry permits ground subsidence and excavation volume to be determined, maps and cross-sections of surface mines to be plotted and cleavage directions to be found. A method of measuring displacements by so-called time paralaxes is described that allows slope instability and dam and tunnel deformations to be detected. Application of stereophotogrammety for mapping underground headings and chamber workings, for recording and documenting mining accidents is discussed as well as application of photogrammetric probes for surveying postexploitation caverns. Other applications considered are: geologic cartography of underground workings, measuring fissures in shaft lining, investigating ventilation air flow, recording conditions at working faces before and after blasting and examining deformations caused in surface structures by mining. Recent research work conducted in Poland on possibilities offered by combining photogrammetric equipment with computers is outlined. 28 refs., 1 tab

Vertebrate Embryonic Cleavage Pattern Determination.

Science.gov (United States)

Hasley, Andrew; Chavez, Shawn; Danilchik, Michael; Wühr, Martin; Pelegri, Francisco

2017-01-01

The pattern of the earliest cell divisions in a vertebrate embryo lays the groundwork for later developmental events such as gastrulation, organogenesis, and overall body plan establishment. Understanding these early cleavage patterns and the mechanisms that create them is thus crucial for the study of vertebrate development. This chapter describes the early cleavage stages for species representing ray-finned fish, amphibians, birds, reptiles, mammals, and proto-vertebrate ascidians and summarizes current understanding of the mechanisms that govern these patterns. The nearly universal influence of cell shape on orientation and positioning of spindles and cleavage furrows and the mechanisms that mediate this influence are discussed. We discuss in particular models of aster and spindle centering and orientation in large embryonic blastomeres that rely on asymmetric internal pulling forces generated by the cleavage furrow for the previous cell cycle. Also explored are mechanisms that integrate cell division given the limited supply of cellular building blocks in the egg and several-fold changes of cell size during early development, as well as cytoskeletal specializations specific to early blastomeres including processes leading to blastomere cohesion. Finally, we discuss evolutionary conclusions beginning to emerge from the contemporary analysis of the phylogenetic distributions of cleavage patterns. In sum, this chapter seeks to summarize our current understanding of vertebrate early embryonic cleavage patterns and their control and evolution.

Presence of UV-endonuclease sensitive sites in daughter DNA of UV-irradiated mammalian cells

International Nuclear Information System (INIS)

D'Ambrosio, S.; Setlow, R.B.

1978-02-01

Asynchronous Chinese hamster cells were irradiated with 10 Jm -2 uv radiation and 0.25 to 4 hours later pulse-labeled with [ 3 H]thymidine. Cells synchronized by shaking off mitotic and G 1 cells were irradiated in either the G 1 -phase or S-phase of the cell cycle and pulse-labeled with [ 3 H]thymidine in the S-phase. After a 12 to 14 hour chase in unlabeled medium, the DNA was extracted, incubated with Micrococcus luteus uv-endonuclease and sedimented in alkaline sucrose. The number of endonuclease sensitive sites decreased as the time between uv irradiation and pulse-labeling of daughter DNA increased. Further, there were significantly less endonuclease sensitive sites in the daughter DNA from cells irradiated in the G 1 -phase than in the S-phase. These data indicate that very few, if any, dimers are transferred from parental DNA to daughter DNA and that the dimers detected in daughter DNA may be due to the irradiation of replicating daughter DNA before labeling

Specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells in vivo

International Nuclear Information System (INIS)

Tanaka, K.; Hayakawa, H.; Sekiguchi, M.; Okada, Y.

1977-01-01

The specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells was examined using an in vivo assay system with hemagglutinating virus of Japan (Sendai virus) inactivated by uv light. A clear dose response was observed between the level of uv-induced unscheduled DNA synthesis of xeroderma pigmentosum cells and the amount of T4 endonuclease V activity added. The T4 enzyme was unstable in human cells, and its half-life was 3 hr. Fractions derived from an extract of Escherichia coli infected with T4v 1 , a mutant defective in the endonuclease V gene, showed no ability to restore the uv-induced unscheduled DNA synthesis of xeroderma pigmentosum cells. However, fractions derived from an extract of T4D-infected E. coli with endonuclease V activity were effective. The T4 enzyme was effective in xeroderma pigmentosum cells on DNA damaged by uv light but not in cells damaged by 4-nitroquinoline 1-oxide. The results of these experiments show that the T4 enzyme has a specific action on human cell DNA in vivo. Treatment with the T4 enzyme increased the survival of group A xeroderma pigmentosum cells after uv irradiation

Journal of Biosciences | Indian Academy of Sciences

Indian Academy of Sciences (India)

These 2′-OMe nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property could enrich our understanding about the endonuclease cleavage mechanism and enhance our ability to regulate the enzymatic cleavage efficiency for applications in synthetic biology.

A putative Type IIS restriction endonuclease GeoICI

Indian Academy of Sciences (India)

As opposed to the unstable prototype, which cleaves DNA at 30°C, GeoICI is highly active at elevated temperatures, up to 73°C and over a very wide salt concentration range. Recognition/cleavage sites were determined by: (i) digestion of plasmid and bacteriophage lambda DNA (λ); (ii) cleavage of custom PCR substrates, ...

Molecular Recognition of DNA Damage Sites by Apurinic/Apyrimidinic Endonucleases

Energy Technology Data Exchange (ETDEWEB)

Braun, W. A.

2005-07-28

The DNA repair/redox factor AP endonuclease 1 (APE1) is a multifunctional protein which is known to to be essential for DNA repair activity in human cells. Structural/functional analyses of the APE activity is thus been an important research field to assess cellular defense mechanisms against ionizing radiation.

A site-specific endonuclease encoded by a typical archaeal intron

DEFF Research Database (Denmark)

Dalgaard, Jacob; Garrett, Roger Antony; Belfort, Malene

1993-01-01

The protein encoded by the archaeal intron in the 23S rRNA gene of the hyperthermophile Desulfurococcus mobilis is a double-strand DNase that, like group I intron homing endonucleases, is capable of cleaving an intronless allele of the gene. This enzyme, I-Dmo I, is unusual among the intron...

Crystal structure and DNA-binding property of the ATPase domain of bacterial mismatch repair endonuclease MutL from Aquifex aeolicus.

Science.gov (United States)

Fukui, Kenji; Iino, Hitoshi; Baba, Seiki; Kumasaka, Takashi; Kuramitsu, Seiki; Yano, Takato

2017-09-01

DNA mismatch repair (MMR) system corrects mismatched bases that are generated mainly by DNA replication errors. The repair system excises the error-containing single-stranded region and enables the re-synthesis of the strand. In the early reactions of MMR, MutL endonuclease incises the newly-synthesized/error-containing strand of the duplex to initiate the downstream excision reaction. MutL endonuclease consists of the N-terminal ATPase and C-terminal endonuclease domains. In this study, we report the crystal structure of the ATPase domain of MutL endonuclease from Aquifex aeolicus. The overall structure of the domain was similar to those of human MutL homologs and Escherichia coli MutL, although E. coli MutL has no endonuclease activity. The ATPase domain was comprised of two subdomains: the N-terminal ATP-binding subdomain and the C-terminal α-β sandwich subdomain. Site-directed mutagenesis experiment identified DNA-interacting eight basic amino acid residues, which were distributed across both the two subdomains and formed a DNA-binding cleft. Docking simulation between the structures of the ATPase and endonuclease domains generated a reliable model structure for the full-length A. aeolicus MutL, which satisfies our previous result of small-angle X-ray scattering analysis. On the basis of the model structure and further experimental results, we concluded that the two separate DNA-binding sites in the full-length A. aeolicus MutL simultaneously bind a dsDNA molecule. Copyright © 2017 Elsevier B.V. All rights reserved.

Cleavage and creep fracture of rock salt

International Nuclear Information System (INIS)

Chan, K.S.; Munson, D.E.; Bodner, S.R.

1996-01-01

The dominant failure mechanism in rock salt at ambient temperature is either cleavage or creep fracture. Since the transition of creep fracture to cleavage in a compressive stress field is not well understood, failure of rock salt by cleavage and creep fracture is analyzed in this paper to elucidate the effect of stress state on the competition between these two fracture mechanisms. For cleavage fracture, a shear crack is assumed to cause the formation and growth of a symmetric pair of wing cracks in a predominantly compressive stress field. The conditions for wing-crack instability are derived and presented as the cleavage fracture boundary in the fracture mechanism map. Using an existing creep fracture model, stress conditions for the onset of creep fracture and isochronous failure curves of specified times-to-rupture are calculated and incorporated into the fracture mechanism map. The regimes of dominance by cleavage and creep fracture are established and compared with experimental data. The result indicates that unstable propagation of cleavage cracks occurs only in the presence of tensile stress. The onset of creep fracture is promoted by a tensile stress, but can be totally suppressed by a high confining pressure. Transition of creep fracture to cleavage occurs when critical conditions of stress difference and tensile stress for crack instability are exceeded

Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

Science.gov (United States)

Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

2009-01-01

Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

Measurement of M. luteus endonuclease-sensitive lesions by alkaline elution

Energy Technology Data Exchange (ETDEWEB)

Fornace, Jr, A J [National Cancer Inst., Bethesda, MD (USA). Lab. for Experimental Pathology

1982-01-01

The UV-endonuclease approach to detect DNA damage has been combined with the alkaline elution technique with a resultant marked increase in sensitivity compared to the conventional method using alkaline sedimentation. DNA from UV-irradiated cells was digested on an inert filter with an extract from Micrococcus luteus and then analyzed by alkaline elution. Endonuclease-sensitive sites (endo-sites) were measured after doses of 0.08-0.7 Jm/sup -2/ of UV-radiation. An estimate of endo-site production with UV radiation, 0.27 endo-sites/10/sup 8/ daltons of DNA/0.1 Jm/sup -2/, was similar to that usually seen at higher doses by others. With repair incubation, approx. 50% of the endo-sites were removed in 4 h by normal human fibroblasts after 0.2 or 0.4 Jm/sup -2/, no appreciable repair was seen in xeroderma pigmentosum fibroblasts from complementation group A after 24 h of repair incubation. No photoreaction of UV damage due to 0.4 Jm/sup -2/ was detected in normal human fibroblasts.

Measurement of M. luteus endonuclease-sensitive lesions by alkaline elution

International Nuclear Information System (INIS)

Fornace, A.J. Jr.

1982-01-01

The UV-endonuclease approach to detect DNA damage has been combined with the alkaline elution technique with a resultant marked increase in sensitivity compared to the conventional method using alkaline sedimentation. DNA from UV-irradiated cells was digested on an inert filter with an extract from Micrococcus luteus and then analyzed by alkaline elution. Endonuclease-sensitive sites (endo-sites) were measured after doses of 0.08-0.7 Jm -2 of UV-radiation. An estimate of endo-site production with UV radiation, 0.27 endo-sites/10 8 daltons of DNA/0.1 Jm -2 , was similar to that usually seen at higher doses by others. With repair incubation, approx. 50% of the endo-sites were removed in 4 h by normal human fibroblasts after 0.2 or 0.4 Jm -2 , no appreciable repair was seen in xeroderma pigmentosum fibroblasts from complementation group A after 24 h of repair incubation. No photoreaction of UV damage due to 0.4 Jm -2 was detected in normal human fibroblasts. (orig./AJ)

CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles.

Science.gov (United States)

Lemos, Brenda R; Kaplan, Adam C; Bae, Ji Eun; Ferrazzoli, Alexander E; Kuo, James; Anand, Ranjith P; Waterman, David P; Haber, James E

2018-02-27

Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5' overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans.

Science.gov (United States)

Manhart, Carol M; Ni, Xiaodan; White, Martin A; Ortega, Joaquin; Surtees, Jennifer A; Alani, Eric

2017-04-01

Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker's yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-Mlh3. In addition, Mlh1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA.

Inroads into base excision repair I. The discovery of apurinic/apyrimidinic (AP) endonuclease. "An endonuclease for depurinated DNA in Escherichia coli B," Canadian Journal of Biochemistry, 1972.

Science.gov (United States)

Lindahl, Tomas; Verly, W G; Paquette Y

2004-11-02

DNA treated with alkylating agents is incised at sites of damage by cell extracts. A key component of this DNA repair function was shown by Verly and co-workers to be an endonuclease acting at secondary lesions, apurinic sites, rather than directly at alkylated nucleotide residues.

Enhancement of ultraviolet-DNA repair in denV gene transfectants and T4 endonuclease V-liposome recipients

International Nuclear Information System (INIS)

Kibitel, J.T.; Yee, V.; Yarosh, D.B.

1991-01-01

The phage T4 denV gene, coding for the pyrimidine-dimer specific T4 endonuclease V, was transfected into human repair-proficient fibroblasts, repair-deficient xeroderma pigmentosum fibroblasts, and wild type CHO hamster cells. Transfectants maintained denV DNA and expressed denV mRNA. Purified T4 endonuclease V encapsulated in liposomes was also used to treat repair-proficient and -deficient human cells. The denV transfected clones and liposome-treated cells showed increased unscheduled DNA synthesis and enhanced removal of pyrimidine dimers compared to controls. Both denV gene transfection and endonuclease V liposome treatment enhanced post-UV survival in xeroderma pigmentosum cells but had no effect on survival in repair-proficient human or hamster cells. The results demonstrate that an exogenous DNA repair enzyme can correct the DNA repair defect in xeroderma pigmentosum cells and enhance DNA repair in normal cells. (author)

Structural analysis and implicit 3D modelling of Jwaneng Mine: Insights into deformation of the Transvaal Supergroup in SE Botswana

Science.gov (United States)

Creus, P. K.; Basson, I. J.; Stoch, B.; Mogorosi, O.; Gabanakgosi, K.; Ramsden, F.; Gaegopolwe, P.

2018-01-01

Country rock at Jwaneng Diamond Mine provides a rare insight into the deformational history of the Transvaal Supergroup in southern Botswana. The ca. 235 Ma kimberlite diatremes intruded into late Archaean to Early Proterozoic, mixed, siliciclastic-carbonate sediments, that were subjected to at least three deformational events. The first deformational event (D1), caused by NW-SE directed compression, is responsible for NE-trending, open folds (F1) with associated diverging, fanning, axial planar cleavage. The second deformational event (D2) is probably progressive, involving a clockwise rotation of the principal stress to NE-SW trends. Early D2, which was N-S directed, involved left-lateral, oblique shearing along cleavage planes that developed around F1 folds, along with the development of antithetic structures. Progressive clockwise rotation of far-field forces saw the development of NW-trending folds (F2) and its associated, weak, axial planar cleavage. D3 is an extensional event in which normal faulting, along pre-existing cleavage planes, created a series of rhomboid-shaped, fault-bounded blocks. Normal faults, which bound these blocks, are the dominant structures at Jwaneng Mine. Combined with block rotation and NW-dipping bedding, a horst-like structure on the northwestern limb of a broad, gentle, NE-trending anticline is indicated. The early compressional and subsequent extensional events are consistent throughout the Jwaneng-Ramotswa-Lobatse-Thabazimbi area, suggesting that a large area records the same fault geometry and, consequently, deformational history. It is proposed that Jwaneng Mine is at or near the northernmost limit of the initial, northwards-directed compressional event.

Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein.

Directory of Open Access Journals (Sweden)

Yaiza Fernández-García

2016-06-01

Full Text Available Andes virus (ANDV is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1-200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure-function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening.

Microstructure and cleavage in lath martensitic steels

International Nuclear Information System (INIS)

Morris, John W Jr; Kinney, Chris; Pytlewski, Ken; Adachi, Y

2013-01-01

In this paper we discuss the microstructure of lath martensitic steels and the mechanisms by which it controls cleavage fracture. The specific experimental example is a 9Ni (9 wt% Ni) steel annealed to have a large prior austenite grain size, then examined and tested in the as-quenched condition to produce a relatively coarse lath martensite. The microstructure is shown to approximate the recently identified ‘classic’ lath martensite structure: prior austenite grains are divided into packets, packets are subdivided into blocks, and blocks contain interleaved laths whose variants are the two Kurjumov–Sachs relations that share the same Bain axis of the transformation. When the steel is fractured in brittle cleavage, the laths in the block share {100} cleavage planes and cleave as a unit. However, cleavage cracks deflect or blunt at the boundaries between blocks with different Bain axes. It follows that, as predicted, the block size governs the effective grain size for cleavage. (paper)

Identification of a mismatch-specific endonuclease in hyperthermophilic Archaea

OpenAIRE

Ishino, Sonoko; Nishi, Yuki; Oda, Soichiro; Uemori, Takashi; Sagara, Takehiro; Takatsu, Nariaki; Yamagami, Takeshi; Shirai, Tsuyoshi; Ishino, Yoshizumi

2016-01-01

The common mismatch repair system processed by MutS and MutL and their homologs was identified in Bacteria and Eukarya. However, no evidence of a functional MutS/L homolog has been reported for archaeal organisms, and it is not known whether the mismatch repair system is conserved in Archaea. Here, we describe an endonuclease that cleaves double-stranded DNA containing a mismatched base pair, from the hyperthermophilic archaeon Pyrococcus furiosus. The corresponding gene revealed that the act...

Creating a monomeric endonuclease TALE-I-SceI with high specificity and low genotoxicity in human cells.

Science.gov (United States)

Lin, Jianfei; Chen, He; Luo, Ling; Lai, Yongrong; Xie, Wei; Kee, Kehkooi

2015-01-01

To correct a DNA mutation in the human genome for gene therapy, homology-directed repair (HDR) needs to be specific and have the lowest off-target effects to protect the human genome from deleterious mutations. Zinc finger nucleases, transcription activator-like effector nuclease (TALEN) and CRISPR-CAS9 systems have been engineered and used extensively to recognize and modify specific DNA sequences. Although TALEN and CRISPR/CAS9 could induce high levels of HDR in human cells, their genotoxicity was significantly higher. Here, we report the creation of a monomeric endonuclease that can recognize at least 33 bp by fusing the DNA-recognizing domain of TALEN (TALE) to a re-engineered homing endonuclease I-SceI. After sequentially re-engineering I-SceI to recognize 18 bp of the human β-globin sequence, the re-engineered I-SceI induced HDR in human cells. When the re-engineered I-SceI was fused to TALE (TALE-ISVB2), the chimeric endonuclease induced the same HDR rate at the human β-globin gene locus as that induced by TALEN, but significantly reduced genotoxicity. We further demonstrated that TALE-ISVB2 specifically targeted at the β-globin sequence in human hematopoietic stem cells. Therefore, this monomeric endonuclease has the potential to be used in therapeutic gene targeting in human cells. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Modeling and inferring cleavage patterns in proliferating epithelia.

Directory of Open Access Journals (Sweden)

Ankit B Patel

2009-06-01

Full Text Available The regulation of cleavage plane orientation is one of the key mechanisms driving epithelial morphogenesis. Still, many aspects of the relationship between local cleavage patterns and tissue-level properties remain poorly understood. Here we develop a topological model that simulates the dynamics of a 2D proliferating epithelium from generation to generation, enabling the exploration of a wide variety of biologically plausible cleavage patterns. We investigate a spectrum of models that incorporate the spatial impact of neighboring cells and the temporal influence of parent cells on the choice of cleavage plane. Our findings show that cleavage patterns generate "signature" equilibrium distributions of polygonal cell shapes. These signatures enable the inference of local cleavage parameters such as neighbor impact, maternal influence, and division symmetry from global observations of the distribution of cell shape. Applying these insights to the proliferating epithelia of five diverse organisms, we find that strong division symmetry and moderate neighbor/maternal influence are required to reproduce the predominance of hexagonal cells and low variability in cell shape seen empirically. Furthermore, we present two distinct cleavage pattern models, one stochastic and one deterministic, that can reproduce the empirical distribution of cell shapes. Although the proliferating epithelia of the five diverse organisms show a highly conserved cell shape distribution, there are multiple plausible cleavage patterns that can generate this distribution, and experimental evidence suggests that indeed plants and fruitflies use distinct division mechanisms.

Type II restriction endonucleases : a historical perspective and more

OpenAIRE

Pingoud, Alfred; Wilson, Geoffrey G.; Wende, Wolfgang

2014-01-01

This article continues the series of Surveys and Summaries on restriction endonucleases (REases) begun this year in Nucleic Acids Research. Here we discuss ‘Type II’ REases, the kind used for DNA analysis and cloning. We focus on their biochemistry: what they are, what they do, and how they do it. Type II REases are produced by prokaryotes to combat bacteriophages. With extreme accuracy, each recognizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nea...

Intermolecular cleavage by UmuD-like mutagenesis proteins

Science.gov (United States)

McDonald, John P.; Frank, Ekaterina G.; Levine, Arthur S.; Woodgate, Roger

1998-01-01

The activity of a number of proteins is regulated by self-processing reactions. Elegant examples are the cleavage of the prokaryotic LexA and λCI transcriptional repressors and the UmuD-like mutagenesis proteins. Various studies support the hypothesis that LexA and λCI cleavage reactions are predominantly intramolecular in nature. The recently described crystal structure of the Escherichia coli UmuD′ protein (the posttranslational cleavage product of the UmuD protein) suggests, however, that the region of the protein corresponding to the cleavage site is at least 50 Å away from the catalytic active site. We considered the possibility, therefore, that the UmuD-like proteins might undergo self-processing that, in contrast to LexA and λCI, occurs via an intermolecular rather than intramolecular reaction. To test this hypothesis, we introduced into E. coli compatible plasmids with mutations at either the cleavage or the catalytic site of three UmuD-like proteins. Cleavage of these proteins only occurs in the presence of both plasmids, indicating that the reaction is indeed intermolecular in nature. Furthermore, this intermolecular reaction is completely dependent upon the multifunctional RecA protein and leads to the restoration of cellular mutagenesis in nonmutable E. coli strains. Intermolecular cleavage of a biotinylated UmuD active site mutant was also observed in vitro in the presence of the wild-type UmuD′ protein, indicating that in addition to the intact UmuD protein, the normal cleavage product (UmuD′) can also act as a classical enzyme. PMID:9465040

Regioselectivity in the Reductive Bond Cleavage of Diarylalkylsulfonium Salts

DEFF Research Database (Denmark)

Kampmeier, Jack; Mansurul Hoque, AKM; D. Saeva, Franklin

2009-01-01

products vary from regiospecific alkyl cleavage to predominant aryl cleavage as a function of the potential of the reducing agent. We conclude that differences between the reductive cleavages of mono- and diarylsulfonium salts are direct consequences of the structures of the sulfuranyl radical......- tolylethylsulfonium and di-4-tolyl-2-phenylethylsulfonium salts by a variety of one-electron reducing agents ranging in potential from -0.77 to +2.5 eV (vs SCE) and including thermal reductants, indirect electrolyses mediated by a series of cyanoaromatics, and excited singlet states. We report that the cleavage...... intermediates and the bond dissociation energies of the alkyl and aryl bonds. Competitions between the rates of cleavage and oxidation of the intermediate sulfuranyl radicals and between concerted and stepwise mechanisms are discussed to explain the variations in bond cleavage products as a function...

Cell-Autonomous Progeroid Changes in Conditional Mouse Models for Repair Endonuclease XPG Deficiency

NARCIS (Netherlands)

S. Barnhoorn (Sander); L.M. Uittenboogaard (Lieneke); D. Jaarsma (Dick); W.P. Vermeij (Wilbert); M. Tresini (Maria); M. Weymaere (Michael); H. Menoni (Hervé); R.M.C. Brandt (Renata); M.C. de Waard (Monique); S.M. Botter (Sander); A.H. Sarker (Altraf); N.G.J. Jaspers (Nicolaas); G.T.J. van der Horst (Gijsbertus); P.K. Cooper (Priscilla K.); J.H.J. Hoeijmakers (Jan); I. van der Pluijm (Ingrid)

2014-01-01

textabstractAs part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG

An analysis of the repair processes in ultraviolet-irradiated Micrococcus luteus using purified ultraviolet-endonuclease

International Nuclear Information System (INIS)

Tomilin, N.V.; Zherebtsov, S.V.

1982-01-01

The measurement of the frequency of endonucleolytic incisions in ultraviolet-irradiated DNA serves as the test for the presence of pyrimidine dimers. In accordance with this approach, the lysates of three Micrococcus luteus strains containing radioactively labeled chromosomes were treated with purified M. luteus ultraviolet-endonuclease to trace segregation of dimers amongst parental and newly synthesized DNA and their removal during postreplication and excision DNA repair. A considerable proportion of the dimers in all strains tested proved to be insensitive to the action of exogenous incising enzyme. The use of chloramphenicol as an inhibitor of postirradiation protein synthesis in combination with ultraviolet-endonuclease treatment of DNA allowed to reveal at least two alternative pathways of postreplication repair: constitutively active recombinational pathway and inducible nonrecombinational one. (Auth.)

Secondary isotope effects on alpha-cleavage reactions

International Nuclear Information System (INIS)

Ingemann, S.; Hammerum, S.

1980-01-01

Kinetic deuterium isotope effects on mass spectral reactions have in several instances been utilized to provide structural information and to answer mechanistic questions. Typically, the influence of the deuterium label on the rate of one of a number of competing reactions has been studied. Secondary isotope effects have usually been assumed to be relatively insignificant in comparison with the observed kinetic effects, even though various workers have shown that secondary isotope effects may indeed exert a considerable influence on the rates of competing simple cleavages. Recent studies have provided quantitative data to show that the mere presence of deuterium atoms up to six bonds away may influence the rate of a simple cleavage reaction. In relation to an investigation of rearrangements accompanying simple cleavage reactions, a semi-quantitative measure was needed of the variation of the secondary isotope effect with the number of bonds between the deuterium label and the point of rupture. The influence has therefore been examined of the presence of remote deuterium atoms on a typical simple cleavage reaction, the α-cleavage of aliphatic amines. As a model compound, N-methyldipentylamine was chosen, systematically labelled with deuterium. (author)

A detailed experimental study of a DNA computer with two endonucleases.

Science.gov (United States)

Sakowski, Sebastian; Krasiński, Tadeusz; Sarnik, Joanna; Blasiak, Janusz; Waldmajer, Jacek; Poplawski, Tomasz

2017-07-14

Great advances in biotechnology have allowed the construction of a computer from DNA. One of the proposed solutions is a biomolecular finite automaton, a simple two-state DNA computer without memory, which was presented by Ehud Shapiro's group at the Weizmann Institute of Science. The main problem with this computer, in which biomolecules carry out logical operations, is its complexity - increasing the number of states of biomolecular automata. In this study, we constructed (in laboratory conditions) a six-state DNA computer that uses two endonucleases (e.g. AcuI and BbvI) and a ligase. We have presented a detailed experimental verification of its feasibility. We described the effect of the number of states, the length of input data, and the nondeterminism on the computing process. We also tested different automata (with three, four, and six states) running on various accepted input words of different lengths such as ab, aab, aaab, ababa, and of an unaccepted word ba. Moreover, this article presents the reaction optimization and the methods of eliminating certain biochemical problems occurring in the implementation of a biomolecular DNA automaton based on two endonucleases.

Survival of Saccharomyces cerevisiae after treatment with the restriction endonuclease Alu I

International Nuclear Information System (INIS)

Winckler, K.; Bach, B.; Obe, G.

1988-01-01

Treatment of yeast cells proficient in the repair of radiation damage (Saccharomyces cervisiae) with the restriction endonuclease Alu I leads to a positive dose-effect relationship between inactivation level and enzyme concentration. The data suggest an uptake of the active restriction enzyme into the cells and a relationship between induction of DNA double-strand breaks and cell killing. (author)

Endonuclease G is a novel determinant of cardiac hypertrophy and mitochondrial function

Czech Academy of Sciences Publication Activity Database

McDermott-Roe, Ch.; Ye, J.; Ahmed, R.; Sun, X. M.; Serafín, A.; Ware, J.; Bottolo, L.; Muckett, P.; Caňas, X.; Zhang, J.; Rowe, G. C.; Buchan, R.; Lu, H.; Braithwaite, A.; Mancini, M.; Hauton, D.; Martí, R.; García-Arumí, E.; Hubner, N.; Jacob, H.; Serikawa, T.; Zídek, Václav; Papoušek, František; Kolář, František; Cardona, M.; Ruiz-Meana, M.; García-Dorado, D.; Comella, J. X.; Felkin, L. E.; Barton, P. J. R.; Arany, Z.; Pravenec, Michal; Petretto, E.; Sanchis, D.; Cook, S.A.

2011-01-01

Roč. 478, č. 7367 (2011), s. 114-118 ISSN 0028-0836 R&D Projects: GA MŠk(CZ) 1M0520; GA ČR(CZ) GA301/08/0166 Institutional research plan: CEZ:AV0Z50110509 Keywords : left ventricular hypertrophy * endonuclease G * mitochondrial dysfunction Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 36.280, year: 2011

Biological significance of facilitated diffusion in protein-DNA interactions. Applications to T4 endonuclease V-initiated DNA repair

International Nuclear Information System (INIS)

Dowd, D.R.; Lloyd, R.S.

1990-01-01

Facilitated diffusion along nontarget DNA is employed by numerous DNA-interactive proteins to locate specific targets. Until now, the biological significance of DNA scanning has remained elusive. T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light. In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli. This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme. However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population. The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E. coli then was assessed. The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion. This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype. These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance

Dataset of cocoa aspartic protease cleavage sites

Directory of Open Access Journals (Sweden)

Katharina Janek

2016-09-01

Full Text Available The data provide information in support of the research article, “The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors” (Janek et al., 2016 [1]. Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans. Keywords: Aspartic protease, Cleavage sites, Cocoa, In-vitro proteolysis, Mass spectrometry, Peptides

Homing endonuclease genes: the rise and fall and rise again of a selfish element.

Science.gov (United States)

Burt, Austin; Koufopanou, Vassiliki

2004-12-01

Homing endonuclease genes (HEGs) are selfish genetic elements that spread by first cleaving chromosomes that do not contain them and then getting copied across to the broken chromosome as a byproduct of the repair process. The success of this strategy will depend on the opportunities for homing--in other words, the frequency with which HEG(+) and HEG(-) chromosomes come into contact--which varies widely among host taxa. HEGs are also unusual in that the selection pressure for endonuclease function disappears if they become fixed in a population, which makes them susceptible to degeneration and imposes a need for regular horizontal transmission between species. HEGs will be selected to reduce the harm done to the host organism, and this is expected to influence the evolution of their sequence specificity and maturase functions. HEGs may also be domesticated by their hosts, and are currently being put to human uses.

Cleavage events and sperm dynamics in chick intrauterine embryos.

Directory of Open Access Journals (Sweden)

Hyung Chul Lee

Full Text Available This study was undertaken to elucidate detailed event of early embryogenesis in chicken embryos using a noninvasive egg retrieval technique before oviposition. White Leghorn intrauterine eggs were retrieved from 95 cyclic hens aged up to 54-56 weeks and morphogenetic observation was made under both bright field and fluorescent image in a time course manner. Differing from mammals, asymmetric cleavage to yield preblastodermal cells was observed throughout early embryogenesis. The first two divisions occurred synchronously and four polarized preblastodermal cells resulted after cruciform cleavage. Then, asynchronous cleavage continued in a radial manner and overall cell size in the initial cleavage region was smaller than that in the distal area. Numerous sperms were visible, regardless of zygotic nuclei formation. Condensed sperm heads were present mainly in the perivitelline space and cytoplasm, and rarely in the yolk region, while decondensed sperm heads were only visible in the yolk. In conclusion, apparent differences in sperm dynamics and early cleavage events compared with mammalian embryos were detected in chick embryo development, which demonstrated polarized cleavage with penetrating supernumerary sperm into multiple regions.

New Insight into the Cleavage Reaction of Nostoc sp. Strain PCC 7120 Carotenoid Cleavage Dioxygenase in Natural and Nonnatural Carotenoids

Science.gov (United States)

Heo, Jinsol; Kim, Se Hyeuk

2013-01-01

Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD of Nostoc sp. strain PCC 7120, in vivo and in vitro using natural and nonnatural carotenoid structures. NSC3 cleaved β-apo-8′-carotenal at 3 positions, C-13C-14, C-15C-15′, and C-13′C-14′, revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4′-diaponeurosporene, 4,4′-diaponeurosporen-4′-al, 4,4′-diaponeurosporen-4′-oic acid, 4,4′-diapotorulene, and 4,4′-diapotorulen-4′-al to generate novel cleavage products (apo-14′-diaponeurosporenal, apo-13′-diaponeurosporenal, apo-10′-diaponeurosporenal, apo-14′-diapotorulenal, and apo-10′-diapotorulenal, respectively). The study of carotenoids with natural or nonnatural structures produced by using synthetic modules could provide information valuable for understanding the cleavage reactions or substrate preferences of other CCDs in vivo and in vitro. PMID:23524669

In vivo analysis of the Notch receptor S1 cleavage.

Directory of Open Access Journals (Sweden)

Robert J Lake

2009-08-01

Full Text Available A ligand-independent cleavage (S1 in the extracellular domain of the mammalian Notch receptor results in what is considered to be the canonical heterodimeric form of Notch on the cell surface. The in vivo consequences and significance of this cleavage on Drosophila Notch signaling remain unclear and contradictory. We determined the cleavage site in Drosophila and examined its in vivo function by a transgenic analysis of receptors that cannot be cleaved. Our results demonstrate a correlation between loss of cleavage and loss of in vivo function of the Notch receptor, supporting the notion that S1 cleavage is an in vivo mechanism of Notch signal control.

In vitro measurement of beta-carotene cleavage activity : methodological considerations and the effect of other carotenoids on beta-carotene cleavage

NARCIS (Netherlands)

Vliet, T. van; Schaik, F. van; Schreurs, W.H.P.; Berg, H. van den

1996-01-01

In view of controversies about assessment of the β-carotene cleavage activity, methodological aspects and problems of the dioxygenase assay are described. Using rat and hamster intestinal preparations the method was optimized on retinal formation, the only cleavage product we could demonstrate. It

Molecular dynamics simulations of deoxyribonucleic acids and repair enzyme T4 endonuclease V

International Nuclear Information System (INIS)

Pinak, Miroslav

1999-01-01

This report describes the results of molecular dynamics (MD) simulation of deoxyribonucleic acids (DNA) and specific repair enzyme T4 endonuclease V. Namely research described here is focused on the examination of specific recognition process, in which this repair enzyme recognizes the damaged site on the DNA molecule-thymine dimer (TD). TD is frequent DNA damage induced by UV radiation in sun light and unless properly repaired it may be mutagenic or lethal for cell, and is also considered among the major causes of skin cancer. T4 endonuclease V is a DNA specific repair enzyme from bacteriophage T4 that catalyzes the first reaction step of TD repair pathway. MD simulations of three molecules - native DNA dodecamer (12 base pairs), DNA of the same sequence of nucleotides as native one but with TD, and repair enzyme T4 endonuclease V - were performed for 1 ns individually for each molecule. Simulations were analyzed to determine the role of electrostatic interaction in the recognition process. It is found that electrostatic energies calculated for amino acids of the enzyme have positive values of around +15 kcal/mol. The electrostatic energy of TD site has negative value of approximately -9 kcal/mol, different from the nearly neutral value of the respective thymines site of the native DNA. The electrostatic interaction of TD site with surrounding water environment differs from the electrostatic interaction of other nucleotides. Differences found between TD site and respective thymines site of native DNA indicate that the electrostatic energy is an important factor contributing to proper recognition of TD site during scanning process in which enzyme scans the DNA. In addition to the electrostatic energy, the important factor in recognition process might be structural complementarity of enzyme and bent DNA with TD. There is significant kink formed around TD site, that is not observed in native DNA. (author)

RPA activates the XPF‐ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks

KAUST Repository

Abdullah, Ummi B

2017-06-13

During replication‐coupled DNA interstrand crosslink (ICL) repair, the XPF‐ERCC1 endonuclease is required for the incisions that release, or “unhook”, ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL. Here, we report that while purified XPF‐ERCC1 incises simple ICL‐containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single‐stranded DNA (ssDNA)‐binding replication protein A (RPA) selectively restores XPF‐ERCC1 endonuclease activity on this structure. The 5′–3′ exonuclease SNM1A can load from the XPF‐ERCC1‐RPA‐induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF‐ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.

RPA activates the XPF‐ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks

KAUST Repository

Abdullah, Ummi B; McGouran, Joanna F; Brolih, Sanja; Ptchelkine, Denis; El‐Sagheer, Afaf H; Brown, Tom; McHugh, Peter J

2017-01-01

During replication‐coupled DNA interstrand crosslink (ICL) repair, the XPF‐ERCC1 endonuclease is required for the incisions that release, or “unhook”, ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL. Here, we report that while purified XPF‐ERCC1 incises simple ICL‐containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single‐stranded DNA (ssDNA)‐binding replication protein A (RPA) selectively restores XPF‐ERCC1 endonuclease activity on this structure. The 5′–3′ exonuclease SNM1A can load from the XPF‐ERCC1‐RPA‐induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF‐ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.

Pripper: prediction of caspase cleavage sites from whole proteomes

Directory of Open Access Journals (Sweden)

Salmi Jussi

2010-06-01

Full Text Available Abstract Background Caspases are a family of proteases that have central functions in programmed cell death (apoptosis and inflammation. Caspases mediate their effects through aspartate-specific cleavage of their target proteins, and at present almost 400 caspase substrates are known. There are several methods developed to predict caspase cleavage sites from individual proteins, but currently none of them can be used to predict caspase cleavage sites from multiple proteins or entire proteomes, or to use several classifiers in combination. The possibility to create a database from predicted caspase cleavage products for the whole genome could significantly aid in identifying novel caspase targets from tandem mass spectrometry based proteomic experiments. Results Three different pattern recognition classifiers were developed for predicting caspase cleavage sites from protein sequences. Evaluation of the classifiers with quality measures indicated that all of the three classifiers performed well in predicting caspase cleavage sites, and when combining different classifiers the accuracy increased further. A new tool, Pripper, was developed to utilize the classifiers and predict the caspase cut sites from an arbitrary number of input sequences. A database was constructed with the developed tool, and it was used to identify caspase target proteins from tandem mass spectrometry data from two different proteomic experiments. Both known caspase cleavage products as well as novel cleavage products were identified using the database demonstrating the usefulness of the tool. Pripper is not restricted to predicting only caspase cut sites, but it gives the possibility to scan protein sequences for any given motif(s and predict cut sites once a suitable cut site prediction model for any other protease has been developed. Pripper is freely available and can be downloaded from http://users.utu.fi/mijopi/Pripper. Conclusions We have developed Pripper, a tool for

Permeabilization of ultraviolet-irradiated chinese hamster cells with polyethylene glycol and introduction of ultraviolet endonuclease from Micrococcus luteus

International Nuclear Information System (INIS)

Yarosh, D.B.; Setlow, R.B.

1981-01-01

Chinese hamster V-79 cells were made permeable by treatment with polyethylene glycol and then incubated with a Micrococcus luteus extract containing ultraviolet-specific endonuclease activity. This treatment introduced nicks in irradiated, but not in unirradiated, deoxyribonucleic acid. The nicks remained open for at least 3 h; there was no loss of endonuclease-sensitive sites, and no excision of dimers as measured by chromatography was detected. In addition, there was no increase in ultraviolet resistance in treated cells. This suggests that the absence of a significant amount of excision repair in rodent cells is due to the lack of both incision and excision capacity

Quantitative characterization of cleavage and hydrogen-assisted quasi-cleavage fracture surfaces with the use of confocal laser scanning microscopy

International Nuclear Information System (INIS)

Merson, E.; Kudrya, A.V.; Trachenko, V.A.; Merson, D.; Danilov, V.; Vinogradov, A.

2016-01-01

“True” cleavage (TC) and quasi-cleavage (QC) fracture surfaces of low-carbon steel specimens tested in liquid nitrogen and after hydrogen charging respectively were investigated by quantitative confocal laser scanning microscopy (CLSM) and conventional scanning electron microscopy (SEM) with electron-backscattered diffraction (EBSD). Topological and crystallographic features of the TC fracture surface are found in good agreement with the generally accepted cleavage mechanism: TC facets diameters correspond to those of grains; the crack path strictly follows the crystallographic orientation of grains and the most of the cleavage cracks are parallel to {100} planes. On the 2D SEM images, the QC facets appeared resembling the TC ones in terms of river line patterns, shapes and sizes. However, the substantial differences between the topography of these two kinds of fracture surfaces were revealed by 3D CLSM: the average misorientation angle between QC facets and the roughness of the QC fracture surface were much lower than those measured for TC. It is demonstrated that all these features are attributed to the specific fracture mechanism operating during hydrogen-assisted cracking.

Quantitative characterization of cleavage and hydrogen-assisted quasi-cleavage fracture surfaces with the use of confocal laser scanning microscopy

Energy Technology Data Exchange (ETDEWEB)

Merson, E. [Institute of Advanced Technologies, Togliatti State University, 445667 (Russian Federation); Kudrya, A.V.; Trachenko, V.A. [Department of Physical Metallurgy and the Physics of Strength, NUST MISiS, Moscow 119490 (Russian Federation); Merson, D. [Institute of Advanced Technologies, Togliatti State University, 445667 (Russian Federation); Laboratory for Advanced Materials, Kazan Federal University, Naberezhnye Chelny 423812, Republic of Tatarstan (Russian Federation); Danilov, V. [Institute of Advanced Technologies, Togliatti State University, 445667 (Russian Federation); Vinogradov, A. [Institute of Advanced Technologies, Togliatti State University, 445667 (Russian Federation); Department of Engineering Design and Materials, Norwegian University of Science and Technology – NTNU, N-7491 Trondheim (Norway)

2016-05-17

“True” cleavage (TC) and quasi-cleavage (QC) fracture surfaces of low-carbon steel specimens tested in liquid nitrogen and after hydrogen charging respectively were investigated by quantitative confocal laser scanning microscopy (CLSM) and conventional scanning electron microscopy (SEM) with electron-backscattered diffraction (EBSD). Topological and crystallographic features of the TC fracture surface are found in good agreement with the generally accepted cleavage mechanism: TC facets diameters correspond to those of grains; the crack path strictly follows the crystallographic orientation of grains and the most of the cleavage cracks are parallel to {100} planes. On the 2D SEM images, the QC facets appeared resembling the TC ones in terms of river line patterns, shapes and sizes. However, the substantial differences between the topography of these two kinds of fracture surfaces were revealed by 3D CLSM: the average misorientation angle between QC facets and the roughness of the QC fracture surface were much lower than those measured for TC. It is demonstrated that all these features are attributed to the specific fracture mechanism operating during hydrogen-assisted cracking.

Structural studies on metal-containing enzymes: T4 endonuclease VII and D. gigas formate dehydrogenase

NARCIS (Netherlands)

Raaijmakers, H.C.A.

2001-01-01

Many biological processes require metal ions, and many of these metal-ion functions involve metalloproteins. The metal ions in metalloproteins are often critical to the protein's function, structure, or stability. This thesis focuses on two of these proteins, bacteriophage T4 endonuclease

DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction*

Science.gov (United States)

Algasaier, Sana I.; Exell, Jack C.; Bennet, Ian A.; Thompson, Mark J.; Gotham, Victoria J. B.; Shaw, Steven J.; Craggs, Timothy D.; Finger, L. David; Grasby, Jane A.

2016-01-01

Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5′-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5′-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5′-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5′-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr40, Asp181, and Arg100 and a reacting duplex 5′-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5′-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage. PMID:26884332

Structural insights from boron tribromide ether cleavage into lignites and low maturity coals from the New Zealand Coal Band

DEFF Research Database (Denmark)

Glombitza, Clemens; Mangelsdorf, Kai; Horsfield, Brian

2011-01-01

structure, boron tribromide (BBr3) ether cleavage was applied to a series of lignite and coal samples of different maturity (R0 0.27–0.80%) obtained from coal mines and natural outcrops from the North and South Island of New Zealand. Terminal ether-bound alcohols rapidly decrease during diagenesis and occur...... over geological time scales. Polyether compounds were detected with chain length up to five carbon atoms. After a small decrease during the diagenetic phase these compounds occur in relatively high concentrations, even in the main catagenetic stage. This suggests that these linkage structures represent...

Functional analysis of coordinated cleavage in V(D)J recombination.

Science.gov (United States)

Kim, D R; Oettinger, M A

1998-08-01

V(D)J recombination in vivo requires a pair of signals with distinct spacer elements of 12 and 23 bp that separate conserved heptamer and nonamer motifs. Cleavage in vitro by the RAG1 and RAG2 proteins can occur at individual signals when the reaction buffer contains Mn2+, but cleavage is restricted to substrates containing two signals when Mg2+ is the divalent cation. By using a novel V(D)J cleavage substrate, we show that while the RAG proteins alone establish a moderate preference for a 12/23 pair versus a 12/12 pair, a much stricter dependence of cleavage on the 12/23 signal pair is produced by the inclusion of HMG1 and competitor double-stranded DNA. The competitor DNA serves to inhibit the cleavage of substrates carrying a 12/12 or 23/23 pair, as well as the cutting at individual signals in 12/23 substrates. We show that a 23/33 pair is more efficiently recombined than a 12/33 pair, suggesting that the 12/23 rule can be generalized to a requirement for spacers that differ from each other by a single helical turn. Furthermore, we suggest that a fixed spatial orientation of signals is required for cleavage. In general, the same signal variants that can be cleaved singly can function under conditions in which a signal pair is required. However, a chemically modified substrate with one noncleavable signal enables us to show that formation of a functional cleavage complex is mechanistically separable from the cleavage reaction itself and that although cleavage requires a pair of signals, cutting does not have to occur simultaneously at both. The implications of these results are discussed with respect to the mechanism of V(D)J recombination and the generation of chromosomal translocations.

Mlh1-Mlh3, a Meiotic Crossover and DNA Mismatch Repair Factor, Is a Msh2-Msh3-stimulated Endonuclease*

Science.gov (United States)

Rogacheva, Maria V.; Manhart, Carol M.; Chen, Cheng; Guarne, Alba; Surtees, Jennifer; Alani, Eric

2014-01-01

Crossing over between homologous chromosomes is initiated in meiotic prophase in most sexually reproducing organisms by the appearance of programmed double strand breaks throughout the genome. In Saccharomyces cerevisiae the double-strand breaks are resected to form three prime single-strand tails that primarily invade complementary sequences in unbroken homologs. These invasion intermediates are converted into double Holliday junctions and then resolved into crossovers that facilitate homolog segregation during Meiosis I. Work in yeast suggests that Msh4-Msh5 stabilizes invasion intermediates and double Holliday junctions, which are resolved into crossovers in steps requiring Sgs1 helicase, Exo1, and a putative endonuclease activity encoded by the DNA mismatch repair factor Mlh1-Mlh3. We purified Mlh1-Mlh3 and showed that it is a metal-dependent and Msh2-Msh3-stimulated endonuclease that makes single-strand breaks in supercoiled DNA. These observations support a direct role for an Mlh1-Mlh3 endonuclease activity in resolving recombination intermediates and in DNA mismatch repair. PMID:24403070

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

International Nuclear Information System (INIS)

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

1987-01-01

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, [ 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 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

Spectroelectrochemical insights into structural and redox properties of immobilized endonuclease III and its catalytically inactive mutant

Science.gov (United States)

Moe, Elin; Rollo, Filipe; Silveira, Célia M.; Sezer, Murat; Hildebrandt, Peter; Todorovic, Smilja

2018-01-01

Endonuclease III is a Fe-S containing bifunctional DNA glycosylase which is involved in the repair of oxidation damaged DNA. Here we employ surface enhanced IR spectroelectrochemistry and electrochemistry to study the enzyme from the highly radiation- and desiccation-resistant bacterium Deinococcus radiodurans (DrEndoIII2). The experiments are designed to shed more light onto specific parameters that are currently proposed to govern damage search and recognition by endonucleases III. We demonstrate that electrostatic interactions required for the redox activation of DrEndoIII2 may result in high electric fields that alter its structural and thermodynamic properties. Analysis of inactive DrEndoIII2 (K132A/D150A double mutant) interacting with undamaged DNA, and the active enzyme interacting with damaged DNA also indicate that the electron transfer is modulated by subtle differences in the protein-DNA complex.

Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

Science.gov (United States)

Smith, Catherine E; Mendillo, Marc L; Bowen, Nikki; Hombauer, Hans; Campbell, Christopher S; Desai, Arshad; Putnam, Christopher D; Kolodner, Richard D

2013-10-01

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.

Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

Directory of Open Access Journals (Sweden)

Catherine E Smith

2013-10-01

Full Text Available Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.

Hydrobiogeochemical interactions in 'anoxic' limestone drains for neutralization of acidic mine drainage

Science.gov (United States)

Robbins, E.I.; Cravotta, C.A.; Savela, C.E.; Nord, G.L.

1999-01-01

Processes affecting neutralization of acidic coal mine drainage were evaluated within 'anoxic' limestone drains (ALDs). Influents had pH???3.5 and dissolved oxygen Al3+ and Fe3+ compounds. Cleavage mounts of calcite and gypsum that were suspended within the ALDs and later examined microscopically showed dissolution features despite coatings by numerous bacteria, biofilms, and Fe-Al-Si precipitates. In the drain exhibiting the greatest flow reduction, Al-hydroxysulfates had accumulated on limestone surfaces and calcite etch points, thus causing the decline in transmissivity and dissolution. Therefore, where Al loadings are high and flow rates are low, a pre-treatment step is indicated to promote Al removal before diverting acidic mine water into alkalinity-producing materials. ?? 1998 Elsevier Science Ltd.

RPA activates the XPF-ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks.

Science.gov (United States)

Abdullah, Ummi B; McGouran, Joanna F; Brolih, Sanja; Ptchelkine, Denis; El-Sagheer, Afaf H; Brown, Tom; McHugh, Peter J

2017-07-14

During replication-coupled DNA interstrand crosslink (ICL) repair, the XPF-ERCC1 endonuclease is required for the incisions that release, or "unhook", ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL Here, we report that while purified XPF-ERCC1 incises simple ICL-containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single-stranded DNA (ssDNA)-binding replication protein A (RPA) selectively restores XPF-ERCC1 endonuclease activity on this structure. The 5'-3' exonuclease SNM1A can load from the XPF-ERCC1-RPA-induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF-ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo . © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Restriction endonuclease analysis of Pasteurella multocida isolates from three California turkey premises.

Science.gov (United States)

Christiansen, K H; Carpenter, T E; Snipes, K P; Hird, D W; Ghazikhanian, G Y

1992-01-01

Three California turkey premises that had repeated outbreaks of fowl cholera were studied for periods of 2 to 4 years. Using biochemical, serologic, plasmid DNA, and restriction endonuclease analyses of isolates of Pasteurella multocida from turkeys and wildlife on the premises, strains of the organism were found to be enzootic on two of the premises. On the third, a variety of strains of P. multocida were isolated from fowl cholera outbreak flocks.

A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments

KAUST Repository

Rodrigo, María J.

2013-09-04

Citrus is the first tree crop in terms of fruit production. The colour of Citrus fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C30 apocarotenoids, mainly ?-citraurin (3-hydroxy-?-apo-8?-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in Citrus fruits, little is known about the formation of C30 apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C30 apocarotenoids. In silico data mining revealed a new family of five CCD4-type genes in Citrus. One gene of this family, CCD4b1, was expressed in reproductive and vegetative tissues of different Citrus species in a pattern correlating with the accumulation of C30 apocarotenoids. Moreover, developmental processes and treatments which alter Citrus fruit peel pigmentation led to changes of ?-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in ?-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and ?-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7?,8? double bond in zeaxanthin and ?-cryptoxanthin, confrming its role in C30 apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7?,8? double bond of cyclic C40 carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C30 apocarotenoids in Citrus which are key pigments in fruit coloration. The Author 2013.

A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments

KAUST Repository

Rodrigo, Marí a J.; Alqué zar, Berta; Aló s, Enriqueta; Medina, Ví ctor; Carmona, Lourdes; Bruno, Mark; Al-Babili, Salim; Zacarí as, Lorenzo

2013-01-01

Citrus is the first tree crop in terms of fruit production. The colour of Citrus fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C30 apocarotenoids, mainly ?-citraurin (3-hydroxy-?-apo-8?-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in Citrus fruits, little is known about the formation of C30 apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C30 apocarotenoids. In silico data mining revealed a new family of five CCD4-type genes in Citrus. One gene of this family, CCD4b1, was expressed in reproductive and vegetative tissues of different Citrus species in a pattern correlating with the accumulation of C30 apocarotenoids. Moreover, developmental processes and treatments which alter Citrus fruit peel pigmentation led to changes of ?-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in ?-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and ?-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7?,8? double bond in zeaxanthin and ?-cryptoxanthin, confrming its role in C30 apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7?,8? double bond of cyclic C40 carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C30 apocarotenoids in Citrus which are key pigments in fruit coloration. The Author 2013.

Looking for exceptions on knowledge rules induced from HIV cleavage data set

Directory of Open Access Journals (Sweden)

Ronaldo Cristiano Prati

2004-01-01

Full Text Available The aim of data mining is to find useful knowledge inout of databases. In order to extract such knowledge, several methods can be used, among them machine learning (ML algorithms. In this work we focus on ML algorithms that express the extracted knowledge in a symbolic form, such as rules. This representation may allow us to ''explain'' the data. Rule learning algorithms are mainly designed to induce classification rules that can predict new cases with high accuracy. However, these sorts of rules generally express common sense knowledge, resulting in many interesting and useful rules not being discovered. Furthermore, the domain independent biases, especially those related to the language used to express the induced knowledge, could induce rules that are difficult to understand. Exceptions might be used in order to overcome these drawbacks. Exceptions are defined as rules that contradict common believebeliefs. This kind of rules can play an important role in the process of understanding the underlying data as well as in making critical decisions. By contradicting the user's common beliefves, exceptions are bound to be interesting. This work proposes a method to find exceptions. In order to illustrate the potential of our approach, we apply the method in a real world data set to discover rules and exceptions in the HIV virus protein cleavage process. A good understanding of the process that generates this data plays an important role oin the research of cleavage inhibitors. We consider believe that the proposed approach may help the domain expert to further understand this process.

Cleavage sites within the poliovirus capsid protein precursors

International Nuclear Information System (INIS)

Larsen, G.R.; Anderson, C.W.; Dorner, A.J.; Semler, B.L.; Wimmer, E.

1982-01-01

Partial amino-terminal sequence analysis was performed on radiolabeled poliovirus capsid proteins VP1, VP2, and VP3. A computer-assisted comparison of the amino acid sequences obtained with that predicted by the nucleotide sequence of the poliovirus genome allows assignment of the amino terminus of each capsid protein to a unique position within the virus polyprotein. Sequence analysis of trypsin-digested VP4, which has a blocked amino terminus, demonstrates that VP4 is encoded at or very near to the amino terminus of the polyprotein. The gene order of the capsid proteins is VP4-VP2-VP3-VP1. Cleavage of VP0 to VP4 and VP2 is shown to occur between asparagine and serine, whereas the cleavages that separate VP2/VP3 and VP3/VP1 occur between glutamine and glycine residues. This finding supports the hypothesis that the cleavage of VP0, which occurs during virion morphogenesis, is distinct from the cleavages that separate functional regions of the polyprotein

A new cultural cleavage in post-modern society

Directory of Open Access Journals (Sweden)

Jan-Erik Lane

2007-09-01

Full Text Available The attitudes towards gender and homosexuality tend to be linked at the micro level (individuals, which explains the political saliency of this newly emerging cleavage. At the macro level (country, the main finding is that the value orientations towards gender and homosexuality are strongly embedded in the basic cultural or civilisation differences among countries. As developing countries modernise and enter post-modernity, they will also experience the gender cleavage, especially when they adhere to an individualistic culture. Cultural cleavages in the post-modern society, whether in rich or developing countries, can only be properly researched by the survey method. It opens up a large area for both micro and macro analyses in the social sciences.

Unexpected tolerance of alpha-cleavage of the prion protein to sequence variations.

Directory of Open Access Journals (Sweden)

José B Oliveira-Martins

Full Text Available The cellular form of the prion protein, PrP(C, undergoes extensive proteolysis at the alpha site (109K [see text]H110. Expression of non-cleavable PrP(C mutants in transgenic mice correlates with neurotoxicity, suggesting that alpha-cleavage is important for PrP(C physiology. To gain insights into the mechanisms of alpha-cleavage, we generated a library of PrP(C mutants with mutations in the region neighbouring the alpha-cleavage site. The prevalence of C1, the carboxy adduct of alpha-cleavage, was determined for each mutant. In cell lines of disparate origin, C1 prevalence was unaffected by variations in charge and hydrophobicity of the region neighbouring the alpha-cleavage site, and by substitutions of the residues in the palindrome that flanks this site. Instead, alpha-cleavage was size-dependently impaired by deletions within the domain 106-119. Almost no cleavage was observed upon full deletion of this domain. These results suggest that alpha-cleavage is executed by an alpha-PrPase whose activity, despite surprisingly limited sequence specificity, is dependent on the size of the central region of PrP(C.

Comparative and phylogenetic perspectives of the cleavage process in tailed amphibians.

Science.gov (United States)

Desnitskiy, Alexey G; Litvinchuk, Spartak N

2015-10-01

The order Caudata includes about 660 species and displays a variety of important developmental traits such as cleavage pattern and egg size. However, the cleavage process of tailed amphibians has never been analyzed within a phylogenetic framework. We use published data on the embryos of 36 species concerning the character of the third cleavage furrow (latitudinal, longitudinal or variable) and the magnitude of synchronous cleavage period (up to 3-4 synchronous cell divisions in the animal hemisphere or a considerably longer series of synchronous divisions followed by midblastula transition). Several species from basal caudate families Cryptobranchidae (Andrias davidianus and Cryptobranchus alleganiensis) and Hynobiidae (Onychodactylus japonicus) as well as several representatives from derived families Plethodontidae (Desmognathus fuscus and Ensatina eschscholtzii) and Proteidae (Necturus maculosus) are characterized by longitudinal furrows of the third cleavage and the loss of synchrony as early as the 8-cell stage. By contrast, many representatives of derived families Ambystomatidae and Salamandridae have latitudinal furrows of the third cleavage and extensive period of synchronous divisions. Our analysis of these ontogenetic characters mapped onto a phylogenetic tree shows that the cleavage pattern of large, yolky eggs with short series of synchronous divisions is an ancestral trait for the tailed amphibians, while the data on the orientation of third cleavage furrows seem to be ambiguous with respect to phylogeny. Nevertheless, the midblastula transition, which is characteristic of the model species Ambystoma mexicanum (Caudata) and Xenopus laevis (Anura), might have evolved convergently in these two amphibian orders.

Ultraviolet-endonuclease activity in cell extracts of Saccharomyces cerevisiae mutants defective in excision of pyrimidine dimers

International Nuclear Information System (INIS)

Bekker, M.L.; Kaboev, O.K.; Akhmedov, A.T.; Luchkina, L.A.

1980-01-01

Cell-free extracts of ultraviolet-sensitive mutants of Saccharomyces cerevisiae defective in excision of pyrimidine dimers, rad1, rad2, rad3, rad4, rad10, and rad16, as well as the extracts of the wild-type strain RAD+, display ultraviolet-endonuclease activity

The structure-specific endonuclease Ercc1-Xpf is required for targeted gene replacement in embryonic stem cells

NARCIS (Netherlands)

L.J. Niedernhofer (Laura); J. Essers (Jeroen); G. Weeda (Geert); H.B. Beverloo (Berna); J. de Wit (Jan); M. Muijtjens (Manja); H. Odijk (Hanny); J.H.J. Hoeijmakers (Jan); R. Kanaar (Roland)

2001-01-01

textabstractThe Ercc1-Xpf heterodimer, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair and homologous recombination. Erccl-Xpf incises

Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. [Haemophilus influenzae, Escherichia coli, Paramecium aurelia

Energy Technology Data Exchange (ETDEWEB)

Lacks, S.; Greenberg, B.

1977-01-01

Restriction endonucleases Dpn I and Dpn II are produced by two distinct strains of Diplococcus pneumoniae. The two enzymes show complementary specificity with respect to methylation of sites in DNA. From the identity of its cleavage site with that of Mbo I, it appears that Dpn II cleaves at the unmodified sequence 5'-G-A-T-C-3'. Dpn I cleaves at the same sequence when the adenine residue is methylated. Both enzymes produce only double-strand breaks in susceptible DNA. Their susceptibility to Dpn I and not Dpn II shows that essentially all the G-A-T-C sequences are methylated in DNA from the pneumococcal strain that produces Dpn II as well as in DNA from Hemophilus influenzae and Escherichia coli. In the dam-3 mutant of E. coli none of these sequences appear to be methylated. Residual adenine methylation in the dam-3 mutant DNA most likely occurs at different sites. Different but characteristic degrees of methylation at G-A-T-C sites are found in the DNA of bacterial viruses grown in E. coli. DNAs from mammalian cells and viruses are not methylated at this sequence. Mitochondrial DNA from Paramecium aurelia is not methylated, but a small proportion of G-A-T-C sequences in the macronuclear DNA of this eukaryote appear to be methylated. Possible roles of sequence-specific methylation in the accommodation of plasmids, in the replication of DNA, in the regulation of gene function and in the restriction of viral infection are discussed.

Kininogen Cleavage Assay: Diagnostic Assistance for Kinin-Mediated Angioedema Conditions.

Directory of Open Access Journals (Sweden)

Rémi Baroso

Full Text Available Angioedema without wheals (AE is a symptom characterised by localised episodes of oedema presumably caused by kinin release from kininogen cleavage. It can result from a hereditary deficiency in C1 Inhibitor (C1Inh, but it can present with normal level of C1Inh. These forms are typically difficult to diagnose although enhanced kinin production is suspected or demonstrated in some cases.We wanted to investigate bradykinin overproduction in all AE condition with normal C1Inh, excluding cases with enhanced kinin catabolism, and to propose this parameter as a disease biomarker.We retrospectively investigated high molecular weight kininogen (HK cleavage pattern, using gel electrophoresis and immunorevelation. Plasma samples were drawn using the same standardised procedure from blood donors or AE patients with normal C1Inh conditions, normal kinin catabolism, and without prophylaxis.Circulating native HK plasma concentrations were similar in the healthy men (interquartile range: 98-175μg/mL, n = 51 and in healthy women (90-176μg/mL, n = 74, while HK cleavage was lower (p14.4% HK cleavage for men; 33.0% HK cleavage for women, with >98% specificity achieved for all parameters. In plasma from patients undergoing recovery two months after oestrogen/progestin combination withdrawal (n = 13 or two weeks after AE attack (n = 2, HK cleavage was not fully restored, suggesting its use as a post-attack assay.As a diagnostic tool, HK cleavage can offer physicians supportive arguments for kinin production in suspected AE cases and improve patient follow-up in clinical trials or prophylactic management.

Detection of nucleic acid sequences by invader-directed cleavage

Science.gov (United States)

Brow, Mary Ann D.; Hall, Jeff Steven Grotelueschen; Lyamichev, Victor; Olive, David Michael; Prudent, James Robert

1999-01-01

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The 5' nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based by charge.

Template-directed addition of nucleosides to DNA by the BfiI restriction enzyme

OpenAIRE

Sasnauskas, Giedrius; Connolly, Bernard A.; Halford, Stephen E.; Siksnys, Virginijus

2008-01-01

Restriction endonucleases catalyse DNA cleavage at specific sites. The BfiI endonuclease cuts DNA to give staggered ends with 1-nt 3′-extensions. We show here that BfiI can also fill in the staggered ends: while cleaving DNA, it can add a 2′-deoxynucleoside to the reaction product to yield directly a blunt-ended DNA. We propose that nucleoside incorporation proceeds through a two-step reaction, in which BfiI first cleaves the DNA to make a covalent enzyme–DNA intermediate and then resolves it...

Controllable laser thermal cleavage of sapphire wafers

Science.gov (United States)

Xu, Jiayu; Hu, Hong; Zhuang, Changhui; Ma, Guodong; Han, Junlong; Lei, Yulin

2018-03-01

Laser processing of substrates for light-emitting diodes (LEDs) offers advantages over other processing techniques and is therefore an active research area in both industrial and academic sectors. The processing of sapphire wafers is problematic because sapphire is a hard and brittle material. Semiconductor laser scribing processing suffers certain disadvantages that have yet to be overcome, thereby necessitating further investigation. In this work, a platform for controllable laser thermal cleavage was constructed. A sapphire LED wafer was modeled using the finite element method to simulate the thermal and stress distributions under different conditions. A guide groove cut by laser ablation before the cleavage process was observed to guide the crack extension and avoid deviation. The surface and cross section of sapphire wafers processed using controllable laser thermal cleavage were characterized by scanning electron microscopy and optical microscopy, and their morphology was compared to that of wafers processed using stealth dicing. The differences in luminous efficiency between substrates prepared using these two processing methods are explained.

Geographically diverse Australian isolates of Melissococcus pluton exhibit minimal genotypic diversity by restriction endonuclease analysis.

Science.gov (United States)

Djordjevic, S P; Smith, L A; Forbes, W A; Hornitzky, M A

1999-04-15

Melissococcus pluton, the causative agent of European foulbrood is an economically significant disease of honey bees (Apis mellifera) across most regions of the world and is prevalent throughout most states of Australia. 49 Isolates of M. pluton recovered from diseased colonies or honey samples in New South Wales, Queensland, South Australia, Tasmania and Victoria were compared using SDS-PAGE, Western immunoblotting and restriction endonuclease analyses. DNA profiles of all 49 geographically diverse isolates showed remarkably similar AluI profiles although four isolates (one each from Queensland, South Australia, New South Wales and Victoria) displayed minor profile variations compared to AluI patterns of all other isolates. DNA from a subset of the 49 Australian and three isolates from the United Kingdom were digested separately with the restriction endonucleases CfoI, RsaI and DraI. Restriction endonuclease fragment patterns generated using these enzymes were also similar although minor variations were noted. SDS-PAGE of whole cell proteins from 13 of the 49 isolates from different states of Australia, including the four isolates which displayed minor profile variations (AluI) produced indistinguishable patterns. Major immunoreactive proteins of approximate molecular masses of 21, 24, 28, 30, 36, 40, 44, 56, 60, 71, 79 and 95 kDa were observed in immunoblots of whole cell lysates of 22 of the 49 isolates and reacted with rabbit hyperimmune antibodies raised against M. pluton whole cells. Neither SDS-PAGE or immunoblotting was capable of distinguishing differences between geographically diverse isolates of M. pluton. Collectively these data confirm that Australian isolates of M. pluton are genetically homogeneous and that this species may be clonal. Plasmid DNA was not detected in whole cell DNA profiles of any isolate resolved using agarose gel electrophoresis.

Activation of Saccharomyces cerevisiae Mlh1-Pms1 Endonuclease in a Reconstituted Mismatch Repair System*

Science.gov (United States)

Smith, Catherine E.; Bowen, Nikki; Graham, William J.; Goellner, Eva M.; Srivatsan, Anjana; Kolodner, Richard D.

2015-01-01

Previous studies reported the reconstitution of an Mlh1-Pms1-independent 5′ nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-Pms1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3′ nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase δ. Both reactions required Mg2+ and Mn2+ for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-Pms1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-Pms1 endonuclease active site, as well as mispair recognition and Mlh1-Pms1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-Pms1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-Pms1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR. PMID:26170454

Activation of Saccharomyces cerevisiae Mlh1-Pms1 Endonuclease in a Reconstituted Mismatch Repair System.

Science.gov (United States)

Smith, Catherine E; Bowen, Nikki; Graham, William J; Goellner, Eva M; Srivatsan, Anjana; Kolodner, Richard D

2015-08-28

Previous studies reported the reconstitution of an Mlh1-Pms1-independent 5' nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-Pms1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3' nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase δ. Both reactions required Mg(2+) and Mn(2+) for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-Pms1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-Pms1 endonuclease active site, as well as mispair recognition and Mlh1-Pms1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-Pms1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-Pms1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Biochemical characterization of recombinant influenza A polymerase heterotrimer complex: Endonuclease activity and evaluation of inhibitors

Czech Academy of Sciences Publication Activity Database

Xing, W.; Barauskas, O.; Kirschberg, T.; Niedziela-Majka, A.; Clarke, M.; Birkuš, Gabriel; Weissburg, P.; Liu, X.; Schultz, B. E.; Sakowicz, R.; Kwon, H. J.; Feng, J. Y.

2017-01-01

Roč. 12, č. 8 (2017), č. článku e0181969. E-ISSN 1932-6203 Institutional support: RVO:61388963 Keywords : virus PA endonuclease * respiratory syncytial virus * RNA synthesis Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 2.806, year: 2016 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0181969

An ultrasensitive colorimeter assay strategy for p53 mutation assisted by nicking endonuclease signal amplification.

Science.gov (United States)

Lin, Zhenyu; Yang, Weiqiang; Zhang, Guiyun; Liu, Qida; Qiu, Bin; Cai, Zongwei; Chen, Guonan

2011-08-28

A novel catalytic colorimetric assay assisted by nicking endonuclease signal amplification (NESA) was developed. With the signal amplification, the detection limit of the p53 target gene can be as low as 1 pM, which is nearly 5 orders of magnitude lower than that of other previously reported colorimetric DNA detection strategies based on catalytic DNAzyme.

CRISPR-Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape

NARCIS (Netherlands)

Wang, Gang; Zhao, Na; Berkhout, Ben; Das, Atze T.

2016-01-01

Several recent studies demonstrated that the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 can be used for guide RNA (gRNA)-directed, sequence-specific cleavage of HIV proviral DNA in infected cells. We here demonstrate profound inhibition of HIV-1

Full Length Research Paper Curcumin induces cleavage of -catenin ...

African Journals Online (AJOL)

β-Catenin/Tcf-4 signaling pathway plays important roles in colorectal tumorigenesis. RT-PCR, western blotting and immunoprecipitation were used to study the effects of curcumin on β-catenin/Tcf-4 signaling pathway in HT-29 cells. Treatment of curcumin could induce cleavage of β-catenin and the cleavage could be ...

Altered cleavage patterns in human tripronuclear embryos and their association to fertilization method

DEFF Research Database (Denmark)

Joergensen, Mette Warming; Agerholm, Inge; Hindkjaer, Johnny

2014-01-01

PURPOSE: To analyze the cleavage patterns in dipronuclear (2PN) and tripronuclear (3PN) embryos in relation to fertilization method. METHOD: Time-lapse analysis. RESULTS: Compared to 2PN, more 3PN IVF embryos displayed early cleavage into 3 cells (p ... stage (p embryos, the 2nd and 3rd cleavage cycles were completed within the expected time frame. However, timing of the cell divisions within the cleavage cycles differed between the two groups. In contrast......, the completion of the 1st, 2nd, and 3rd cleavage cycle was delayed, but with a similar division pattern for 3PN ICSI compared with the 2PN ICSI embryos. 3PN, more often than 2PN ICSI embryos, displayed early cleavage into 3 cells (p = 0.03) and arrested development from the compaction stage and onwards (p = 0...

RecA-mediated cleavage reaction of Lambda repressor and DNA ...

African Journals Online (AJOL)

PRECIOUS

2010-01-11

Jan 11, 2010 ... hydrolyze ATP at all, but fulfills RecA functions such as cleavage of Lambda repressor and strand .... DNA binding properties of RecA and may result in an in- .... AMP-PNP there is no cleavage of Lambda repressor (Figure.

Creation of targeted inversion mutations in plants using an RNA-guided endonuclease

Institute of Scientific and Technical Information of China (English)

Congsheng Zhang; Changlin Liu; Jianfeng Weng; Beijiu Cheng; Fang Liu; Xinhai Li; Chuanxiao Xie

2017-01-01

Inversions are DNA rearrangements that are essential for plant gene evolution and adaptation to environmental changes. We demonstrate the creation of targeted inversions and previously reported targeted deletion mutations via delivery of a pair of RNA-guided endonucleases (RGENs) of CRISPR/Cas9. The efficiencies of the targeted inversions were 2.6%and 2.2%in the Arabidopsis FLOWERING TIME (AtFT) and TERMINAL FLOWER 1 (AtTFL1) loci, respectively. Thus, we successfully established an approach that can potentially be used to introduce targeted DNA inversions of interest for functional studies and crop improvement.

Alteration of Sequence Specificity of the Type IIS Restriction Endonuclease BtsI

OpenAIRE

Guan, Shengxi; Blanchard, Aine; Zhang, Penghua; Zhu, Zhenyu

2010-01-01

The Type IIS restriction endonuclease BtsI recognizes and digests at GCAGTG(2/0). It comprises two subunits: BtsIA and BtsIB. The BtsIB subunit contains the recognition domain, one catalytic domain for bottom strand nicking and part of the catalytic domain for the top strand nicking. BtsIA has the rest of the catalytic domain that is responsible for the DNA top strand nicking. BtsIA alone has no activity unless it mixes with BtsIB to reconstitute the BtsI activity. During characterization of ...

Acquisition of a novel eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site confers intracellular cleavage of an H7N7 influenza virus hemagglutinin

International Nuclear Information System (INIS)

Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik; Whittaker, Gary R.

2012-01-01

A critical feature of highly pathogenic avian influenza viruses (H5N1 and H7N7) is the efficient intracellular cleavage of the hemagglutinin (HA) protein. H7N7 viruses also exist in equine species, and a unique feature of the equine H7N7 HA is the presence of an eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site. Here, we show that three histidine residues within the unique insertion of the equine H7N7 HA are essential for intracellular cleavage. An asparagine residue within the insertion-derived glycosylation site was also found to be essential for intracellular cleavage. The presence of the histidine residues also appear to be involved in triggering fusion, since mutation of the histidine residues resulted in a destabilizing effect. Importantly, the addition of a tetrabasic site and the eleven amino acid insertion conferred efficient intracellular cleavage to the HA of an H7N3 low pathogenicity avian influenza virus. Our studies show that acquisition of the eleven amino acid insertion offers an alternative mechanism for intracellular cleavage of influenza HA.

Acquisition of a novel eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site confers intracellular cleavage of an H7N7 influenza virus hemagglutinin

Energy Technology Data Exchange (ETDEWEB)

Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik [Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca NY 14853 (United States); New York Center of Excellence for Influenza Research and Surveillance, University of Rochester Medical Center, Rochester NY 14627 (United States); Whittaker, Gary R., E-mail: grw7@cornell.edu [Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca NY 14853 (United States); New York Center of Excellence for Influenza Research and Surveillance, University of Rochester Medical Center, Rochester NY 14627 (United States)

2012-12-05

A critical feature of highly pathogenic avian influenza viruses (H5N1 and H7N7) is the efficient intracellular cleavage of the hemagglutinin (HA) protein. H7N7 viruses also exist in equine species, and a unique feature of the equine H7N7 HA is the presence of an eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site. Here, we show that three histidine residues within the unique insertion of the equine H7N7 HA are essential for intracellular cleavage. An asparagine residue within the insertion-derived glycosylation site was also found to be essential for intracellular cleavage. The presence of the histidine residues also appear to be involved in triggering fusion, since mutation of the histidine residues resulted in a destabilizing effect. Importantly, the addition of a tetrabasic site and the eleven amino acid insertion conferred efficient intracellular cleavage to the HA of an H7N3 low pathogenicity avian influenza virus. Our studies show that acquisition of the eleven amino acid insertion offers an alternative mechanism for intracellular cleavage of influenza HA.

Short RNA guides cleavage by eukaryotic RNase III.

Directory of Open Access Journals (Sweden)

Bruno Lamontagne

Full Text Available In eukaryotes, short RNAs guide a variety of enzymatic activities that range from RNA editing to translation repression. It is hypothesized that pre-existing proteins evolved to bind and use guide RNA during evolution. However, the capacity of modern proteins to adopt new RNA guides has never been demonstrated. Here we show that Rnt1p, the yeast orthologue of the bacterial dsRNA-specific RNase III, can bind short RNA transcripts and use them as guides for sequence-specific cleavage. Target cleavage occurred at a constant distance from the Rnt1p binding site, leaving the guide RNA intact for subsequent cleavage. Our results indicate that RNase III may trigger sequence-specific RNA degradation independent of the RNAi machinery, and they open the road for a new generation of precise RNA silencing tools that do not trigger a dsRNA-mediated immune response.

A RecB-family nuclease motif in the Type I restriction endonuclease EcoR124I

Czech Academy of Sciences Publication Activity Database

Šišáková, Eva; Stanley, L. K.; Weiserová, Marie; Szczelkun, M. D.

2008-01-01

Roč. 36, č. 12 (2008), s. 1-11 ISSN 0305-1048 R&D Projects: GA ČR GA204/07/0325 Grant - others:XE(XE) BioNano-Switch 043288 Institutional research plan: CEZ:AV0Z50200510 Keywords : restriction endonuclease * mutagenesis * dsdna Subject RIV: EE - Microbiology, Virology Impact factor: 6.878, year: 2008

Atypical myxomatosis--virus isolation, experimental infection of rabbits and restriction endonuclease analysis of the isolate.

Science.gov (United States)

Psikal, I; Smíd, B; Rodák, L; Valícek, L; Bendová, J

2003-08-01

Atypical form of myxomatosis, which caused non-lethal and clinically mild disease in domestic rabbits 1 month after immunization with a commercially available vaccine MXT, is described. The isolated myxoma virus designated as Litovel 2 (Li-2) did not induce systemic disease following subcutaneous and intradermal applications in susceptible experimental rabbits but led to the immune response demonstrated by ELISA. No severe disease was induced in those Li-2 inoculated rabbits by challenge with the virulent strains Lausanne (Lu) or Sanar (SA), while the control animals showed nodular form of myxomatosis with lethal course of the illness. Restriction fragment length polymorphism (RFLP) of genomic DNA with KpnI and BamHI endonucleases was used for genetic characterization of the Li-2 isolate, the vaccine strain MXT and both virulent strains Lu and SA, respectively. In general, RFLP analysis has shown to be informative for inferring genetic relatedness between myxoma viruses. Based on restriction endonuclease DNA fragment size distribution, it was evident that the pathogenic strain SA is genetically related to the reference strain Lu and the isolate Li-2 is more related, but not identical, to the vaccination strain MXT.

Single substitution in bacteriophage T4 RNase H alters the ratio between its exo- and endonuclease activities.

Science.gov (United States)

Kholod, Natalia; Sivogrivov, Dmitry; Latypov, Oleg; Mayorov, Sergey; Kuznitsyn, Rafail; Kajava, Andrey V; Shlyapnikov, Mikhail; Granovsky, Igor

2015-11-01

The article describes substitutions in bacteriophage T4 RNase H which provide so called das-effect. Phage T4 DNA arrest suppression (das) mutations have been described to be capable of partially suppressing the phage DNA arrest phenotype caused by a dysfunction in genes 46 and/or 47 (also known as Mre11/Rad50 complex). Genetic mapping of das13 (one of the das mutations) has shown it to be in the region of the rnh gene encoding RNase H. Here we report that Das13 mutant of RNase H has substitutions of valine 43 and leucine 242 with isoleucines. To investigate the influence of these mutations on RNase H nuclease properties we have designed a novel in vitro assay that allows us to separate and quantify exo- or endonuclease activities of flap endonuclease. The nuclease assay in vitro showed that V43I substitution increased the ratio between exonuclease/endonuclease activities of RNase H whereas L242I substitution did not affect the nuclease activity of RNase H in vitro. However, both mutations were necessary for the full das effect in vivo. Molecular modelling of the nuclease structure suggests that V43I substitution may lead to disposition of H4 helix, responsible for the interaction with the first base pairs of 5'end of branched DNA. These structural changes may affect unwinding of the first base pairs of gapped or nicked DNA generating a short flap and therefore may stabilize the DNA-enzyme complex. L242I substitution did not affect the structure of RNase H and its role in providing das-effect remains unclear. Copyright © 2015 Elsevier B.V. All rights reserved.

An AP endonuclease 1-DNA polymerase beta complex: theoretical prediction of interacting surfaces.

Directory of Open Access Journals (Sweden)

Alexej Abyzov

2008-04-01

Full Text Available Abasic (AP sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1 cleaves the phosphodiester backbone 5' to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-beta. While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-beta, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-beta based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-beta located downstream of APEX1 (3' to the damaged site and three with pol-beta located upstream of APEX1 (5' to the damaged site. Molecular dynamics (MD simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (approximately -10.0 kcal/mol to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-beta at the 3'-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-beta in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-beta in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3' side. The method described here can be used for analysis in

The oxidation of acid azo dye AY 36 by a manganese oxide containing mine waste

International Nuclear Information System (INIS)

Clarke, Catherine E.; Kielar, Filip; Johnson, Karen L.

2013-01-01

Highlights: ► This study looks at the oxidative breakdown of the amine containing dye acid yellow 36 by a Mn oxide containing mine waste. ► The oxidation proceeds by successive one electron transfers between the dye molecule and the Mn oxide minerals. ► The initial decolorization of the dye is rapid, but does not involve the cleavage of the azo bond. -- Abstract: The oxidative breakdown of acid azo dye acid yellow 36 (AY 36) by a Mn oxide containing mine tailings is demonstrated. The oxidation reaction is pH dependent with the rate of decolorization increasing with decreasing pH. The oxidation reaction mechanism is initiated at the amino moiety and proceeds via successive, one electron transfers from the dye to the Mn oxide minerals. The reaction pathway involves the formation of a number of colorless intermediate products, some of which hydrolyze in a Mn oxide-independent step. Decolorization of the dye is rapid and is observed before the cleavage of the azo-bond, which is a slower process. The terminal oxidation products were observed to be p-benzoquinone and 3-hydroxybenzenesulfonate. The reaction order of the initial decolorization was determined to be pseudo fractional order with respect to pH and pseudo first order with respect to dye concentration and Mn tailings’ surface area

Selective metal binding to Cys-78 within endonuclease V causes an inhibition of catalytic activities without altering nontarget and target DNA binding

International Nuclear Information System (INIS)

Prince, M.A.; Friedman, B.; Gruskin, E.A.; Schrock, R.D. III; Lloyd, R.S.

1991-01-01

T4 endonuclease V is a pyrimidine dimer-specific DNA repair enzyme which has been previously shown not to require metal ions for either of its two catalytic activities or its DNA binding function. However, we have investigated whether the single cysteine within the enzyme was able to bind metal salts and influence the various activities of this repair enzyme. A series of metals (Hg2+, Ag+, Cu+) were shown to inactivate both endonuclease Vs pyrimidine dimer-specific DNA glycosylase activity and the subsequent apurinic nicking activity. The binding of metal to endonuclease V did not interfere with nontarget DNA scanning or pyrimidine dimer-specific binding. The Cys-78 codon within the endonuclease V gene was changed by oligonucleotide site-directed mutagenesis to Thr-78 and Ser-78 in order to determine whether the native cysteine was directly involved in the enzyme's DNA catalytic activities and whether the cysteine was primarily responsible for the metal binding. The mutant enzymes were able to confer enhanced ultraviolet light (UV) resistance to DNA repair-deficient Escherichia coli at levels equal to that conferred by the wild type enzyme. The C78T mutant enzyme was purified to homogeneity and shown to be catalytically active on pyrimidine dimer-containing DNA. The catalytic activities of the C78T mutant enzyme were demonstrated to be unaffected by the addition of Hg2+ or Ag+ at concentrations 1000-fold greater than that required to inhibit the wild type enzyme. These data suggest that the cysteine is not required for enzyme activity but that the binding of certain metals to that amino acid block DNA incision by either preventing a conformational change in the enzyme after it has bound to a pyrimidine dimer or sterically interfering with the active site residue's accessibility to the pyrimidine dimer

Apurinic/apyrimidinic endonucleases of Mycobacterium tuberculosis protect against DNA damage but are dispensable for the growth of the pathogen in guinea pigs.

Science.gov (United States)

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.

Apurinic/apyrimidinic endonucleases of Mycobacterium tuberculosis protect against DNA damage but are dispensable for the growth of the pathogen in guinea pigs.

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Rupangi Verma Puri

Full Text Available 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.

A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease

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The Cas9 endonuclease of the Type II-a clustered regularly interspersed short palindromic repeats (CRISPR), of Streptococcus pyogenes (SpCas9) has been adapted as a widely used tool for genome editing and genome engineering. Herein, we describe a gene encoding a novel Cas9 ortholog (BpsuCas9) and th...

Effect of microstructure on the cleavage fracture strength of low carbon Mn-Ni-Mo bainitic steels

International Nuclear Information System (INIS)

Im, Young-Roc; Lee, Byeong-Joo; Oh, Yong Jun; Hong, Jun Hwa; Lee, Hu-Chul

2004-01-01

The effects of the microstructure on the cleavage fracture strength of low carbon Mn-Ni-Mo bainitic steels were examined. A four-point bend test and double-notched bend specimens were used to measure the cleavage fracture strength of the alloys and identify the cleavage initiating micro-cracks, respectively. The cleavage fracture strength and DBTT of Mn-Ni-Mo bainitic steels were strongly affected by the alloy carbon content. The decrease in the alloy carbon content resulted in a decrease in the inter-lath cementite-crowded layers and higher cleavage fracture strength. Micro-cracks that formed across the inter-lath cementite-crowded layers were observed to initiate cleavage fracture. The width of these inter-lath cementite-crowded layers was accepted as a cleavage initiating micro-crack size in the micro-mechanical modeling of the cleavage fracture, and the measured cleavage strength values of the bainitic Mn-Ni-Mo steels were well represented by the modified Griffith relationship

Characterization of SNARE Cleavage Products Generated by Formulated Botulinum Neurotoxin Type-A Drug Products

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Jack Xie

2010-08-01

Full Text Available The study evaluated substrate cleavage product(s generated by three botulinum neurotoxin serotype A (BoNT/A medicinal drug products utilizing a novel and highly specific, light-chain activity, high-performance liquid chromatography (LCA-HPLC method. Samples were reacted with a commercially available BoNT/A fluorescent substrate derived from the SNAP-25 sequence. Reaction products were separated by reversed-phase HPLC. The method detected an atypical cleavage pattern by one of the formulated drug products. IncobotulinumtoxinA produced two cleavage fragments rather than the single fragment typically generated by BoNT/A. Identification confirmed the secondary cleavage at a position corresponding to SNAP-25 Arg198–Ala199 (normal BoNT/A cleavage is Gln197–Arg198. Arg198–Ala199 is also the cleavage site for trypsin and serotype C toxin. Normal cleavage was observed for all other BoNT/A drug product samples, as well as 900-kD and 150-kD bulk toxin BoNT/A. The reason for this unexpected secondary cleavage pattern by one formulated BoNT/A drug product is unknown. Possible explanations include a contaminating protease and/or damage to the 150-kD type-A toxin causing nonspecific substrate recognition and subsequent cleavage uncharacteristic of type-A toxin. The BoNT/A drug products were also analyzed via the LCA-HPLC assay using a commercial BoNT/C fluorescent substrate derived from the syntaxin sequence. Cleavage of the serotype C substrate by incobotulinumtoxinA was also confirmed whilst neither of the other drug products cleaved the syntaxin substrate.

Post-transcription cleavage generates the 3' end of F17R transcripts in vaccinia virus

International Nuclear Information System (INIS)

D'Costa, Susan M.; Antczak, James B.; Pickup, David J.; Condit, Richard C.

2004-01-01

Most vaccinia virus intermediate and late mRNAs possess 3' ends that are extremely heterogeneous in sequence. However, late mRNAs encoding the cowpox A-type inclusion protein (ATI), the second largest subunit of the RNA polymerase, and the late telomeric transcripts possess homogeneous 3' ends. In the case of the ATI mRNA, it has been shown that the homogeneous 3' end is generated by a post-transcriptional endoribonucleolytic cleavage event. We have determined that the F17R gene also produces homogeneous transcripts generated by a post-transcriptional cleavage event. Mapping of in vivo mRNA shows that the major 3' end of the F17R transcript maps 1262 nt downstream of the F17R translational start site. In vitro transcripts spanning the in vivo 3' end are cleaved in an in vitro reaction using extracts from virus infected cells, and the site of cleavage is the same both in vivo and in vitro. Cleavage is not observed using extract from cells infected in the presence of hydroxyurea; therefore, the cleavage factor is either virus-coded or virus-induced during the post-replicative phase of virus replication. The cis-acting sequence responsible for cleavage is orientation specific and the factor responsible for cleavage activity has biochemical properties similar to the factor required for cleavage of ATI transcripts. Partially purified cleavage factor generates cleavage products of expected size when either the ATI or F17R substrates are used in vitro, strongly suggesting that cleavage of both transcripts is mediated by the same factor

Analysis of Endonuclease R·EcoRI Fragments of DNA from Lambdoid Bacteriophages and Other Viruses by Agarose-Gel Electrophoresis

Science.gov (United States)

Helling, Robert B.; Goodman, Howard M.; Boyer, Herbert W.

1974-01-01

By means of agarose-gel electrophoresis, endonuclease R·EcoRI-generated fragments of DNA from various viruses were separated, their molecular weights were determined, and complete or partial fragment maps for lambda, φ80, and hybrid phages were constructed. Images PMID:4372397

Coronavirus 3CLpro proteinase cleavage sites: Possible relevance to SARS virus pathology

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Blom Nikolaj

2004-06-01

Full Text Available Abstract Background Despite the passing of more than a year since the first outbreak of Severe Acute Respiratory Syndrome (SARS, efficient counter-measures are still few and many believe that reappearance of SARS, or a similar disease caused by a coronavirus, is not unlikely. For other virus families like the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection. Prompted by this, we set out to analyse and predict cleavage by the coronavirus main proteinase using computational methods. Results We retrieved sequence data on seven fully sequenced coronaviruses and identified the main 3CL proteinase cleavage sites in polyproteins using alignments. A neural network was trained to recognise the cleavage sites in the genomes obtaining a sensitivity of 87.0% and a specificity of 99.0%. Several proteins known to be cleaved by other viruses were submitted to prediction as well as proteins suspected relevant in coronavirus pathology. Cleavage sites were predicted in proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR, transcription factors CREB-RP and OCT-1, and components of the ubiquitin pathway. Conclusions Our prediction method NetCorona predicts coronavirus cleavage sites with high specificity and several potential cleavage candidates were identified which might be important to elucidate coronavirus pathology. Furthermore, the method might assist in design of proteinase inhibitors for treatment of SARS and possible future diseases caused by coronaviruses. It is made available for public use at our website: http://www.cbs.dtu.dk/services/NetCorona/.

Prediction of proteasome cleavage motifs by neural networks

DEFF Research Database (Denmark)

Kesimir, C.; Nussbaum, A.K.; Schild, H.

2002-01-01

physiological conditions. Our algorithm has been trained not only on in vitro data, but also on MHC Class I ligand data, which reflect a combination of immunoproteasome and constitutive proteasome specificity. This feature, together with the use of neural networks, a non-linear classification technique, make...... the prediction of MHC Class I ligand boundaries more accurate: 65% of the cleavage sites and 85% of the non-cleavage sites are correctly determined. Moreover, we show that the neural networks trained on the constitutive proteasome data learns a specificity that differs from that of the networks trained on MHC...

Remarkable weakness against cleavage stress for YBCO-coated conductors and its effect on the YBCO coil performance

International Nuclear Information System (INIS)

Yanagisawa, Y.; Nakagome, H.; Takematsu, T.; Takao, T.; Sato, N.; Takahashi, M.; Maeda, H.

2011-01-01

Cleavage strength for YBCO-coated conductor is extremely low, typically 0.5 MPa. The remarkable weakness is due to cracks on the slit edge of the conductor. The cleavage stress appears on YBCO double pancake coils impregnated with epoxy. The cleavage stress should be avoided in the coil winding. Cleavage strength for an YBCO-coated conductor at 77 K was investigated with a model experiment. The nominal cleavage strength for an YBCO-coated conductor is extremely low, typically 0.5 MPa. This low nominal cleavage strength is due to stress concentration on a small part of the YBCO-coated conductor in cleavage fracture. Debonding by the cleavage stress occurs at the interface between the buffer layer and the Hastelloy substrate. The nominal cleavage strength for a slit edge of the conductor is 2.5-times lower than that for the original edge of the conductor; cracks and micro-peel existing over the slit edge reduce the cleavage strength for the slit edge. Cleavage stress and peel stress should be avoided in coil winding, as they easily delaminate the YBCO-coated conductor, resulting in substantial degradation of coil performance. These problems are especially important for epoxy impregnated YBCO-coated conductor coils. It appears that effect of cleavage stress and peel stress are mostly negligible for paraffin impregnated YBCO-coated conductor coils or dry wound YBCO-coated conductor coils.

Microinjection of Micrococcus luteus UV-endonuclease restores UV-induced unscheduled DNA synthesis in cells of 9 xeroderma pigmentosum complementation groups.

NARCIS (Netherlands)

A.J.R. de Jonge; W. Vermeulen (Wim); W. Keijzer; J.H.J. Hoeijmakers (Jan); D. Bootsma (Dirk)

1985-01-01

textabstractThe UV-induced unscheduled DNA synthesis (UDS) in cultured cells of excision-deficient xeroderma pigmentosum (XP) complementation groups A through I was assayed after injection of Micrococcus luteus UV-endonuclease using glass microneedles. In all complementation groups a restoration of

Flanking signal and mature peptide residues influence signal peptide cleavage

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Ranganathan Shoba

2008-12-01

Full Text Available Abstract Background Signal peptides (SPs mediate the targeting of secretory precursor proteins to the correct subcellular compartments in prokaryotes and eukaryotes. Identifying these transient peptides is crucial to the medical, food and beverage and biotechnology industries yet our understanding of these peptides remains limited. This paper examines the most common type of signal peptides cleavable by the endoprotease signal peptidase I (SPase I, and the residues flanking the cleavage sites of three groups of signal peptide sequences, namely (i eukaryotes (Euk (ii Gram-positive (Gram+ bacteria, and (iii Gram-negative (Gram- bacteria. Results In this study, 2352 secretory peptide sequences from a variety of organisms with amino-terminal SPs are extracted from the manually curated SPdb database for analysis based on physicochemical properties such as pI, aliphatic index, GRAVY score, hydrophobicity, net charge and position-specific residue preferences. Our findings show that the three groups share several similarities in general, but they display distinctive features upon examination in terms of their amino acid compositions and frequencies, and various physico-chemical properties. Thus, analysis or prediction of their sequences should be separated and treated as distinct groups. Conclusion We conclude that the peptide segment recognized by SPase I extends to the start of the mature protein to a limited extent, upon our survey of the amino acid residues surrounding the cleavage processing site. These flanking residues possibly influence the cleavage processing and contribute to non-canonical cleavage sites. Our findings are applicable in defining more accurate prediction tools for recognition and identification of cleavage site of SPs.

Staggering in the cleavage pattern of E. coli ABC-excinuclease

International Nuclear Information System (INIS)

Myles, G.M.; Van Houten, B.; Sancar, A.

1986-01-01

E. coli ABC excinuclease is a complex of three proteins encoded by the uvrA, uvrB, and uvrC genes. The enzyme repairs DNA mono and diadducts by the single strand cleavage of DNA eight phosphodiester bond 5' and four or five phosphodiester bonds 3' to a DNA lesion and facilitates the removal of the resulting twelve or thirteen nucleotide fragment. In this study, the authors have investigated the excision pattern for ultraviolet (UV) induced diadducts, i.e. cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6-4) photoproducts. Terminally (5' or 3') labeled DNA was irradiated with 254nm UV and treated with ABC excinuclease before and after photoreactivation of cyclobutane dimers by E. coli DNA photolyase. In this way, the authors were able to differentiate between the cleavage pattern of pyrimidine dimers and of (6-4) photoproducts. Their results show that certain TT cyclobutane dimers and rare TT (6-4) photoproducts are excised by cleavage seven and, less frequently, six phosphodiester bonds to the 5' side of the DNA lesion in addition to the primary cutting site at the eight 5' phosphodiester bond. The 3' cleavage sites are maintained at the fourth and fifth phosphodiester bonds for the these UV induced lesions. These data indicate that the cleavage pattern of the ABC excinuclease may be dependent upon both the type of DNA lesion as well as it surrounding nucleotide sequence. In addition, the authors analysis shows that (6-4) photoproducts are much better substrates for ABC excinuclease than are pyrimidine dimers

Identification of flap structure-specific endonuclease 1 as a factor involved in long-term memory formation of aversive learning.

Science.gov (United States)

Saavedra-Rodríguez, Lorena; Vázquez, Adrinel; Ortiz-Zuazaga, Humberto G; Chorna, Nataliya E; González, Fernando A; Andrés, Lissette; Rodríguez, Karen; Ramírez, Fernando; Rodríguez, Alan; Peña de Ortiz, Sandra

2009-05-06

We previously proposed that DNA recombination/repair processes play a role in memory formation. Here, we examined the possible role of the fen-1 gene, encoding a flap structure-specific endonuclease, in memory consolidation of conditioned taste aversion (CTA). Quantitative real-time PCR showed that amygdalar fen-1 mRNA induction was associated to the central processing of the illness experience related to CTA and to CTA itself, but not to the central processing resulting from the presentation of a novel flavor. CTA also increased expression of the Fen-1 protein in the amygdala, but not the insular cortex. In addition, double immunofluorescence analyses showed that amygdalar Fen-1 expression is mostly localized within neurons. Importantly, functional studies demonstrated that amygdalar antisense knockdown of fen-1 expression impaired consolidation, but not short-term memory, of CTA. Overall, these studies define the fen-1 endonuclease as a new DNA recombination/repair factor involved in the formation of long-term memories.

Downstream element determines RNase Y cleavage of the saePQRS operon in Staphylococcus aureus.

Science.gov (United States)

Marincola, Gabriella; Wolz, Christiane

2017-06-02

In gram-positive bacteria, RNase J1, RNase J2 and RNase Y are thought to be major contributors to mRNA degradation and maturation. In Staphylococcus aureus, RNase Y activity is restricted to regulating the mRNA decay of only certain transcripts. Here the saePQRS operon was used as a model to analyze RNase Y specificity in living cells. A RNase Y cleavage site is located in an intergenic region between saeP and saeQ. This cleavage resulted in rapid degradation of the upstream fragment and stabilization of the downstream fragment. Thereby, the expression ratio of the different components of the operon was shifted towards saeRS, emphasizing the regulatory role of RNase Y activity. To assess cleavage specificity different regions surrounding the sae CS were cloned upstream of truncated gfp, and processing was analyzed in vivo using probes up- and downstream of CS. RNase Y cleavage was not determined by the cleavage site sequence. Instead a 24-bp double-stranded recognition structure was identified that was required to initiate cleavage 6 nt upstream. The results indicate that RNase Y activity is determined by secondary structure recognition determinants, which guide cleavage from a distance. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Protein cleavage strategies for an improved analysis of the membrane proteome

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Poetsch Ansgar

2006-03-01

Full Text Available Abstract Background Membrane proteins still remain elusive in proteomic studies. This is in part due to the distribution of the amino acids lysine and arginine, which are less frequent in integral membrane proteins and almost absent in transmembrane helices. As these amino acids are cleavage targets for the commonly used protease trypsin, alternative cleavage conditions, which should improve membrane protein analysis, were tested by in silico digestion for the three organisms Saccharomyces cerevisiae, Halobacterium sp. NRC-1, and Corynebacterium glutamicum as hallmarks for eukaryotes, archea and eubacteria. Results For the membrane proteomes from all three analyzed organisms, we identified cleavage conditions that achieve better sequence and proteome coverage than trypsin. Greater improvement was obtained for bacteria than for yeast, which was attributed to differences in protein size and GRAVY. It was demonstrated for bacteriorhodopsin that the in silico predictions agree well with the experimental observations. Conclusion For all three examined organisms, it was found that a combination of chymotrypsin and staphylococcal peptidase I gave significantly better results than trypsin. As some of the improved cleavage conditions are not more elaborate than trypsin digestion and have been proven useful in practice, we suppose that the cleavage at both hydrophilic and hydrophobic amino acids should facilitate in general the analysis of membrane proteins for all organisms.

Dissociation from DNA of Type III Restriction–Modification enzymes during helicase-dependent motion and following endonuclease activity

Science.gov (United States)

Tóth, Júlia; van Aelst, Kara; Salmons, Hannah; Szczelkun, Mark D.

2012-01-01

DNA cleavage by the Type III Restriction–Modification (RM) enzymes requires the binding of a pair of RM enzymes at two distant, inversely orientated recognition sequences followed by helicase-catalysed ATP hydrolysis and long-range communication. Here we addressed the dissociation from DNA of these enzymes at two stages: during long-range communication and following DNA cleavage. First, we demonstrated that a communicating species can be trapped in a DNA domain without a recognition site, with a non-specific DNA association lifetime of ∼200 s. If free DNA ends were present the lifetime became too short to measure, confirming that ends accelerate dissociation. Secondly, we observed that Type III RM enzymes can dissociate upon DNA cleavage and go on to cleave further DNA molecules (they can ‘turnover’, albeit inefficiently). The relationship between the observed cleavage rate and enzyme concentration indicated independent binding of each site and a requirement for simultaneous interaction of at least two enzymes per DNA to achieve cleavage. In light of various mechanisms for helicase-driven motion on DNA, we suggest these results are most consistent with a thermally driven random 1D search model (i.e. ‘DNA sliding’). PMID:22523084

Cleavage and Cell Adhesion Properties of Human Epithelial Cell Adhesion Molecule (HEPCAM)*

Science.gov (United States)

Tsaktanis, Thanos; Kremling, Heidi; Pavšič, Miha; von Stackelberg, Ricarda; Mack, Brigitte; Fukumori, Akio; Steiner, Harald; Vielmuth, Franziska; Spindler, Volker; Huang, Zhe; Jakubowski, Jasmine; Stoecklein, Nikolas H.; Luxenburger, Elke; Lauber, Kirsten; Lenarčič, Brigita; Gires, Olivier

2015-01-01

Human epithelial cell adhesion molecule (HEPCAM) is a tumor-associated antigen frequently expressed in carcinomas, which promotes proliferation after regulated intramembrane proteolysis. Here, we describe extracellular shedding of HEPCAM at two α-sites through a disintegrin and metalloprotease (ADAM) and at one β-site through BACE1. Transmembrane cleavage by γ-secretase occurs at three γ-sites to generate extracellular Aβ-like fragments and at two ϵ-sites to release human EPCAM intracellular domain HEPICD, which is efficiently degraded by the proteasome. Mapping of cleavage sites onto three-dimensional structures of HEPEX cis-dimer predicted conditional availability of α- and β-sites. Endocytosis of HEPCAM warrants acidification in cytoplasmic vesicles to dissociate protein cis-dimers required for cleavage by BACE1 at low pH values. Intramembrane cleavage sites are accessible and not part of the structurally important transmembrane helix dimer crossing region. Surprisingly, neither chemical inhibition of cleavage nor cellular knock-out of HEPCAM using CRISPR-Cas9 technology impacted the adhesion of carcinoma cell lines. Hence, a direct function of HEPCAM as an adhesion molecule in carcinoma cells is not supported and appears to be questionable. PMID:26292218

Characterization of a Non-Canonical Signal Peptidase Cleavage Site in a Replication Protein from Tomato Ringspot Virus.

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Ting Wei

Full Text Available The NTB-VPg polyprotein from tomato ringspot virus is an integral membrane replication protein associated with endoplasmic reticulum membranes. A signal peptidase (SPase cleavage was previously detected in the C-terminal region of NTB-VPg downstream of a 14 amino acid (aa-long hydrophobic region (termed TM2. However, the exact location of the cleavage site was not determined. Using in vitro translation assays, we show that the SPase cleavage site is conserved in the NTB-VPg protein from various ToRSV isolates, although the rate of cleavage varies from one isolate to another. Systematic site-directed mutagenesis of the NTB-VPg SPase cleavage sites of two ToRSV isolates allowed the identification of sequences that affect cleavage efficiency. We also present evidence that SPase cleavage in the ToRSV-Rasp2 isolate occurs within a GAAGG sequence likely after the AAG (GAAG/G. Mutation of a downstream MAAV sequence to AAAV resulted in SPase cleavage at both the natural GAAG/G and the mutated AAA/V sequences. Given that there is a distance of seven aa between the two cleavage sites, this indicates that there is flexibility in the positioning of the cleavage sites relative to the inner surface of the membrane and the SPase active site. SPase cleavage sites are typically located 3-7 aa downstream of the hydrophobic region. However, the NTB-VPg GAAG/G cleavage site is located 17 aa downstream of the TM2 hydrophobic region, highlighting unusual features of the NTB-VPg SPase cleavage site. A putative 11 aa-long amphipathic helix was identified immediately downstream of the TM2 region and five aa upstream of the GAAG/G cleavage site. Based on these results, we present an updated topology model in which the hydrophobic and amphipathic domains form a long tilted helix or a bent helix in the membrane lipid bilayer, with the downstream cleavage site(s oriented parallel to the membrane inner surface.

Spectrophotometric, colorimetric and visually detection of Pseudomonas aeruginosa ETA gene based gold nanoparticles DNA probe and endonuclease enzyme

Science.gov (United States)

Amini, Bahram; Kamali, Mehdi; Salouti, Mojtaba; Yaghmaei, Parichehreh

2018-06-01

Colorimetric DNA detection is preferred over other methods for clinical molecular diagnosis because it does not require expensive equipment. In the present study, the colorimetric method based on gold nanoparticles (GNPs) and endonuclease enzyme was used for the detection of P. aeruginosa ETA gene. Firstly, the primers and probe for P. aeruginosa exotoxin A (ETA) gene were designed and checked for specificity by the PCR method. Then, GNPs were synthesized using the citrate reduction method and conjugated with the prepared probe to develop the new nano-biosensor. Next, the extracted target DNA of the bacteria was added to GNP-probe complex to check its efficacy for P. aeruginosa ETA gene diagnosis. A decrease in absorbance was seen when GNP-probe-target DNA cleaved into the small fragments of BamHI endonuclease due to the weakened electrostatic interaction between GNPs and the shortened DNA. The right shift of the absorbance peak from 530 to 562 nm occurred after adding the endonuclease. It was measured using a UV-VIS absorption spectroscopy that indicates the existence of the P. aeruginosa ETA gene. Sensitivity was determined in the presence of different concentrations of target DNA of P. aeruginosa. The results obtained from the optimized conditions showed that the absorbance value has linear correlation with concentration of target DNA (R: 0.9850) in the range of 10-50 ng mL-1 with the limit detection of 9.899 ng mL-1. Thus, the specificity of the new method for detection of P. aeruginosa was established in comparison with other bacteria. Additionally, the designed assay was quantitatively applied to detect the P. aeruginosa ETA gene from 103 to 108 CFU mL-1 in real samples with a detection limit of 320 CFU mL-1.

Molecular mechanisms involved in the production of chromosomal aberrations. I. Utilization of Neurospora endonuclease for the study of aberration production in G2 stage of the cell cycle

Energy Technology Data Exchange (ETDEWEB)

Natarajan, A T; Obe, G [Rijksuniversiteit Leiden (Netherlands). J.A. Cohen Inst. voor Radiopathologie en Stralingsbescherming

1978-10-01

Chinese hamster ovary cells (CHO) were X-irradiated in G2 stage of the cell cycle and immediately treated, in the presence of inactivated Sendai virus, with Neurospora endonuclease (E.C. 3.1.4.), an enzyme which is specific for cleaving single-stranded DNA. With this treatment, the frequencies of all types of chromosome aberrations increased when compared to X-irradiated controls. These results are interpreted as due to the conversion of some of the X-ray induced single-stranded DNA breaks into double-strand breaks by this enzyme. Similar enhancement due to this enzyme was found following treatment with methyl methanesulfonate (MMS) and bleomycin, but not following UV and mitomycin C. Addition of Micrococcus endonuclease and Neurospora endonuclease to the cells did not alter the frequencies of aberrations induced by UV. The introduction of enzymes with specific DNA-repair function offers possibilities to probe into the molecular events involved in the formation of structural chromosome aberrations induced by different classes of physical and chemical mutagens.

Specificity of binding to four-way junctions in DNA by bacteriophage T7 endonuclease I.

OpenAIRE

Parsons, C A; West, S C

1990-01-01

T7 endonuclease I binds specifically to four-way junctions in duplex DNA and promotes their resolution into linear duplexes. Under conditions in which the nuclease activity is blocked by the absence of divalent cations, the enzyme forms a distinct protein-DNA complex with the junction, as detected by gel retardation and filter binding assays. The formation of this complex is structure-specific and contrasts with the short-lived binding complexes formed on linear duplex DNA. The binding comple...

An ultra-sensitive colorimetric Hg(2+)-sensing assay based on DNAzyme-modified Au NP aggregation, MNPs and an endonuclease.

Science.gov (United States)

Li, Chao; Dai, Peiqing; Rao, Xinyi; Shao, Lin; Cheng, Guifang; He, Pingang; Fang, Yuzhi

2015-01-01

This paper reports the development of an ultra-sensitive colorimetric method for the detection of trace mercury ions involving DNAzymes, Au nanoparticle aggregation, magnetic nanoparticles and an endonuclease. DNAzyme-sensing elements are conjugated to the surface of Au nanoparticle-2, which can crosslink with the T-rich strands coated on Au nanoparticle-1 to form Au nanoparticle aggregation. Other T-rich stands are immobilized on the surface of MNPs. The specific hybridization of these two T-rich strands depends on the presence of Hg(2+), resulting in the formation of a T-Hg(2+)-T structure. Added endonuclease then digests the hybridized strands, and DNAzyme-modified Au NP aggregation is released, catalysing the conversion of the colourless ABTS into a blue-green product by H2O2-mediated oxidation. The increase in the adsorption spectrum of ABTS(+) at 421 nm is related to the concentration of Hg(2+). This assay was validated by detecting mercury ion concentrations in river water. The colorimetric responses were not significantly altered in the presence of 100-fold excesses of other metal ions such as Zn(2+), Pb(2+), Cd(2+), Mn(2+), Ca(2+) and Ni(2+). The inclusion of both Au NP aggregation and an endonuclease enables the assay to eliminate interference from the magnetic nanoparticles with colorimetric detection, decrease the background and improve the detection sensitivity. The calibration curve of the assay was linear over the range of Hg(2+) concentrations from 1 to 30 nM, and the detection limit was 0.8 nM, which is far lower than the 10 nM US EPA limit for drinking water. Copyright © 2014 Elsevier B.V. All rights reserved.

Calicivirus 3C-like proteinase inhibits cellular translation by cleavage of poly(A)-binding protein.

Science.gov (United States)

Kuyumcu-Martinez, Muge; Belliot, Gaël; Sosnovtsev, Stanislav V; Chang, Kyeong-Ok; Green, Kim Y; Lloyd, Richard E

2004-08-01

Caliciviruses are single-stranded RNA viruses that cause a wide range of diseases in both humans and animals, but little is known about the regulation of cellular translation during infection. We used two distinct calicivirus strains, MD145-12 (genus Norovirus) and feline calicivirus (FCV) (genus Vesivirus), to investigate potential strategies used by the caliciviruses to inhibit cellular translation. Recombinant 3C-like proteinases (r3CL(pro)) from norovirus and FCV were found to cleave poly(A)-binding protein (PABP) in the absence of other viral proteins. The norovirus r3CL(pro) PABP cleavage products were indistinguishable from those generated by poliovirus (PV) 3C(pro) cleavage, while the FCV r3CL(pro) products differed due to cleavage at an alternate cleavage site 24 amino acids downstream of one of the PV 3C(pro) cleavage sites. All cleavages by calicivirus or PV proteases separated the C-terminal domain of PABP that binds translation factors eIF4B and eRF3 from the N-terminal RNA-binding domain of PABP. The effect of PABP cleavage by the norovirus r3CL(pro) was analyzed in HeLa cell translation extracts, and the presence of r3CL(pro) inhibited translation of both endogenous and exogenous mRNAs. Translation inhibition was poly(A) dependent, and replenishment of the extracts with PABP restored translation. Analysis of FCV-infected feline kidney cells showed that the levels of de novo cellular protein synthesis decreased over time as virus-specific proteins accumulated, and cleavage of PABP occurred in virus-infected cells. Our data indicate that the calicivirus 3CL(pro), like PV 3C(pro), mediates the cleavage of PABP as part of its strategy to inhibit cellular translation. PABP cleavage may be a common mechanism among certain virus families to manipulate cellular translation.

The potato carotenoid cleavage dioxygenase 4 catalyzes a single cleavage of β-ionone ring-containing carotenes and non-epoxidated xanthophylls

KAUST Repository

Bruno, Mark

2015-04-01

Down-regulation of the potato carotenoid cleavage dioxygenase 4 (StCCD4) transcript level led to tubers with altered morphology and sprouting activity, which also accumulated higher levels of violaxanthin and lutein leading to elevated carotenoid amounts. This phenotype indicates a role of this enzyme in tuber development, which may be exerted by a cleavage product. In this work, we investigated the enzymatic activity of StCCD4, by expressing the corresponding cDNA in carotenoid accumulating Escherichia coli strains and by performing in vitro assays with heterologously expressed enzyme. StCCD4 catalyzed the cleavage of all-. trans-β-carotene at the C9\\'-C10\\' double bond, leading to β-ionone and all-. trans-β-apo-10\\'-carotenal, both in vivo and in vitro. The enzyme also cleaved β,β-cryptoxanthin, zeaxanthin and lutein either at the C9\\'-C10\\' or the C9-C10 double bond in vitro. In contrast, we did not observe any conversion of violaxanthin and only traces of activity with 9-. cis-β-carotene, which led to 9-. cis-β-apo-10\\'-carotenal. Our data indicate that all-. trans-β-carotene is the likely substrate of StCCD4 in planta, and that this carotene may be precursor of an unknown compound involved in tuber development.

Restoration of ultraviolet-induced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendai virus)

International Nuclear Information System (INIS)

Tanaka, K.; Sekiguchi, M.; Okada, Y.

1975-01-01

Ultraviolet (uv)-induced unscheduled DNA synthesis of xeroderma pigmentosum cells, belonging to complementation groups, A, B, C, D, and E, was restored to the normal level by concomitant treatment of the cells with T4 endonuclease V and uv-inactivated HVJ (Sendai virus). The present results suggest that T4 endonuclease molecules were inserted effectively into the cells by the interaction of HVJ with the cell membranes, the enzyme was functional on human chromosomal DNA which had been damaged by uv irradiation in the viable cells, all the studied groups of xeroderma pigmentosum (variant was not tested) were defective in the first step (incision) of excision repair

Mine drivage in hydraulic mines

Energy Technology Data Exchange (ETDEWEB)

Ehkber, B Ya

1983-09-01

From 20 to 25% of labor cost in hydraulic coal mines falls on mine drivage. Range of mine drivage is high due to the large number of shortwalls mined by hydraulic monitors. Reducing mining cost in hydraulic mines depends on lowering drivage cost by use of new drivage systems or by increasing efficiency of drivage systems used at present. The following drivage methods used in hydraulic mines are compared: heading machines with hydraulic haulage of cut rocks and coal, hydraulic monitors with hydraulic haulage, drilling and blasting with hydraulic haulage of blasted rocks. Mining and geologic conditions which influence selection of the optimum mine drivage system are analyzed. Standardized cross sections of mine roadways driven by the 3 methods are shown in schemes. Support systems used in mine roadways are compared: timber supports, roof bolts, roof bolts with steel elements, and roadways driven in rocks without a support system. Heading machines (K-56MG, GPKG, 4PU, PK-3M) and hydraulic monitors (GMDTs-3M, 12GD-2) used for mine drivage are described. Data on mine drivage in hydraulic coal mines in the Kuzbass are discussed. From 40 to 46% of roadways are driven by heading machines with hydraulic haulage and from 12 to 15% by hydraulic monitors with hydraulic haulage.

A Mismatch EndoNuclease Array-Based Methodology (MENA) for Identifying Known SNPs or Novel Point Mutations.

Science.gov (United States)

Comeron, Josep M; Reed, Jordan; Christie, Matthew; Jacobs, Julia S; Dierdorff, Jason; Eberl, Daniel F; Manak, J Robert

2016-04-05

Accurate and rapid identification or confirmation of single nucleotide polymorphisms (SNPs), point mutations and other human genomic variation facilitates understanding the genetic basis of disease. We have developed a new methodology (called MENA (Mismatch EndoNuclease Array)) pairing DNA mismatch endonuclease enzymology with tiling microarray hybridization in order to genotype both known point mutations (such as SNPs) as well as identify previously undiscovered point mutations and small indels. We show that our assay can rapidly genotype known SNPs in a human genomic DNA sample with 99% accuracy, in addition to identifying novel point mutations and small indels with a false discovery rate as low as 10%. Our technology provides a platform for a variety of applications, including: (1) genotyping known SNPs as well as confirming newly discovered SNPs from whole genome sequencing analyses; (2) identifying novel point mutations and indels in any genomic region from any organism for which genome sequence information is available; and (3) screening panels of genes associated with particular diseases and disorders in patient samples to identify causative mutations. As a proof of principle for using MENA to discover novel mutations, we report identification of a novel allele of the beethoven (btv) gene in Drosophila, which encodes a ciliary cytoplasmic dynein motor protein important for auditory mechanosensation.

[Recent knowledge about intestinal absorption and cleavage of carotenoids].

Science.gov (United States)

Borel, P; Drai, J; Faure, H; Fayol, V; Galabert, C; Laromiguière, M; Le Moël, G

2005-01-01

Our knowledge about intestinal absorption and cleavage of carotenoids has rapidly grown during the last years. New facts about carotenoid absorption have emerged while some controversies about cleavage are close to end. The knowledge of the absorption and conversion processes is indispensable to understand and interpret the perturbations that can occur in the metabolism of carotenoids and vitamin A. Recently, it has been shown that the absorption of certain carotenoids is not passive - as believed for a long time - but is a facilitated process that requires, at least for lutein, the class B-type 1 scavenger receptor (SR-B1). Various epidemiological and clinical studies have shown wide variations in carotenoid absorption from one subject to another, such differences are now explained by the structure of the concerned carotenoid, by the nature of the food that is absorbed with the carotenoid, by diverse exogenous factors like the intake of medicines or interfering components, by diet factors, by genetic factors, and by the nutritional status of the subject. Recently, the precise mechanism of beta-carotene cleavage by betabeta-carotene 15,15' monooxygenase (EC 1.14.99.36) - formerly called beta-carotene 15,15' dioxygenase (ex EC 1.13.11.21) - has been discovered, and a second enzyme which cleaves asymmetrically the beta-carotene molecule has been found. beta-carotene 15,15' monooxygenase only acts on the 15,15' bond, thus forming two molecules of retinal from one molecule of beta-carotene by central cleavage. Even though the betabeta-carotene 15,15' monooxygenase is much more active on the beta-carotene molecule, a study has shown that it can act on all carotenoids. Searchers now agree that other enzymes that can catalyse an eccentric cleavage of carotenoids probably exist, but under physiological conditions the betabeta-carotene 15,15' monooxygenase is by far the most active, and it is mainly effective in the small bowel mucosa and in the liver. However the

Endocytic down-regulation of ErbB2 is stimulated by cleavage of its C-terminus

DEFF Research Database (Denmark)

Lerdrup, Mads; Bruun, Silas; Grandal, Michael Vibo

2007-01-01

inhibition of HSP90 with geldanamycin this cleavage is accompanied by proteasome-dependent endocytosis of ErbB2. However, it is unknown whether C-terminal cleavage is linked to endocytosis. To study ErbB2 cleavage and endocytic trafficking, we fused yellow fluorescent protein (YFP) and cyan fluorescent...

Effects of Cysteamine on Sheep Embryo Cleavage Rates

Directory of Open Access Journals (Sweden)

Sinem Ö. ENGİNLER

2015-01-01

Full Text Available Oxidative stress during in vitro culture leads to defects in development of gametes and embryos. Several antioxidants such as cysteamine, L-ascorbic acid, beta mercaptoethanol, cysteine, glutathione, proteins, vitamins have been used to supplement culture media to counter the oxidative stress. This study was conducted to detect the effect of adding cysteamine to the maturation medium to subsequent cleavage rates of sheep embryos. Totally 604 ovaries were obtained by ten replica and 2060 oocytes were collected. The cumulus oocyte complexes were recovered by the slicing method. A total of 1818 selected oocytes were divided into two groups and used for maturation (88.25%. The first group was created as supplemented with cysteamine (Group A and second group (Group B, control without cysteamine in TCM-199. The two groups were incubated for 24 h at 38.8 °C in an atmosphere of 5% CO2 in humidified air for in vitro maturation (IVM. After IVM, oocytes were fertilized with 50 x 107 / mL fresh ram semen in BSOF medium for 18 h. After fertilization, maturation groups were divided into two subgroups with different culture media: Group AI-SOF (Synthetic Oviduct Fluid medium, Group AII-CR1aa (Charles Rosencrans medium, Group BI-SOF and Group BII-CR1aa were achieved. Cleavage rates were evaluated at day 2. post insemination. The rates of cleavage were detected as 59.54% (184/309, 55.44% (173/312, 65.34% (215/329, 59.34% (200/337 respectively, with showing no statistically significant difference between the groups at the level of P>0.05. In conclusion, supplementing cysteamine to maturation media in TCM-199 did not affect the cleavage rates of sheep embryos in SOF and CR1aa culture media.

TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting

Science.gov (United States)

Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

2013-01-01

Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity. PMID:24349308

TALE-PvuII fusion proteins--novel tools for gene targeting.

Science.gov (United States)

Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

2013-01-01

Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.

Variable context Markov chains for HIV protease cleavage site prediction.

Science.gov (United States)

Oğul, Hasan

2009-06-01

Deciphering the knowledge of HIV protease specificity and developing computational tools for detecting its cleavage sites in protein polypeptide chain are very desirable for designing efficient and specific chemical inhibitors to prevent acquired immunodeficiency syndrome. In this study, we developed a generative model based on a generalization of variable order Markov chains (VOMC) for peptide sequences and adapted the model for prediction of their cleavability by certain proteases. The new method, called variable context Markov chains (VCMC), attempts to identify the context equivalence based on the evolutionary similarities between individual amino acids. It was applied for HIV-1 protease cleavage site prediction problem and shown to outperform existing methods in terms of prediction accuracy on a common dataset. In general, the method is a promising tool for prediction of cleavage sites of all proteases and encouraged to be used for any kind of peptide classification problem as well.

Changes in Contact Area in Meniscus Horizontal Cleavage Tears Subjected to Repair and Resection.

Science.gov (United States)

Beamer, Brandon S; Walley, Kempland C; Okajima, Stephen; Manoukian, Ohan S; Perez-Viloria, Miguel; DeAngelis, Joseph P; Ramappa, Arun J; Nazarian, Ara

2017-03-01

To assess the changes in tibiofemoral contact pressure and contact area in human knees with a horizontal cleavage tear before and after treatment. Ten human cadaveric knees were tested. Pressure sensors were placed under the medial meniscus and the knees were loaded at twice the body weight for 20 cycles at 0°, 10°, and 20° of flexion. Contact area and pressure were recorded for the intact meniscus, the meniscus with a horizontal cleavage tear, after meniscal repair, after partial meniscectomy (single leaflet), and after subtotal meniscectomy (double leaflet). The presence of a horizontal cleavage tear significantly increased average peak contact pressure and reduced effective average tibiofemoral contact area at all flexion angles tested compared with the intact state (P contact pressure after creation of the horizontal cleavage tear. Repairing the horizontal cleavage tear restored peak contact pressures and areas to within 15% of baseline, statistically similar to the intact state at all angles tested (P contact pressure and reduced average contact area at all degrees of flexion compared with the intact state (P contact area and a significant elevation in contact pressure. These changes may accelerate joint degeneration. A suture-based repair of these horizontal cleavage tears returns the contact area and contact pressure to nearly normal, whereas both partial and subtotal meniscectomy lead to significant reductions in contact area and significant elevations in contact pressure within the knee. Repairing horizontal cleavage tears may lead to improved clinical outcomes by preserving meniscal tissue and the meniscal function. Understanding contact area and peak contact pressure resulting from differing strategies for treating horizontal cleavage tears will allow the surgeon to evaluate the best strategy for treating his or her patients who present with this meniscal pathology. Copyright © 2016 Arthroscopy Association of North America. Published by Elsevier

Oxidative cleavage of erucic acid for the synthesis of brassylic acid

Energy Technology Data Exchange (ETDEWEB)

Mohammed J. Nasrullah; Pooja Thapliyal; Erica N. Pfarr; Nicholas S. Dusek; Kristofer L. Schiele; James A. Bahr

2010-10-29

The main focus of this work is to synthesize Brassylic Acid (BA) using oxidative cleavage of Erucic Acid (EA). Crambe (Crambe abyssinica) is an industrial oilseed grown in North Dakota. Crambe has potential as an industrial fatty acid feedstock as a source of Erucic acid (EA). It has approximately 50-60 % of EA, a C{sub 22} monounsaturated fatty acid. Oxidative cleavage of unsaturated fatty acids derived from oilseeds produces long chain (9, 11, and 13 carbon atoms) dibasic and monobasic acids. These acids are known commercial feedstocks for the preparation of nylons, polyesters, waxes, surfactants, and perfumes. Other sources of EA are Rapeseed seed oil which 50-60 % of EA. Rapeseed is grown outside USA. The oxidative cleavage of EA was done using a high throughput parallel pressure reactor system. Kinetics of the reaction shows that BA yields reach a saturation at 12 hours. H{sub 2}WO{sub 4} was found to be the best catalyst for the oxidative cleavage of EA. High yields of BA were obtained at 80 C with bubbling of O{sub 2} or 10 bar of O{sub 2} for 12 hours.

Caspase-Dependent Apoptosis Induced by Telomere Cleavage and TRF2 Loss

Directory of Open Access Journals (Sweden)

Asha S. Multani

2000-07-01

Full Text Available Chromosomal abnormalities involving telomeric associations (TAs often precede replicative senescence and abnormal chromosome configurations. We report here that telomere cleavage following exposure to proapoptotic agents is an early event in apoptosis. Exposure of human and murine cancer cells to a variety of pro-apoptotic stimuli (staurosporine, thapsigargin, anti-Fas antibody, cancer chemotherapeutic agents resulted in telomere cleavage and aggregation, finally their extrusion from the nuclei. Telomere loss was associated with arrest of cells in G2/M phase and preceded DNA fragmentation. Telomere erosion and subsequent large-scale chromatin cleavage were inhibited by overexpression of the anti -apoptotic protein, bcl-2, two peptide caspase inhibitors (BACMK and zVADfmk, indicating that both events are regulated by caspase activation. The results demonstrate that telomere cleavage is an early chromatin alteration detected in various cancer cell lines leading to drug-induced apoptosis, suggest that this event contributes to mitotic catastrophe and induction of cell death. Results also suggest that the decrease of telomeric-repeat binding factor 2 (TRF2 may be the earliest event in the ara-C-induced telomere shortening, induction of endoreduplication and chromosomal fragmentation leading to cell death.

The potato carotenoid cleavage dioxygenase 4 catalyzes a single cleavage of β-ionone ring-containing carotenes and non-epoxidated xanthophylls.

Science.gov (United States)

Bruno, Mark; Beyer, Peter; Al-Babili, Salim

2015-04-15

Down-regulation of the potato carotenoid cleavage dioxygenase 4 (StCCD4) transcript level led to tubers with altered morphology and sprouting activity, which also accumulated higher levels of violaxanthin and lutein leading to elevated carotenoid amounts. This phenotype indicates a role of this enzyme in tuber development, which may be exerted by a cleavage product. In this work, we investigated the enzymatic activity of StCCD4, by expressing the corresponding cDNA in carotenoid accumulating Escherichia coli strains and by performing in vitro assays with heterologously expressed enzyme. StCCD4 catalyzed the cleavage of all-trans-β-carotene at the C9'-C10' double bond, leading to β-ionone and all-trans-β-apo-10'-carotenal, both in vivo and in vitro. The enzyme also cleaved β,β-cryptoxanthin, zeaxanthin and lutein either at the C9'-C10' or the C9-C10 double bond in vitro. In contrast, we did not observe any conversion of violaxanthin and only traces of activity with 9-cis-β-carotene, which led to 9-cis-β-apo-10'-carotenal. Our data indicate that all-trans-β-carotene is the likely substrate of StCCD4 in planta, and that this carotene may be precursor of an unknown compound involved in tuber development. Copyright © 2015 Elsevier Inc. All rights reserved.

Pressure modulates the self-cleavage step of the hairpin ribozyme

Science.gov (United States)

Schuabb, Caroline; Kumar, Narendra; Pataraia, Salome; Marx, Dominik; Winter, Roland

2017-03-01

The ability of certain RNAs, denoted as ribozymes, to not only store genetic information but also catalyse chemical reactions gave support to the RNA world hypothesis as a putative step in the development of early life on Earth. This, however, might have evolved under extreme environmental conditions, including the deep sea with pressures in the kbar regime. Here we study pressure-induced effects on the self-cleavage of hairpin ribozyme by following structural changes in real-time. Our results suggest that compression of the ribozyme leads to an accelerated transesterification reaction, being the self-cleavage step, although the overall process is retarded in the high-pressure regime. The results reveal that favourable interactions between the reaction site and neighbouring nucleobases are strengthened under pressure, resulting therefore in an accelerated self-cleavage step upon compression. These results suggest that properly engineered ribozymes may also act as piezophilic biocatalysts in addition to their hitherto known properties.

Mining and mining authorities in Saarland 2016. Mining economy, mining technology, occupational safety, environmental protection, statistics, mining authority activities. Annual report

International Nuclear Information System (INIS)

2016-01-01

The annual report of the Saarland Upper Mining Authority provides an insight into the activities of mining authorities. Especially, the development of the black coal mining, safety and technology of mining as well as the correlation between mining and environment are stressed.

Restriction endonucleases from invasive Neisseria gonorrhoeae cause double-strand breaks and distort mitosis in epithelial cells during infection.

Directory of Open Access Journals (Sweden)

Linda Weyler

Full Text Available The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

Restriction endonucleases from invasive Neisseria gonorrhoeae cause double-strand breaks and distort mitosis in epithelial cells during infection.

Science.gov (United States)

Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

2014-01-01

The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

Computational analysis and modeling of cleavage by the immunoproteasome and the constitutive proteasome

Directory of Open Access Journals (Sweden)

Lafuente Esther M

2010-09-01

Full Text Available Abstract Background Proteasomes play a central role in the major histocompatibility class I (MHCI antigen processing pathway. They conduct the proteolytic degradation of proteins in the cytosol, generating the C-terminus of CD8 T cell epitopes and MHCI-peptide ligands (P1 residue of cleavage site. There are two types of proteasomes, the constitutive form, expressed in most cell types, and the immunoproteasome, which is constitutively expressed in mature dendritic cells. Protective CD8 T cell epitopes are likely generated by the immunoproteasome and the constitutive proteasome, and here we have modeled and analyzed the cleavage by these two proteases. Results We have modeled the immunoproteasome and proteasome cleavage sites upon two non-overlapping sets of peptides consisting of 553 CD8 T cell epitopes, naturally processed and restricted by human MHCI molecules, and 382 peptides eluted from human MHCI molecules, respectively, using N-grams. Cleavage models were generated considering different epitope and MHCI-eluted fragment lengths and the same number of C-terminal flanking residues. Models were evaluated in 5-fold cross-validation. Judging by the Mathew's Correlation Coefficient (MCC, optimal cleavage models for the proteasome (MCC = 0.43 ± 0.07 and the immunoproteasome (MCC = 0.36 ± 0.06 were obtained from 12-residue peptide fragments. Using an independent dataset consisting of 137 HIV1-specific CD8 T cell epitopes, the immunoproteasome and proteasome cleavage models achieved MCC values of 0.30 and 0.18, respectively, comparatively better than those achieved by related methods. Using ROC analyses, we have also shown that, combined with MHCI-peptide binding predictions, cleavage predictions by the immunoproteasome and proteasome models significantly increase the discovery rate of CD8 T cell epitopes restricted by different MHCI molecules, including A*0201, A*0301, A*2402, B*0702, B*2705. Conclusions We have developed models that are specific

A new restriction endonuclease-based method for highly-specific detection of DNA targets from methicillin-resistant Staphylococcus aureus.

Directory of Open Access Journals (Sweden)

Maria W Smith

Full Text Available PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP. At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i any mutation of the restriction site reduced the signal to zero; (ii double and triple point mutations of sequences flanking the restriction site reduced restriction to 50-80% of the positive control; and (iii a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same

On the temperature independence of statistical model parameters for cleavage fracture in ferritic steels

Science.gov (United States)

Qian, Guian; Lei, Wei-Sheng; Niffenegger, M.; González-Albuixech, V. F.

2018-04-01

The work relates to the effect of temperature on the model parameters in local approaches (LAs) to cleavage fracture. According to a recently developed LA model, the physical consensus of plastic deformation being a prerequisite to cleavage fracture enforces any LA model of cleavage fracture to observe initial yielding of a volume element as its threshold stress state to incur cleavage fracture in addition to the conventional practice of confining the fracture process zone within the plastic deformation zone. The physical consistency of the new LA model to the basic LA methodology and the differences between the new LA model and other existing models are interpreted. Then this new LA model is adopted to investigate the temperature dependence of LA model parameters using circumferentially notched round tensile specimens. With the published strength data as input, finite element (FE) calculation is conducted for elastic-perfectly plastic deformation and the realistic elastic-plastic hardening, respectively, to provide stress distributions for model calibration. The calibration results in temperature independent model parameters. This leads to the establishment of a 'master curve' characteristic to synchronise the correlation between the nominal strength and the corresponding cleavage fracture probability at different temperatures. This 'master curve' behaviour is verified by strength data from three different steels, providing a new path to calculate cleavage fracture probability with significantly reduced FE efforts.

Fractographic observations of cleavage initiation in the ductile-brittle transition region of a reactor-pressure-vessel steel

International Nuclear Information System (INIS)

Rosenfield, A.R.; Shetty, D.K.; Skidmore, A.J.

1983-01-01

This note reports the results of a fractographic study conducted on a group of 1T compact fracture toughness specimens of a heavy-section A508 steel denoted TSE6 tested in the ductile-brittle transition region (22 and 82 0 C). The fatigue-precracked specimens were loaded at a rapid rate (760 or 550 mm per second) to promote cleavage-crack growth and lower-bound toughness behavior. All specimens experienced unstable cleavage fracture prior to reaching a maximum in the load displacement curve. Some ductile crack growth occurred in half of the specimens. The objective of fractographic examinations was to understand the observed statistical variations in cleavage initiation by (a) locating the origins of unstable cleavage fracture in the vicinity of the fatigue-precrack or ductilerupture crack fronts, (b) identifying microstructural features associated with the triggering of cleavage, and (c) documenting characteristic fracture surface dimensions such as the extent of stable-crack growth prior to unstable cleavage (Δα) and the distance of the cleavage origin from the ductilerupture front, /chi/ (or fatigue-crack front when Δα = 0)

Sensitive and fast mutation detection by solid phase chemical cleavage

DEFF Research Database (Denmark)

Hansen, Lise Lotte; Justesen, Just; Kruse, Torben A

1996-01-01

We have developed a solid phase chemical cleavage method (SpCCM) for screening large DNA fragments for mutations. All reactions can be carried out in microtiterwells from the first amplification of the patient (or test) DNA through the search for mutations. The reaction time is significantly...... reduced compared to the conventional chemical cleavage method (CCM), and even by using a uniformly labelled probe, the exact position and nature of the mutation can be revealed. The SpCCM is suitable for automatization using a workstation to carry out the reactions and a fluorescent detection-based DNA...

On the formation and nature of quasi-cleavage fracture surfaces in hydrogen embrittled steels

Energy Technology Data Exchange (ETDEWEB)

Martin, May L.; Fenske, Jamey A.; Liu, Grace S.; Sofronis, Petros [University of Illinois, Dept. of Materials Science and Engineering, 1304 W. Green St., Urbana, IL 61801 (United States); Robertson, Ian M., E-mail: ianr@illinois.edu [University of Illinois, Dept. of Materials Science and Engineering, 1304 W. Green St., Urbana, IL 61801 (United States)

2011-02-15

Quasi-cleavage, a common feature of hydrogen-induced fracture surfaces, is generally taken as being cleavage-like but not along a known cleavage plane. Despite the frequency with which this surface is observed, the relationship to the underlying microstructure remains unknown. Through a combination of topographical reconstruction of secondary electron microscope fractographs and a transmission electron microscopy study of the microstructure from site-specific locations, it will be shown that the features on quasi-cleavage surfaces are ridges that can be correlated with sub-surface intense and highly localized deformation bands. It will be demonstrated that the fracture surface arises from the growth and coalescence of voids that initiate at and extend along slip band intersections. This mechanism and process is fully consistent with hydrogen enhancing and localizing plastic processes.

The potato carotenoid cleavage dioxygenase 4 catalyzes a single cleavage of β-ionone ring-containing carotenes and non-epoxidated xanthophylls

KAUST Repository

Bruno, Mark; Beyer, Peter D.; Al-Babili, Salim

2015-01-01

amounts. This phenotype indicates a role of this enzyme in tuber development, which may be exerted by a cleavage product. In this work, we investigated the enzymatic activity of StCCD4, by expressing the corresponding cDNA in carotenoid accumulating

Mine Water Treatment in Hongai Coal Mines

Science.gov (United States)

Dang, Phuong Thao; Dang, Vu Chi

2018-03-01

Acid mine drainage (AMD) is recognized as one of the most serious environmental problem associated with mining industry. Acid water, also known as acid mine drainage forms when iron sulfide minerals found in the rock of coal seams are exposed to oxidizing conditions in coal mining. Until 2009, mine drainage in Hongai coal mines was not treated, leading to harmful effects on humans, animals and aquatic ecosystem. This report has examined acid mine drainage problem and techniques for acid mine drainage treatment in Hongai coal mines. In addition, selection and criteria for the design of the treatment systems have been presented.

Numerical modeling of ductile tearing effects on cleavage fracture toughness

International Nuclear Information System (INIS)

Dodds, R.H. Jr.; Tang, M.; Anderson, T.L.

1994-05-01

Experimental studies demonstrate a significant effect of specimen size, a/W ratio and prior ductile tearing on cleavage fracture toughness values (J c ) measured in the ductile-to-brittle transition region of ferritic materials. In the lower-transition region, cleavage fracture often occurs under conditions of large-scale yielding but without prior ductile crack extension. The increased toughness develops when plastic zones formed at the crack tip interact with nearby specimen surfaces which relaxes crack-tip constraint (stress triaxiality). In the mid-to-upper transition region, small amounts of ductile crack extension (often c -values. Previous work by the authors described a micromechanics fracture model to correct measured J c -values for the mechanistic effects of large-scale yielding. This new work extends the model to also include the influence of ductile crack extension prior to cleavage. The paper explores development of the new model, provides necessary graphs and procedures for its application and demonstrates the effects of the model on fracture data sets for two pressure vessel steels (A533B and A515)

Mine Water Treatment in Hongai Coal Mines

OpenAIRE

Dang Phuong Thao; Dang Vu Chi

2018-01-01

Acid mine drainage (AMD) is recognized as one of the most serious environmental problem associated with mining industry. Acid water, also known as acid mine drainage forms when iron sulfide minerals found in the rock of coal seams are exposed to oxidizing conditions in coal mining. Until 2009, mine drainage in Hongai coal mines was not treated, leading to harmful effects on humans, animals and aquatic ecosystem. This report has examined acid mine drainage problem and techniques for acid mine ...

Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein.

Science.gov (United States)

Welch, Brett D; Liu, Yuanyuan; Kors, Christopher A; Leser, George P; Jardetzky, Theodore S; Lamb, Robert A

2012-10-09

The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein.

Critical cleavage fracture stress characterization of A508 nuclear pressure vessel steels

International Nuclear Information System (INIS)

Wu, Sujun; Jin, Huijin; Sun, Yanbin; Cao, Luowei

2014-01-01

The critical cleavage fracture stress of SA508 Gr.4N and SA508 Gr.3 low alloy reactor pressure vessel (RPV) steels was studied through the combination of experiments and finite element method (FEM) analysis. The results showed that the value of the local cleavage fracture stress, σ F , of SA508 Gr.4N steel was significantly higher than that of SA508 Gr.3 steel. Detailed microstructural analysis was carried out using FEGSEM which revealed much smaller grains, finer and more homogenous carbide particles formed in SA508 Gr.4N steel. Compared with the SA508 Gr.3 steel currently used in the nuclear industry, the SA508 Gr.4N steel possesses higher strength and notch toughness as well as improved cleavage fracture behavior, and is considered a better candidate RPV steel for the next generation nuclear reactors. - Highlights: • Critical cleavage fracture stress was calculated through experiments and FEM. • Effects of both grain and carbide particle sizes on σ F were discussed. • The SA508 Gr.4N steel is a better candidate for the next generation nuclear reactors

A set of simple cell processes is sufficient to model spiral cleavage.

Science.gov (United States)

Brun-Usan, Miguel; Marín-Riera, Miquel; Grande, Cristina; Truchado-Garcia, Marta; Salazar-Ciudad, Isaac

2017-01-01

During cleavage, different cellular processes cause the zygote to become partitioned into a set of cells with a specific spatial arrangement. These processes include the orientation of cell division according to: an animal-vegetal gradient; the main axis (Hertwig's rule) of the cell; and the contact areas between cells or the perpendicularity between consecutive cell divisions (Sachs' rule). Cell adhesion and cortical rotation have also been proposed to be involved in spiral cleavage. We use a computational model of cell and tissue biomechanics to account for the different existing hypotheses about how the specific spatial arrangement of cells in spiral cleavage arises during development. Cell polarization by an animal-vegetal gradient, a bias to perpendicularity between consecutive cell divisions (Sachs' rule), cortical rotation and cell adhesion, when combined, reproduce the spiral cleavage, whereas other combinations of processes cannot. Specifically, cortical rotation is necessary at the 8-cell stage to direct all micromeres in the same direction. By varying the relative strength of these processes, we reproduce the spatial arrangement of cells in the blastulae of seven different invertebrate species. © 2017. Published by The Company of Biologists Ltd.

DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme

NARCIS (Netherlands)

Zaremba, M.; Lyubchenko, Y.L.; Laurens, N.; van den Broek, B.; Wuite, G.J.L.; Siksnys, V.

2010-01-01

To cut DNA at their target sites, restriction enzymes assemble into different oligomeric structures. The Ecl18kI endonuclease in the crystal is arranged as a tetramer made of two dimers each bound to a DNA copy. However, free in solution Ecl18kI is a dimer. To find out whether the Ecl18kI dimer or

Functional Coupling of Duplex Translocation to DNA Cleavage in a Type I Restriction Enzyme

Czech Academy of Sciences Publication Activity Database

Cséfalvay, Eva; Lapkouski, Mikalai; Guzanová, Alena; Cséfalvay, Ladislav; Baikova, T.; Shevelev, Igor; Bialevich, V.; Shamayeva, Katerina; Janščák, Pavel; Kutá-Smatanová, Ivana; Panjikar, S.; Carey, J.; Weiserová, Marie; Ettrich, Rüdiger

2015-01-01

Roč. 10, č. 6 (2015), e0128700 E-ISSN 1932-6203 R&D Projects: GA ČR GAP207/12/2323; GA ČR GAP305/10/0281 Institutional support: RVO:67179843 ; RVO:61388971 ; RVO:68378050 Keywords : Escherichia-Coli * Endonuclease ecor1241 * HSDR subunit * RECBCD enzyme * proteins * genes * helicase * sequence * family * domain Subject RIV: CE - Biochemistry Impact factor: 3.057, year: 2015

Boron-doped diamond electrodes for the electrochemical oxidation and cleavage of peptides.

Science.gov (United States)

Roeser, Julien; Alting, Niels F A; Permentier, Hjalmar P; Bruins, Andries P; Bischoff, Rainer

2013-07-16

Electrochemical oxidation of peptides and proteins is traditionally performed on carbon-based electrodes. Adsorption caused by the affinity of hydrophobic and aromatic amino acids toward these surfaces leads to electrode fouling. We compared the performance of boron-doped diamond (BDD) and glassy carbon (GC) electrodes for the electrochemical oxidation and cleavage of peptides. An optimal working potential of 2000 mV was chosen to ensure oxidation of peptides on BDD by electron transfer processes only. Oxidation by electrogenerated OH radicals took place above 2500 mV on BDD, which is undesirable if cleavage of a peptide is to be achieved. BDD showed improved cleavage yield and reduced adsorption for a set of small peptides, some of which had been previously shown to undergo electrochemical cleavage C-terminal to tyrosine (Tyr) and tryptophan (Trp) on porous carbon electrodes. Repeated oxidation with BDD electrodes resulted in progressively lower conversion yields due to a change in surface termination. Cathodic pretreatment of BDD at a negative potential in an acidic environment successfully regenerated the electrode surface and allowed for repeatable reactions over extended periods of time. BDD electrodes are a promising alternative to GC electrodes in terms of reduced adsorption and fouling and the possibility to regenerate them for consistent high-yield electrochemical cleavage of peptides. The fact that OH-radicals can be produced by anodic oxidation of water at elevated positive potentials is an additional advantage as they allow another set of oxidative reactions in analogy to the Fenton reaction, thus widening the scope of electrochemistry in protein and peptide chemistry and analytics.

Mine Water Treatment in Hongai Coal Mines

Directory of Open Access Journals (Sweden)

Dang Phuong Thao

2018-01-01

Full Text Available Acid mine drainage (AMD is recognized as one of the most serious environmental problem associated with mining industry. Acid water, also known as acid mine drainage forms when iron sulfide minerals found in the rock of coal seams are exposed to oxidizing conditions in coal mining. Until 2009, mine drainage in Hongai coal mines was not treated, leading to harmful effects on humans, animals and aquatic ecosystem. This report has examined acid mine drainage problem and techniques for acid mine drainage treatment in Hongai coal mines. In addition, selection and criteria for the design of the treatment systems have been presented.

Complex group-I introns in nuclear SSU rDNA of red and green algae: evidence of homing-endonuclease pseudogenes in the Bangiophyceae

DEFF Research Database (Denmark)

Haugen, P; Huss, V A; Nielsen, Henrik

1999-01-01

on the complementary strand. A comparison between related group-I introns in the Bangiophyceae revealed homing-endonuclease-like pseudogenes due to frame-shifts and deletions in Porphyra and Bangia. The Scenedesmus and Porphyra introns provide new insights into the evolution and possible novel functions of nuclear...

Cleavage mechanoluminescence in elemental and III-V semiconductors

International Nuclear Information System (INIS)

Chandra, B.P.; Patel, R.P.; Gour, Anubha S.; Chandra, V.K.; Gupta, R.K.

2003-01-01

The present paper reports the theory of mechanoluminescence (ML) produced during cleavage of elemental and III-V semiconductors. It seems that the formation of crack-induced localized states is responsible for the ML excitation produced during the cleavage of elemental and III-V semiconductors. According to this mechanism, as the atoms are drawn away from each other in an advancing crack tip, the decreasing wave function overlap across the crack may result in localized states which is associated with increasing electron energy. If the energy of these localized states approach that of the conduction band, transition to the conduction band via tunnelling would be possible, creating minority carriers, and consequently the electron-hole recombination may give rise to mechanoluminescence. When an elemental or III-V semiconductor is cleaved, initially the ML intensity increases with time, attains a peak value I m at the time t m corresponding to completion of the cleavage of the semiconductor, and then it decreases following power law decay. Expressions are derived for the ML intensity I m corresponding to the peak of the ML intensity versus time curve and for the total ML intensity I T . It is shown that both I m and I T should increase directly with the area of the newly created surfaces of the crystals. From the measurements of the ML intensity, the velocity of crack propagation in material can be determined by using the relation v=H/t m

Implementation of a combinatorial cleavage and deprotection scheme

DEFF Research Database (Denmark)

Nielsen, John; Rasmussen, Palle H.

1996-01-01

Phthalhydrazide libraries are synthesized in solution from substituted hydrazines and phthalimides in several different library formats including single compounds, indexed sub-libraries and a full library. When carried out during solid-phase synthesis, this combinatorial cleavage and deprotection...

Mutation in Spike Protein Cleavage Site and Pathogenesis of Feline Coronavirus

Science.gov (United States)

Licitra, Beth N.; Millet, Jean K.; Regan, Andrew D.; Hamilton, Brian S.; Rinaldi, Vera D.; Duhamel, Gerald E.

2013-01-01

Feline coronaviruses (FCoV) exist as 2 biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV causes subclinical infections; FIPV causes feline infectious peritonitis (FIP), a systemic and fatal disease. It is thought that mutations in FECV enable infection of macrophages, causing FIP. However, the molecular basis for this biotype switch is unknown. We examined a furin cleavage site in the region between receptor-binding (S1) and fusion (S2) domains of the spike of serotype 1 FCoV. FECV sequences were compared with FIPV sequences. All FECVs had a conserved furin cleavage motif. For FIPV, there was a correlation with the disease and >1 substitution in the S1/S2 motif. Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage. We document a functionally relevant S1/S2 mutation that arises when FIP develops in a cat. These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses. PMID:23763835

Trichomonas vaginalis Metalloproteinase Induces mTOR Cleavage of SiHa Cells

Science.gov (United States)

Quan, Juan-Hua; Choi, In-Wook; Yang, Jung-Bo; Zhou, Wei; Cha, Guang-Ho; Zhou, Yu; Ryu, Jae-Sook

2014-01-01

Trichomonas vaginalis secretes a number of proteases which are suspected to be the cause of pathogenesis; however, little is understood how they manipulate host cells. The mammalian target of rapamycin (mTOR) regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. We detected various types of metalloproteinases including GP63 protein from T. vaginalis trophozoites, and T. vaginalis GP63 metalloproteinase was confirmed by sequencing and western blot. When SiHa cells were stimulated with live T. vaginalis, T. vaginalis excretory-secretory products (ESP) or T. vaginalis lysate, live T. vaginalis and T. vaginalis ESP induced the mTOR cleavage in both time- and parasite load-dependent manner, but T. vaginalis lysate did not. Pretreatment of T. vaginalis with a metalloproteinase inhibitor, 1,10-phenanthroline, completely disappeared the mTOR cleavage in SiHa cells. Collectively, T. vaginalis metallopeptidase induces host cell mTOR cleavage, which may be related to survival of the parasite. PMID:25548410

Condensed tannins: A novel rearrangement of procyanidins and prodelphinidins in thiolytic cleavage

Science.gov (United States)

G. Wayne McGraw; Jan P. Steynberg; Richard W. Hemingway

1993-01-01

Conditions commonly used for the thiolytic cleavage of interflavanoid bonds of condensed tannins also result in cleavage of the C4 to C10 bond of flavan units. Subsequenet lectrophilic attack of the C4 carbocation on the C2' or C6' of the B-ring, and loss of phloroglucinol (the A-ring), result in the formation of a mixture of 1,3-dithiobenzyl-2,4,s,6-...

Yeast ribonuclease III uses a network of multiple hydrogen bonds for RNA binding and cleavage.

Science.gov (United States)

Lavoie, Mathieu; Abou Elela, Sherif

2008-08-19

Members of the bacterial RNase III family recognize a variety of short structured RNAs with few common features. It is not clear how this group of enzymes supports high cleavage fidelity while maintaining a broad base of substrates. Here we show that the yeast orthologue of RNase III (Rnt1p) uses a network of 2'-OH-dependent interactions to recognize substrates with different structures. We designed a series of bipartite substrates permitting the distinction between binding and cleavage defects. Each substrate was engineered to carry a single or multiple 2'- O-methyl or 2'-fluoro ribonucleotide substitutions to prevent the formation of hydrogen bonds with a specific nucleotide or group of nucleotides. Interestingly, introduction of 2'- O-methyl ribonucleotides near the cleavage site increased the rate of catalysis, indicating that 2'-OH are not required for cleavage. Substitution of nucleotides in known Rnt1p binding site with 2'- O-methyl ribonucleotides inhibited cleavage while single 2'-fluoro ribonucleotide substitutions did not. This indicates that while no single 2'-OH is essential for Rnt1p cleavage, small changes in the substrate structure are not tolerated. Strikingly, several nucleotide substitutions greatly increased the substrate dissociation constant with little or no effect on the Michaelis-Menten constant or rate of catalysis. Together, the results indicate that Rnt1p uses a network of nucleotide interactions to identify its substrate and support two distinct modes of binding. One mode is primarily mediated by the dsRNA binding domain and leads to the formation of stable RNA/protein complex, while the other requires the presence of the nuclease and N-terminal domains and leads to RNA cleavage.

Apoptotic DNA Degradation into Oligonucleosomal Fragments, but Not Apoptotic Nuclear Morphology, Relies on a Cytosolic Pool of DFF40/CAD Endonuclease*

Science.gov (United States)

Iglesias-Guimarais, Victoria; Gil-Guiñon, Estel; Gabernet, Gisela; García-Belinchón, Mercè; Sánchez-Osuna, María; Casanelles, Elisenda; Comella, Joan X.; Yuste, Victor J.

2012-01-01

Apoptotic cell death is characterized by nuclear fragmentation and oligonucleosomal DNA degradation, mediated by the caspase-dependent specific activation of DFF40/CAD endonuclease. Here, we describe how, upon apoptotic stimuli, SK-N-AS human neuroblastoma-derived cells show apoptotic nuclear morphology without displaying concomitant internucleosomal DNA fragmentation. Cytotoxicity afforded after staurosporine treatment is comparable with that obtained in SH-SY5Y cells, which exhibit a complete apoptotic phenotype. SK-N-AS cell death is a caspase-dependent process that can be impaired by the pan-caspase inhibitor q-VD-OPh. The endogenous inhibitor of DFF40/CAD, ICAD, is correctly processed, and dff40/cad cDNA sequence does not reveal mutations altering its amino acid composition. Biochemical approaches show that both SH-SY5Y and SK-N-AS resting cells express comparable levels of DFF40/CAD. However, the endonuclease is poorly expressed in the cytosolic fraction of healthy SK-N-AS cells. Despite this differential subcellular distribution of DFF40/CAD, we find no differences in the subcellular localization of both pro-caspase-3 and ICAD between the analyzed cell lines. After staurosporine treatment, the preferential processing of ICAD in the cytosolic fraction allows the translocation of DFF40/CAD from this fraction to a chromatin-enriched one. Therefore, the low levels of cytosolic DFF40/CAD detected in SK-N-AS cells determine the absence of DNA laddering after staurosporine treatment. In these cells DFF40/CAD cytosolic levels can be restored by the overexpression of their own endonuclease, which is sufficient to make them proficient at degrading their chromatin into oligonucleosome-size fragments after staurosporine treatment. Altogether, the cytosolic levels of DFF40/CAD are determinants in achieving a complete apoptotic phenotype, including oligonucleosomal DNA degradation. PMID:22253444

Hierarchical recruitment of ribosomal proteins and assembly factors remodels nucleolar pre-60S ribosomes.

Science.gov (United States)

Biedka, Stephanie; Micic, Jelena; Wilson, Daniel; Brown, Hailey; Diorio-Toth, Luke; Woolford, John L

2018-04-24

Ribosome biogenesis involves numerous preribosomal RNA (pre-rRNA) processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Removal of the internal transcribed spacer 2 spacer RNA is the final step in large subunit pre-rRNA processing and begins with endonucleolytic cleavage at the C 2 site of 27SB pre-rRNA. C 2 cleavage requires the hierarchical recruitment of 11 ribosomal proteins and 14 ribosome assembly factors. However, the function of these proteins in C 2 cleavage remained unclear. In this study, we have performed a detailed analysis of the effects of depleting proteins required for C 2 cleavage and interpreted these results using cryo-electron microscopy structures of assembling 60S subunits. This work revealed that these proteins are required for remodeling of several neighborhoods, including two major functional centers of the 60S subunit, suggesting that these remodeling events form a checkpoint leading to C 2 cleavage. Interestingly, when C 2 cleavage is directly blocked by depleting or inactivating the C 2 endonuclease, assembly progresses through all other subsequent steps. © 2018 Biedka et al.

Functional Insights Revealed by the Kinetic Mechanism of CRISPR/Cas9.

Science.gov (United States)

Raper, Austin T; Stephenson, Anthony A; Suo, Zucai

2018-02-28

The discovery of prokaryotic adaptive immunity prompted widespread use of the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) endonuclease Cas9 for genetic engineering. However, its kinetic mechanism remains undefined, and details of DNA cleavage are poorly characterized. Here, we establish a kinetic mechanism of Streptococcus pyogenes Cas9 from guide-RNA binding through DNA cleavage and product release. Association of DNA to the binary complex of Cas9 and guide-RNA is rate-limiting during the first catalytic turnover, while DNA cleavage from a pre-formed ternary complex of Cas9, guide-RNA, and DNA is rapid. Moreover, an extremely slow release of DNA products essentially restricts Cas9 to be a single-turnover enzyme. By simultaneously measuring the contributions of the HNH and RuvC nuclease activities of Cas9 to DNA cleavage, we also uncovered the kinetic basis by which HNH conformationally regulates the RuvC cleavage activity. Together, our results provide crucial kinetic and functional details regarding Cas9 which will inform gene-editing experiments, guide future research to understand off-target DNA cleavage by Cas9, and aid in the continued development of Cas9 as a biotechnological tool.

International mining forum 2004, new technologies in underground mining, safety in mines proceedings

Energy Technology Data Exchange (ETDEWEB)

Jerzy Kicki; Eugeniusz Sobczyk (eds.)

2004-01-15

The book comprises technical papers that were presented at the International Mining Forum 2004. This event aims to bring together scientists and engineers in mining, rock mechanics, and computer engineering, with a view to explore and discuss international developments in the field. Topics discussed in this book are: trends in the mining industry; new solutions and tendencies in underground mines; rock engineering problems in underground mines; utilization and exploitation of methane; prevention measures for the control of rock bursts in Polish mines; and current problems in Ukrainian coal mines.

Polycystin-1 C-terminal Cleavage Is Modulated by Polycystin-2 Expression*

Science.gov (United States)

Bertuccio, Claudia A.; Chapin, Hannah C.; Cai, Yiqiang; Mistry, Kavita; Chauvet, Veronique; Somlo, Stefan; Caplan, Michael J.

2009-01-01

Autosomal dominant polycystic kidney disease is caused by mutations in the genes encoding polycystin-1 (PC-1) and polycystin-2 (PC-2). PC-1 cleavage releases its cytoplasmic C-terminal tail (CTT), which enters the nucleus. To determine whether PC-1 CTT cleavage is influenced by PC-2, a quantitative cleavage assay was utilized, in which the DNA binding and activation domains of Gal4 and VP16, respectively, were appended to PC-1 downstream of its CTT domain (PKDgalvp). Cells cotransfected with the resultant PKDgalvp fusion protein and PC-2 showed an increase in luciferase activity and in CTT expression, indicating that the C-terminal tail of PC-1 is cleaved and enters the nucleus. To assess whether CTT cleavage depends upon Ca2+ signaling, cells transfected with PKDgalvp alone or together with PC-2 were incubated with several agents that alter intracellular Ca2+ concentrations. PC-2 enhancement of luciferase activity was not altered by any of these treatments. Using a series of PC-2 C-terminal truncated mutations, we identified a portion of the PC-2 protein that is required to stimulate PC-1 CTT accumulation. These data demonstrate that release of the CTT from PC-1 is influenced and stabilized by PC-2. This effect is independent of Ca2+ but is regulated by sequences contained within the PC-2 C-terminal tail, suggesting a mechanism through which PC-1 and PC-2 may modulate a novel signaling pathway. PMID:19491093

Control of extracellular cleavage of ProBDNF by high frequency neuronal activity

OpenAIRE

Nagappan, Guhan; Zaitsev, Eugene; Senatorov, Vladimir V.; Yang, Jianmin; Hempstead, Barbara L.; Lu, Bai

2009-01-01

Pro- and mature neurotrophins often elicit opposing biological effects. For example, mature brain-derived neurotrophic factor (mBDNF) is critical for long-term potentiation induced by high-frequency stimulation, whereas proBDNF facilitate long-term depression induced by low-frequency stimulation. Because mBDNF is derived from proBDNF by endoproteolytic cleavage, mechanisms regulating the cleavage of proBDNF may control the direction of BDNF regulation. Using methods that selectively detect pr...

Germline excision of transgenes in Aedes aegypti by homing endonucleases.

Science.gov (United States)

Aryan, Azadeh; Anderson, Michelle A E; Myles, Kevin M; Adelman, Zach N

2013-01-01

Aedes (Ae.) aegypti is the primary vector for dengue viruses (serotypes1-4) and chikungunya virus. Homing endonucleases (HEs) are ancient selfish elements that catalyze double-stranded DNA breaks (DSB) in a highly specific manner. In this report, we show that the HEs Y2-I-AniI, I-CreI and I-SceI are all capable of catalyzing the excision of genomic segments from the Ae. aegypti genome in a heritable manner. Y2-I-AniI demonstrated the highest efficiency at two independent genomic targets, with 20-40% of Y2-I-AniI-treated individuals producing offspring that had lost the target transgene. HE-induced DSBs were found to be repaired via the single-strand annealing (SSA) and non-homologous end-joining (NHEJ) pathways in a manner dependent on the availability of direct repeat sequences in the transgene. These results support the development of HE-based gene editing and gene drive strategies in Ae. aegypti, and confirm the utility of HEs in the manipulation and modification of transgenes in this important vector.

Effects of Dimerization of Serratia marcescens Endonuclease on Water Dynamics.

Energy Technology Data Exchange (ETDEWEB)

Chen, Chuanying; Beck, Brian W.; Krause, Kurt; Weksberg, Tiffany E.; Pettitt, Bernard M.

2007-02-15

The research described in this product was performed in part in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. The dynamics and structure of Serratia marcescens endonuclease and its neighboring solvent are investigated by molecular dynamics (MD). Comparisons are made with structural and biochemical experiments. The dimer form is physiologic and functions more processively than the monomer. We previously found a channel formed by connected clusters of waters from the active site to the dimer interface. Here, we show that dimerization clearly changes correlations in the water structure and dynamics in the active site not seen in the monomer. Our results indicate that water at the active sites of the dimer is less affected compared with bulk solvent than in the monomer where it has much slower characteristic relaxation times. Given that water is a required participant in the reaction, this gives a clear advantage to dimerization in the absence of an apparent ability to use both active sites simultaneously.

Computational study of hydration at the TD damaged site of DNA in complex with repair enzyme T4 endonuclease V

International Nuclear Information System (INIS)

Pinak, Miroslav

2000-02-01

An analysis of the distribution of water around DNA surface focusing on the role of the distribution of water molecules in the proper recognition of damaged site by repair enzyme T4 Endonuclease V was performed. The native DNA dodecamer, dodecamer with the thymine dimer (TD) and complex of DNA and part of repair enzyme T4 Endonuclease V were examined throughout the 500 ps of molecular dynamics simulation. During simulation the number of water molecules close to the DNA atoms and the residence time were calculated. There is an increase in number of water molecules lying in the close vicinity to TD if compared with those lying close to two native thymines (TT). Densely populated area with water molecules around TD is one of the factors detected by enzyme during scanning process. The residence time was found higher for molecule of the complex and the six water molecules were found occupying the stabile positions between the TD and catalytic center close to atoms P, C3' and N3. These molecules originate water mediated hydrogen bond network that contribute to the stability of complex required for the onset of repair process. (author)

Computational study of hydration at the TD damaged site of DNA in complex with repair enzyme T4 endonuclease V

Energy Technology Data Exchange (ETDEWEB)

Pinak, Miroslav [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

2000-02-01

An analysis of the distribution of water around DNA surface focusing on the role of the distribution of water molecules in the proper recognition of damaged site by repair enzyme T4 Endonuclease V was performed. The native DNA dodecamer, dodecamer with the thymine dimer (TD) and complex of DNA and part of repair enzyme T4 Endonuclease V were examined throughout the 500 ps of molecular dynamics simulation. During simulation the number of water molecules close to the DNA atoms and the residence time were calculated. There is an increase in number of water molecules lying in the close vicinity to TD if compared with those lying close to two native thymines (TT). Densely populated area with water molecules around TD is one of the factors detected by enzyme during scanning process. The residence time was found higher for molecule of the complex and the six water molecules were found occupying the stabile positions between the TD and catalytic center close to atoms P, C3' and N3. These molecules originate water mediated hydrogen bond network that contribute to the stability of complex required for the onset of repair process. (author)

Sustainable Mining Environment: Technical Review of Post-mining Plans

Directory of Open Access Journals (Sweden)

Restu Juniah

2017-12-01

Full Text Available The mining industry exists because humans need mining commodities to meet their daily needs such as motor vehicles, mobile phones, electronic equipment and others. Mining commodities as mentioned in Government Regulation No. 23 of 2010 on Implementation of Mineral and Coal Mining Business Activities are radioactive minerals, metal minerals, nonmetallic minerals, rocks and coal. Mineral and coal mining is conducted to obtain the mining commodities through production operations. Mining and coal mining companies have an obligation to ensure that the mining environment in particular after the post production operation or post mining continues. The survey research aims to examine technically the post-mining plan in coal mining of PT Samantaka Batubara in Indragiri Hulu Regency of Riau Province towards the sustainability of the mining environment. The results indicate that the post-mining plan of PT Samantaka Batubara has met the technical aspects required in post mining planning for a sustainable mining environment. Postponement of post-mining land of PT Samantaka Batubara for garden and forest zone. The results of this study are expected to be useful and can be used by stakeholders, academics, researchers, practitioners and associations of mining, and the environment.

Stimulation of NADH-dependent microsomal DNA strand cleavage by rifamycin SV.

Science.gov (United States)

Kukiełka, E; Cederbaum, A I

1995-04-15

Rifamycin SV is an antibiotic anti-bacterial agent used in the treatment of tuberculosis. This drug can autoxidize, especially in the presence of metals, and generate reactive oxygen species. A previous study indicated that rifamycin SV can increase NADH-dependent microsomal production of reactive oxygen species. The current study evaluated the ability of rifamycin SV to interact with iron and increase microsomal production of hydroxyl radical, as detected by conversion of supercoiled plasmid DNA into the relaxed open circular state. The plasmid used was pBluescript II KS(-), and the forms of DNA were separated by agarose-gel electrophoresis. Incubation of rat liver microsomes with plasmid plus NADH plus ferric-ATP caused DNA strand cleavage. The addition of rifamycin SV produced a time- and concentration-dependent increase in DNA-strand cleavage. No stimulation by rifamycin SV occurred in the absence of microsomes, NADH or ferric-ATP. Stimulation occurred with other ferric complexes besides ferric-ATP, e.g. ferric-histidine, ferric-citrate, ferric-EDTA, and ferric-(NH4)2SO4. Rifamycin SV did not significantly increase the high rates of DNA strand cleavage found with NADPH as the microsomal reductant. The stimulation of NADH-dependent microsomal DNA strand cleavage was completely blocked by catalase, superoxide dismutase, GSH and a variety of hydroxyl-radical-scavenging agents, but not by anti-oxidants that prevent microsomal lipid peroxidation. Redox cycling agents, such as menadione and paraquat, in contrast with rifamycin SV, stimulated the NADPH-dependent reaction; menadione and rifamycin SV were superior to paraquat in stimulating the NADH-dependent reaction. These results indicate that rifamycin SV can, in the presence of an iron catalyst, increase microsomal production of reactive oxygen species which can cause DNA-strand cleavage. In contrast with other redox cycling agents, the stimulation by rifamycin SV is more pronounced with NADH than with NADPH as the

Contract Mining versus Owner Mining

African Journals Online (AJOL)

Owner

mining companies can concentrate on their core businesses while using specialists for ... 2 Definition of Contract and Owner. Mining ... equipment maintenance, scheduling and budgeting ..... No. Region. Amount Spent on. Contract Mining. ($ billion). Percent of. Total. 1 ... cost and productivity data based on a large range.

Optimization of mining design of Hongwei uranium mine

International Nuclear Information System (INIS)

Wu Sanmao; Yuan Baixiang

2012-01-01

Combined with the mining conditions of Hongwei uranium mine, optimization schemes for hoisting cage, mine drainge,ore transport, mine wastewater treatment, power-supply system,etc are put forward in the mining design of the mine. Optimized effects are analyzed from the aspects of technique, economy, and energy saving and reducing emissions. (authors)

Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage

Science.gov (United States)

Adam Smith, R; Sewell, Sarah L; Giorgio, Todd D

2008-01-01

The development and in vitro performance of a modular nanoscale system capable of specific structural modification by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fluorescent species including a quantum dot base particle and fluorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fluorometric analysis. The model proximity-activated nanoparticles studied here exhibit significant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7) at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specific to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specific modification is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure. PMID:18488420

PROSPER: an integrated feature-based tool for predicting protease substrate cleavage sites.

Directory of Open Access Journals (Sweden)

Jiangning Song

Full Text Available The ability to catalytically cleave protein substrates after synthesis is fundamental for all forms of life. Accordingly, site-specific proteolysis is one of the most important post-translational modifications. The key to understanding the physiological role of a protease is to identify its natural substrate(s. Knowledge of the substrate specificity of a protease can dramatically improve our ability to predict its target protein substrates, but this information must be utilized in an effective manner in order to efficiently identify protein substrates by in silico approaches. To address this problem, we present PROSPER, an integrated feature-based server for in silico identification of protease substrates and their cleavage sites for twenty-four different proteases. PROSPER utilizes established specificity information for these proteases (derived from the MEROPS database with a machine learning approach to predict protease cleavage sites by using different, but complementary sequence and structure characteristics. Features used by PROSPER include local amino acid sequence profile, predicted secondary structure, solvent accessibility and predicted native disorder. Thus, for proteases with known amino acid specificity, PROSPER provides a convenient, pre-prepared tool for use in identifying protein substrates for the enzymes. Systematic prediction analysis for the twenty-four proteases thus far included in the database revealed that the features we have included in the tool strongly improve performance in terms of cleavage site prediction, as evidenced by their contribution to performance improvement in terms of identifying known cleavage sites in substrates for these enzymes. In comparison with two state-of-the-art prediction tools, PoPS and SitePrediction, PROSPER achieves greater accuracy and coverage. To our knowledge, PROSPER is the first comprehensive server capable of predicting cleavage sites of multiple proteases within a single substrate

Fe-Catalyzed Oxidative Cleavage of Unsaturated Fatty Acids

NARCIS (Netherlands)

Spannring, P.

2013-01-01

The oxidative cleavage of unsaturated fatty acids into aldehydes or carboxylic acids gives access to valuable products. The products can be used as chemical building blocks, as emulsifiers or in the paint or polymer industry. Ozonolysis is applied industrially to cleave the fatty acid oleic acid

Mechanisms of DNA Packaging by Large Double-Stranded DNA Viruses

Science.gov (United States)

Rao, Venigalla B.; Feiss, Michael

2016-01-01

Translocation of viral double-stranded DNA (dsDNA) into the icosahedral prohead shell is catalyzed by TerL, a motor protein that has ATPase, endonuclease, and translocase activities. TerL, following endonucleolytic cleavage of immature viral DNA concatemer recognized by TerS, assembles into a pentameric ring motor on the prohead’s portal vertex and uses ATP hydrolysis energy for DNA translocation. TerL’s N-terminal ATPase is connected by a hinge to the C-terminal endonuclease. Inchworm models propose that modest domain motions accompanying ATP hydrolysis are amplified, through changes in electrostatic interactions, into larger movements of the C-terminal domain bound to DNA. In phage φ29, four of the five TerL subunits sequentially hydrolyze ATP, each powering translocation of 2.5 bp. After one viral genome is encapsidated, the internal pressure signals termination of packaging and ejection of the motor. Current focus is on the structures of packaging complexes and the dynamics of TerL during DNA packaging, endonuclease regulation, and motor mechanics. PMID:26958920

Small-angle X-ray scattering analysis reveals the ATP-bound monomeric state of the ATPase domain from the homodimeric MutL endonuclease, a GHKL phosphotransferase superfamily protein.

Science.gov (United States)

Iino, Hitoshi; Hikima, Takaaki; Nishida, Yuya; Yamamoto, Masaki; Kuramitsu, Seiki; Fukui, Kenji

2015-05-01

DNA mismatch repair is an excision system that removes mismatched bases chiefly generated by replication errors. In this system, MutL endonucleases direct the excision reaction to the error-containing strand of the duplex by specifically incising the newly synthesized strand. Both bacterial homodimeric and eukaryotic heterodimeric MutL proteins belong to the GHKL ATPase/kinase superfamily that comprises the N-terminal ATPase and C-terminal dimerization regions. Generally, the GHKL proteins show large ATPase cycle-dependent conformational changes, including dimerization-coupled ATP binding of the N-terminal domain. Interestingly, the ATPase domain of human PMS2, a subunit of the MutL heterodimer, binds ATP without dimerization. The monomeric ATP-bound state of the domain has been thought to be characteristic of heterodimeric GHKL proteins. In this study, we characterized the ATP-bound state of the ATPase domain from the Aquifex aeolicus MutL endonuclease, which is a homodimeric GHKL protein unlike the eukaryotic MutL. Gel filtration, dynamic light scattering, and small-angle X-ray scattering analyses clearly showed that the domain binds ATP in a monomeric form despite its homodimeric nature. This indicates that the uncoupling of dimerization and ATP binding is a common feature among bacterial and eukaryotic MutL endonucleases, which we suggest is closely related to the molecular mechanisms underlying mismatch repair.

Signal peptide discrimination and cleavage site identification using SVM and NN.

Science.gov (United States)

Kazemian, H B; Yusuf, S A; White, K

2014-02-01

About 15% of all proteins in a genome contain a signal peptide (SP) sequence, at the N-terminus, that targets the protein to intracellular secretory pathways. Once the protein is targeted correctly in the cell, the SP is cleaved, releasing the mature protein. Accurate prediction of the presence of these short amino-acid SP chains is crucial for modelling the topology of membrane proteins, since SP sequences can be confused with transmembrane domains due to similar composition of hydrophobic amino acids. This paper presents a cascaded Support Vector Machine (SVM)-Neural Network (NN) classification methodology for SP discrimination and cleavage site identification. The proposed method utilises a dual phase classification approach using SVM as a primary classifier to discriminate SP sequences from Non-SP. The methodology further employs NNs to predict the most suitable cleavage site candidates. In phase one, a SVM classification utilises hydrophobic propensities as a primary feature vector extraction using symmetric sliding window amino-acid sequence analysis for discrimination of SP and Non-SP. In phase two, a NN classification uses asymmetric sliding window sequence analysis for prediction of cleavage site identification. The proposed SVM-NN method was tested using Uni-Prot non-redundant datasets of eukaryotic and prokaryotic proteins with SP and Non-SP N-termini. Computer simulation results demonstrate an overall accuracy of 0.90 for SP and Non-SP discrimination based on Matthews Correlation Coefficient (MCC) tests using SVM. For SP cleavage site prediction, the overall accuracy is 91.5% based on cross-validation tests using the novel SVM-NN model. © 2013 Published by Elsevier Ltd.

Surface Mines, Other - Longwall Mining Panels

Data.gov (United States)

NSGIC Education | GIS Inventory — Coal mining has occurred in Pennsylvania for over a century. A method of coal mining known as Longwall Mining has become more prevalent in recent decades. Longwall...

DNA Cleavage Activity of Diazonium Salts: Chemical Nucleases

OpenAIRE

KIZIL, Murat

2014-01-01

4-Fenoldiazonium tetrafluoroborate and 4-benzoicaciddiazonium tetrafluoroborate was prepared and was shown to be an effective DNA cleavage agent in the presence of the 1-electron donor copper(II) chloride. Its mechanism involves the generation of the aryl radical cleaving DNA by hydrogen atom abstraction from deoxyribose sugar.

Autoactivation of mouse trypsinogens is regulated by chymotrypsin C via cleavage of the autolysis loop.

Science.gov (United States)

Németh, Balázs Csaba; Wartmann, Thomas; Halangk, Walter; Sahin-Tóth, Miklós

2013-08-16

Chymotrypsin C (CTRC) is a proteolytic regulator of trypsinogen autoactivation in humans. CTRC cleavage of the trypsinogen activation peptide stimulates autoactivation, whereas cleavage of the calcium binding loop promotes trypsinogen degradation. Trypsinogen mutations that alter these regulatory cleavages lead to increased intrapancreatic trypsinogen activation and cause hereditary pancreatitis. The aim of this study was to characterize the regulation of autoactivation of mouse trypsinogens by mouse Ctrc. We found that the mouse pancreas expresses four trypsinogen isoforms to high levels, T7, T8, T9, and T20. Only the T7 activation peptide was cleaved by mouse Ctrc, causing negligible stimulation of autoactivation. Surprisingly, mouse Ctrc poorly cleaved the calcium binding loop in all mouse trypsinogens. In contrast, mouse Ctrc readily cleaved the Phe-150-Gly-151 peptide bond in the autolysis loop of T8 and T9 and inhibited autoactivation. Mouse chymotrypsin B also cleaved the same peptide bond but was 7-fold slower. T7 was less sensitive to chymotryptic regulation, which involved slow cleavage of the Leu-149-Ser-150 peptide bond in the autolysis loop. Modeling indicated steric proximity of the autolysis loop and the activation peptide in trypsinogen, suggesting the cleaved autolysis loop may directly interfere with activation. We conclude that autoactivation of mouse trypsinogens is under the control of mouse Ctrc with some notable differences from the human situation. Thus, cleavage of the trypsinogen activation peptide or the calcium binding loop by Ctrc is unimportant. Instead, inhibition of autoactivation via cleavage of the autolysis loop is the dominant mechanism that can mitigate intrapancreatic trypsinogen activation.

Analysis by the reductive-cleavage method of linkage positions in a polysaccharide containing 4-linked D-glucopyranosyluronic residues.

Science.gov (United States)

Vodonik, S A; Gray, G R

1988-04-01

The fate of 4-linked D-glucopyranosyluronic residues under reductive-cleavage conditions was investigated by using the Klebsiella aerogenes type 54 strain A3 capsular polysaccharide. Treatment of the fully methylated polysaccharide with triethylsilane and trimethylsilyl trifluoromethanesulfonate in dichloromethane, followed by in situ acetylation, yielded 1,5-anhydro-2,3,4,6-tetra-O-methyl-D-glucitol, 3,4-di-O-acetyl-1,5-anhydro-2,6-di-O-methyl-D-glucitol, and 3-O-acetyl-1,5-anhydro-2,4-di-O-methyl-L-fucitol, as expected, but the expected product of reductive cleavage of the 4-linked D-glucopyranosyluronic residue, namely, methyl 3-O-acetyl-2,6-anhydro-4,5-di-O-methyl-L-gulonate, was not observed. Instead, methyl 2-O-acetyl-3,6-anhydro-4,5-di-O-methyl-L-gulonate (6) was identified as the sole product of reductive cleavage of the 4-linked D-glucopyranosyluronic residue. That compound 6 arose as a result of rearrangement during reductive cleavage rather than by reductive cleavage of a 5-linked D-glucofuranosyluronic residue, was established by reductive cleavage of the fully methylated polysaccharide following reduction of its ester groups with either lithium aluminum hydride or lithium aluminum deuteride. The products of the latter reductive cleavage were the same as before, except for the absence of 6 and the presence of 4,6-di-O-acetyl-1,5-anhydro-2,3-di-O-methyl-D-glucitol, or its 6,6-dideuterio isomer. Although the reductive-cleavage technique is suitable for the direct analysis of polysaccharides containing 4-linked D-glucopyranosyluronic residues, it does not establish whether the uronic residue is a 4-linked pyranoside or a 5-linked furanoside. The expected product is, however, derived from the 4-linked D-glucopyranosyluronic residue after sequential methylation, reduction of its ester group and reductive cleavage.

Application for trackless mining technique in Benxi uranium mine

International Nuclear Information System (INIS)

Chen Bingguo

1998-01-01

The author narrates the circumstances achieving constructional target in Benxi Uranium Mine under relying on advance of science and technology and adopting small trackless mining equipment, presents the application of trackless mining equipment at mining small mine and complex mineral deposit and discusses the unique superiority of trackless mining technique in development work, mining preparation work and backstoping

Mining engineer requirements in a German coal mine

Energy Technology Data Exchange (ETDEWEB)

Rauhut, F J

1985-10-01

Basic developments in German coal mines, new definitions of working areas of mining engineers, and groups of requirements in education are discussed. These groups include: requirements of hard-coal mining at great depth and in extended collieries; application of process technology and information systems in semi-automated mines; thinking in processes and systems; organizational changes; future requirements of mining engineers; responsibility of the mining engineer for employees and society.

Conservation of a proteinase cleavage site between an insect retrovirus (gypsy) Env protein and a baculovirus envelope fusion protein

International Nuclear Information System (INIS)

Pearson, Margot N.; Rohrmann, George F.

2004-01-01

The predicted Env protein of insect retroviruses (errantiviruses) is related to the envelope fusion protein of a major division of the Baculoviridae. The highest degree of homology is found in a region that contains a furin cleavage site in the baculovirus proteins and an adjacent sequence that has the properties of a fusion peptide. In this investigation, the homologous region in the Env protein of the gypsy retrovirus of Drosophila melanogaster (DmegypV) was investigated. Alteration of the predicted DmegypV Env proteinase cleavage site from RIAR to AIAR significantly reduced cleavage of Env in both Spodoptera frugiperda (Sf-9) and D. melanogaster (S2) cell lines. When the predicted DmegypV Env cleavage site RIAR was substituted for the cleavage sequence RRKR in the Lymantria dispar nucleopolyhedrovirus fusion protein (LD130) sequence, cleavage of the hybrid LD130 molecules still occurred, although at a reduced level. The conserved 21-amino acid sequence just downstream of the cleavage site, which is thought to be the fusion peptide in LD130, was also characterized. When this sequence from DmegypV Env was substituted for the homologous sequence in LD130, cleavage still occurred, but no fusion was observed in either cell type. In addition, although a DmegypV-Env-green fluorescent protein construct localized to cell membranes, no cell fusion was observed

Drosha regulates gene expression independently of RNA cleavage function

DEFF Research Database (Denmark)

Gromak, Natalia; Dienstbier, Martin; Macias, Sara

2013-01-01

Drosha is the main RNase III-like enzyme involved in the process of microRNA (miRNA) biogenesis in the nucleus. Using whole-genome ChIP-on-chip analysis, we demonstrate that, in addition to miRNA sequences, Drosha specifically binds promoter-proximal regions of many human genes in a transcription......-dependent manner. This binding is not associated with miRNA production or RNA cleavage. Drosha knockdown in HeLa cells downregulated nascent gene transcription, resulting in a reduction of polyadenylated mRNA produced from these gene regions. Furthermore, we show that this function of Drosha is dependent on its N......-terminal protein-interaction domain, which associates with the RNA-binding protein CBP80 and RNA Polymerase II. Consequently, we uncover a previously unsuspected RNA cleavage-independent function of Drosha in the regulation of human gene expression....

Polycystin-1 Cleavage and the Regulation of Transcriptional Pathways

Science.gov (United States)

Merrick, David; Bertuccio, Claudia A.; Chapin, Hannah C.; Lal, Mark; Chauvet, Veronique; Caplan, Michael J.

2013-01-01

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end stage renal disease, affecting ~1 in 1,000 people. The disease is characterized by the development of numerous large fluid filled renal cysts over the course of decades. These cysts compress the surrounding renal parenchyma and impair its function. Mutations in two genes are responsible for ADPKD. The protein products of both of these genes, polycystin-1 and polycystin-2, localize to the primary cilium and participate in a wide variety of signaling pathways. Polycystin-1 undergoes several proteolytic cleavages that produce fragments that manifest biological activities. Recent results suggest that the production of polycystin-1 cleavage fragments is necessary and sufficient to account for at least some, although certainly not all, of the physiological functions of the parent protein. PMID:23824180

Cell-surface acceleration of urokinase-catalyzed receptor cleavage

DEFF Research Database (Denmark)

Høyer-Hansen, G; Ploug, M; Behrendt, N

1997-01-01

by a prior incubation of the cells with uPA inactivated by diisopropyl fluorophosphate, demonstrating a requirement for specific receptor binding of the active uPA to obtain the high-efficiency cleavage of cell-bound uPAR. Furthermore, amino-terminal sequence analysis revealed that uPAR(2+3), purified from U...

Fetal hemoglobin is much less prone to DNA cleavage compared to the adult protein

Directory of Open Access Journals (Sweden)

Sandeep Chakane

2017-08-01

Full Text Available Hemoglobin (Hb is well protected inside the red blood cells (RBCs. Upon hemolysis and when free in circulation, Hb can be involved in a range of radical generating reactions and may thereby attack several different biomolecules. In this study, we have examined the potential damaging effects of cell-free Hb on plasmid DNA (pDNA. Hb induced cleavage of supercoiled pDNA (sc pDNA which was proportional to the concentration of Hb applied. Almost 70% of sc pDNA was converted to open circular or linear DNA using 10 µM of Hb in 12 h. Hb can be present in several different forms. The oxy (HbO2 and met forms are most reactive, while the carboxy-protein shows only low hydrolytic activity. Hemoglobin A (HbA could easily induce complete pDNA cleavage while fetal hemoglobin (HbF was three-fold less reactive. By inserting, a redox active cysteine residue on the surface of the alpha chain of HbF by site-directed mutagenesis, the DNA cleavage reaction was enhanced by 82%. Reactive oxygen species were not directly involved in the reaction since addition of superoxide dismutase and catalase did not prevent pDNA cleavage. The reactivity of Hb with pDNA can rather be associated with the formation of protein based radicals. Keywords: Adult hemoglobin, Fetal hemoglobin, Supercoiled plasmid DNA, DNA cleavage, Cysteine, Protein radicals

Abyssal fiction: common shares, colonial cleavages

Directory of Open Access Journals (Sweden)

Alexandre Montaury

2016-12-01

Full Text Available The paper aims to develop a reflection on the interaction between the legacies of colonialism and traditional symbolic and cultural practices in African Portuguese-speaking spaces. From a preliminary analysis of fictional texts of wide circulation in Brazil, aims to examine the cleavages, or “abyssal lines” that constitute experiences printed in the daily life of the former Portuguese colony of Cape Verde, Mozambique and Angola.---DOI: http://dx.doi.org/10.21881/abriluff.2016n17a378

Transcriptional down-regulation and rRNA cleavage in Dictyostelium discoideum mitochondria during Legionella pneumophila infection.

Directory of Open Access Journals (Sweden)

Chenyu Zhang

2009-05-01

Full Text Available Bacterial pathogens employ a variety of survival strategies when they invade eukaryotic cells. The amoeba Dictyostelium discoideum is used as a model host to study the pathogenic mechanisms that Legionella pneumophila, the causative agent of Legionnaire's disease, uses to kill eukaryotic cells. Here we show that the infection of D. discoideum by L. pneumophila results in a decrease in mitochondrial messenger RNAs, beginning more than 8 hours prior to detectable host cell death. These changes can be mimicked by hydrogen peroxide treatment, but not by other cytotoxic agents. The mitochondrial large subunit ribosomal RNA (LSU rRNA is also cleaved at three specific sites during the course of infection. Two LSU rRNA fragments appear first, followed by smaller fragments produced by additional cleavage events. The initial LSU rRNA cleavage site is predicted to be on the surface of the large subunit of the mitochondrial ribosome, while two secondary sites map to the predicted interface with the small subunit. No LSU rRNA cleavage was observed after exposure of D. discoideum to hydrogen peroxide, or other cytotoxic chemicals that kill cells in a variety of ways. Functional L. pneumophila type II and type IV secretion systems are required for the cleavage, establishing a correlation between the pathogenesis of L. pneumophila and D. discoideum LSU rRNA destruction. LSU rRNA cleavage was not observed in L. pneumophila infections of Acanthamoeba castellanii or human U937 cells, suggesting that L. pneumophila uses distinct mechanisms to interrupt metabolism in different hosts. Thus, L. pneumophila infection of D. discoideum results in dramatic decrease of mitochondrial RNAs, and in the specific cleavage of mitochondrial rRNA. The predicted location of the cleavage sites on the mitochondrial ribosome suggests that rRNA destruction is initiated by a specific sequence of events. These findings suggest that L. pneumophila specifically disrupts mitochondrial

Antiviral Goes Viral: Harnessing CRISPR/Cas9 to Combat Viruses in Humans.

Science.gov (United States)

Soppe, Jasper Adriaan; Lebbink, Robert Jan

2017-10-01

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are RNA-guided sequence-specific prokaryotic antiviral immune systems. In prokaryotes, small RNA molecules guide Cas effector endonucleases to invading foreign genetic elements in a sequence-dependent manner, resulting in DNA cleavage by the endonuclease upon target binding. A rewired CRISPR/Cas9 system can be used for targeted and precise genome editing in eukaryotic cells. CRISPR/Cas has also been harnessed to target human pathogenic viruses as a potential new antiviral strategy. Here, we review recent CRISPR/Cas9-based approaches to combat specific human viruses in humans and discuss challenges that need to be overcome before CRISPR/Cas9 may be used in the clinic as an antiviral strategy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mining Together : Large-Scale Mining Meets Artisanal Mining, A Guide for Action

OpenAIRE

World Bank

2009-01-01

The present guide mining together-when large-scale mining meets artisanal mining is an important step to better understanding the conflict dynamics and underlying issues between large-scale and small-scale mining. This guide for action not only points to some of the challenges that both parties need to deal with in order to build a more constructive relationship, but most importantly it sh...

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

KAUST Repository

Rashid, Fahad; Harris, Paul D.; Zaher, Manal S.; Sobhy, Mohamed Abdelmaboud; Joudeh, Luay I; Yan, Chunli; Piwonski, Hubert Marek; Tsutakawa, Susan E; Ivanov, Ivaylo; Tainer, John A; Habuchi, Satoshi; Hamdan, Samir

2017-01-01

that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually 'locks' protein and DNA conformation and enables substrate verification with extreme

Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

International Nuclear Information System (INIS)

Kennedy, Edward M.; Cullen, Bryan R.

2015-01-01

CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

Energy Technology Data Exchange (ETDEWEB)

Kennedy, Edward M.; Cullen, Bryan R., E-mail: bryan.cullen@duke.edu

2015-05-15

CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

Cleavage strain in the Variscan fold belt, County Cork, Ireland, estimated from stretched arsenopyrite rosettes

Science.gov (United States)

Ford, M.; Ferguson, C.C.

1985-01-01

In south-west Ireland, hydrothermally formed arsenopyrite crystals in a Devonian mudstone have responded to Variscan deformation by brittle extension fracture and fragment separation. The interfragment gaps and terminal extension zones of each crystal are infilled with fibrous quartz. Stretches within the cleavage plane have been calculated by the various methods available, most of which can be modified to incorporate terminal extension zones. The Strain Reversal Method is the most accurate currently available but still gives a minimum estimate of the overall strain. The more direct Hossain method, which gives only slightly lower estimates with this data, is more practical for field use. A strain ellipse can be estimated from each crystal rosette composed of three laths (assuming the original interlimb angles were all 60??) and, because actual rather than relative stretches are estimated, this provides a lower bound to the area increase in the plane of cleavage. Based on the average of our calculated strain ellipses this area increase is at least 114% and implies an average shortening across the cleavage of at least 53%. However, several lines of evidence suggest that the cleavage deformation was more intense and more oblate than that calculated, and we argue that a 300% area increase in the cleavage plane and 75% shortening across the cleavage are more realistic estimates of the true strain. Furthermore, the along-strike elongation indicated is at least 80%, which may be regionally significant. Estimates of orogenic contraction derived from balanced section construction should therefore take into account the possibility of a substantial strike elongation, and tectonic models that can accommodate such elongations need to be developed. ?? 1985.

The Helicobacter pylori HpyAXII restriction–modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components

Science.gov (United States)

Humbert, Olivier; Salama, Nina R.

2008-01-01

The naturally competent organism Helicobacter pylori encodes a large number of restriction–modification (R–M) systems that consist of a restriction endonuclease and a DNA methyltransferase. R–M systems are not only believed to limit DNA exchange among bacteria but may also have other cellular functions. We report a previously uncharacterized H. pylori type II R–M system, M.HpyAXII/R.HpyAXII. We show that this system targets GTAC sites, which are rare in the H. pylori chromosome but numerous in ribosomal RNA genes. As predicted, this type II R–M system showed attributes of a selfish element. Deletion of the methyltransferase M.HpyAXII is lethal when associated with an active endonuclease R.HpyAXII unless compensated by adaptive mutation or gene amplification. R.HpyAXII effectively restricted both unmethylated plasmid and chromosomal DNA during natural transformation and was predicted to belong to the novel ‘half pipe’ structural family of endonucleases. Analysis of a panel of clinical isolates revealed that R.HpyAXII was functional in a small number of H. pylori strains (18.9%, n = 37), whereas the activity of M.HpyAXII was highly conserved (92%, n = 50), suggesting that GTAC methylation confers a selective advantage to H. pylori. However, M.HpyAXII activity did not enhance H. pylori fitness during stomach colonization of a mouse infection model. PMID:18978016

Comparative studies of the endonucleases from two related Xenopus laevis retrotransposons, Tx1L and Tx2L: target site specificity and evolutionary implications.

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Christensen, S; Pont-Kingdon, G; Carroll, D

2000-01-01

In the genome of the South African frog, Xenopus laevis, there are two complex families of transposable elements, Tx1 and Tx2, that have identical overall structures, but distinct sequences. In each family there are approximately 1500 copies of an apparent DNA-based element (Tx1D and Tx2D). Roughly 10% of these elements in each family are interrupted by a non-LTR retrotransposon (Tx1L and Tx2L). Each retrotransposon is flanked by a 23-bp target duplication of a specific D element sequence. In earlier work, we showed that the endonuclease domain (Tx1L EN) located in the second open reading frame (ORF2) of Tx1L encodes a protein that makes a single-strand cut precisely at the expected site within its target sequence, supporting the idea that Tx1L is a site-specific retrotransposon. In this study, we express the endonuclease domain of Tx2L (Tx2L EN) and compare the target preferences of the two enzymes. Each endonuclease shows some preference for its cognate target, on the order of 5-fold over the non-cognate target. The observed discrimination is not sufficient, however, to explain the observation that no cross-occupancy is observed - that is, L elements of one family have never been found within D elements of the other family. Possible sources of additional specificity are discussed. We also compare two hypotheses regarding the genome duplication event that led to the contemporary pseudotetraploid character of Xenopus laevis in light of the Tx1L and Tx2L data.

Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs using PICS with proteome-derived peptide libraries.

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Olivier Barré

Full Text Available Type II transmembrane serine proteases (TTSPs are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS. Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin to simultaneously determine sequence preferences on the N-terminal non-prime (P and C-terminal prime (P' sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1' position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived.Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1' positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.

Change in radiosensitivity of sea-urchin eggs during early cleavage stages

International Nuclear Information System (INIS)

Nakamura, I.

1977-01-01

When sea-urchin eggs were irradiated with 137 Cs γ-rays, their radiosensitivity, expressed by the percentage which formed pluteus larvae, fluctuated during the early cleavage cycle. Split-dose irradiations were made both in the sensitive and resistant phases. For eggs in the sensitive phase, the effect of the first exposure of 500 rad was not diminished during the interval before the second exposure. Eggs irradiated in the resistant phase were only slightly damaged. Results implied that fluctuations in radiosensitivity of sea-urchin eggs were caused mainly by different degrees of non-repairable damage in each phase of cleavage rather than by different recovery abilities. (author)

Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis*

Science.gov (United States)

Leroy, Catherine; Belkina, Natalya V.; Long, Thavy; Deruy, Emeric; Dissous, Colette; Shaw, Stephen; Tulasne, David

2016-01-01

The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK−/− and LOK+/− lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments. PMID:26945071

Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis.

Science.gov (United States)

Leroy, Catherine; Belkina, Natalya V; Long, Thavy; Deruy, Emeric; Dissous, Colette; Shaw, Stephen; Tulasne, David

2016-05-06

The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK(-/-) and LOK(+/-) lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Caspase-2 cleavage of tau reversibly impairs memory.

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Zhao, Xiaohui; Kotilinek, Linda A; Smith, Benjamin; Hlynialuk, Chris; Zahs, Kathleen; Ramsden, Martin; Cleary, James; Ashe, Karen H

2016-11-01

In Alzheimer's disease (AD) and other tauopathies, the tau protein forms fibrils, which are believed to be neurotoxic. However, fibrillar tau has been dissociated from neuron death and network dysfunction, suggesting the involvement of nonfibrillar species. Here we describe a novel pathological process in which caspase-2 cleavage of tau at Asp314 impairs cognitive and synaptic function in animal and cellular models of tauopathies by promoting the missorting of tau to dendritic spines. The truncation product, Δtau314, resists fibrillation and is present at higher levels in brains from cognitively impaired mice and humans with AD. The expression of tau mutants that resisted caspase-2 cleavage prevented tau from infiltrating spines, dislocating glutamate receptors and impairing synaptic function in cultured neurons, and it prevented memory deficits and neurodegeneration in mice. Decreasing the levels of caspase-2 restored long-term memory in mice that had existing deficits. Our results suggest an overall treatment strategy for re-establishing synaptic function and restoring memory in patients with AD by preventing tau from accumulating in dendritic spines.

Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand*

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Teasley, Daniel C.; Parajuli, Shankar; Nguyen, Mai; Moore, Hayley R.; Alspach, Elise; Lock, Ying Jie; Honaker, Yuchi; Saharia, Abhishek; Piwnica-Worms, Helen; Stewart, Sheila A.

2015-01-01

The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer. PMID:25922071

AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination.

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Kobayashi, Maki; Aida, Masatoshi; Nagaoka, Hitoshi; Begum, Nasim A; Kitawaki, Yoko; Nakata, Mikiyo; Stanlie, Andre; Doi, Tomomitsu; Kato, Lucia; Okazaki, Il-mi; Shinkura, Reiko; Muramatsu, Masamichi; Kinoshita, Kazuo; Honjo, Tasuku

2009-12-29

To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5' to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the Smu region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.

Carotenoid-cleavage activities of crude enzymes from Pandanous amryllifolius.

Science.gov (United States)

Ningrum, Andriati; Schreiner, Matthias

2014-11-01

Carotenoid degradation products, known as norisoprenoids, are aroma-impact compounds in several plants. Pandan wangi is a common name of the shrub Pandanus amaryllifolius. The genus name 'Pandanus' is derived from the Indonesian name of the tree, pandan. In Indonesia, the leaves from the plant are used for several purposes, e.g., as natural colorants and flavor, and as traditional treatments. The aim of this study was to determine the cleavage of β-carotene and β-apo-8'-carotenal by carotenoid-cleavage enzymes isolated from pandan leaves, to investigate dependencies of the enzymatic activities on temperature and pH, to determine the enzymatic reaction products by using Headspace Solid Phase Microextraction Gas Chromatography/Mass Spectrophotometry (HS-SPME GC/MS), and to investigate the influence of heat treatment and addition of crude enzyme on formation of norisoprenoids. Crude enzymes from pandan leaves showed higher activity against β-carotene than β-apo-8'-carotenal. The optimum temperature of crude enzymes was 70°, while the optimum pH value was 6. We identified β-ionone as the major volatile reaction product from the incubations of two different carotenoid substrates, β-carotene and β-apo-8'-carotenal. Several treatments, e.g., heat treatment and addition of crude enzymes in pandan leaves contributed to the norisoprenoid content. Our findings revealed that the crude enzymes from pandan leaves with carotenoid-cleavage activity might provide a potential application, especially for biocatalysis, in natural-flavor industry. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.

Caspase activation increases beta-amyloid generation independently of caspase cleavage of the beta-amyloid precursor protein (APP).

Science.gov (United States)

Tesco, Giuseppina; Koh, Young Ho; Tanzi, Rudolph E

2003-11-14

The amyloid precursor protein (APP) undergoes "alternative" proteolysis mediated by caspases. Three major caspase recognition sites have been identified in the APP, i.e. one at the C terminus (Asp720) and two at the N terminus (Asp197 and Asp219). Caspase cleavage at Asp720 has been suggested as leading to increased production of Abeta. Thus, we set out to determine which putative caspase sites in APP, if any, are cleaved in Chinese hamster ovary cell lines concurrently with the increased Abeta production that occurs during apoptosis. We found that cleavage at Asp720 occurred concurrently with caspase 3 activation and the increased production of total secreted Abeta and Abeta1-42 in association with staurosporine- and etoposide-induced apoptosis. To investigate the contribution of caspase cleavage of APP to Abeta generation, we expressed an APP mutant truncated at Asp720 that mimics APP caspase cleavage at the C-terminal site. This did not increase Abeta generation but, in contrast, dramatically decreased Abeta production in Chinese hamster ovary cells. Furthermore, the ablation of caspase-dependent cleavage at Asp720, Asp197, and Asp219 (by site-directed mutagenesis) did not prevent enhanced Abeta production following etoposide-induced apoptosis. These findings indicate that the enhanced Abeta generation associated with apoptosis does not require cleavage of APP at its C-terminal (Asp720) and/or N-terminal caspase sites.

Implementation of Paste Backfill Mining Technology in Chinese Coal Mines

Science.gov (United States)

Chang, Qingliang; Zhou, Huaqiang; Bai, Jianbiao

2014-01-01

Implementation of clean mining technology at coal mines is crucial to protect the environment and maintain balance among energy resources, consumption, and ecology. After reviewing present coal clean mining technology, we introduce the technology principles and technological process of paste backfill mining in coal mines and discuss the components and features of backfill materials, the constitution of the backfill system, and the backfill process. Specific implementation of this technology and its application are analyzed for paste backfill mining in Daizhuang Coal Mine; a practical implementation shows that paste backfill mining can improve the safety and excavation rate of coal mining, which can effectively resolve surface subsidence problems caused by underground mining activities, by utilizing solid waste such as coal gangues as a resource. Therefore, paste backfill mining is an effective clean coal mining technology, which has widespread application. PMID:25258737

The DNA repair endonuclease XPG interacts directly and functionally with the WRN helicase defective in Werner syndrome

Energy Technology Data Exchange (ETDEWEB)

Trego, Kelly S.; Chernikova, Sophia B.; Davalos, Albert R.; Perry, J. Jefferson P.; Finger, L. David; Ng, Cliff; Tsai, Miaw-Sheue; Yannone, Steven M.; Tainer, John A.; Campisi, Judith; Cooper, Priscilla K.

2011-04-20

XPG is a structure-specific endonuclease required for nucleotide excision repair (NER). XPG incision defects result in the cancer-prone syndrome xeroderma pigmentosum, whereas truncating mutations of XPG cause the severe postnatal progeroid developmental disorder Cockayne syndrome. We show that XPG interacts directly with WRN protein, which is defective in the premature aging disorder Werner syndrome, and that the two proteins undergo similar sub-nuclear redistribution in S-phase and co-localize in nuclear foci. The co-localization was observed in mid- to late-S-phase, when WRN moves from nucleoli to nuclear foci that have been shown to contain protein markers of both stalled replication forks and telomeric proteins. We mapped the interaction between XPG and WRN to the C-terminal domains of each and show that interaction with the C-terminal domain of XPG strongly stimulates WRN helicase activity. WRN also possesses a competing DNA single-strand annealing activity that, combined with unwinding, has been shown to coordinate regression of model replication forks to form Holliday junction/chicken foot intermediate structures. We tested whether XPG stimulated WRN annealing activity and found that XPG itself has intrinsic strand annealing activity that requires the unstructured R- and C-terminal domains, but not the conserved catalytic core or endonuclease activity. Annealing by XPG is cooperative, rather than additive, with WRN annealing. Taken together, our results suggest a novel function for XPG in S-phase that is at least in part carried out coordinately with WRN, and which may contribute to the severity of the phenotypes that occur upon loss of XPG.

Structural insights of the ssDNA binding site in the multifunctional endonuclease AtBFN2 from Arabidopsis thaliana.

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Tsung-Fu Yu

Full Text Available The multi S1/P1 nuclease AtBFN2 (EC 3.1.30.1 encoded by the Arabidopsis thaliana At1g68290 gene is a glycoprotein that digests RNA, ssDNA, and dsDNA. AtBFN2 depends on three zinc ions for cleaving DNA and RNA at 3'-OH to yield 5'-nucleotides. In addition, AtBFN2's enzymatic activity is strongly glycan dependent. Plant Zn(2+-dependent endonucleases present a unique fold, and belong to the Phospholipase C (PLC/P1 nuclease superfamily. In this work, we present the first complete, ligand-free, AtBFN2 crystal structure, along with sulfate, phosphate and ssDNA co-crystal structures. With these, we were able to provide better insight into the glycan structure and possible enzymatic mechanism. In comparison with other nucleases, the AtBFN2/ligand-free and AtBFN2/PO4 models suggest a similar, previously proposed, catalytic mechanism. Our data also confirm that the phosphate and vanadate can inhibit the enzyme activity by occupying the active site. More importantly, the AtBFN2/A5T structure reveals a novel and conserved secondary binding site, which seems to be important for plant Zn(2+-dependent endonucleases. Based on these findings, we propose a rational ssDNA binding model, in which the ssDNA wraps itself around the protein and the attached surface glycan, in turn, reinforces the binding complex.

SOCIAL CLEAVAGES IN THE AMERICAN SOCIETY AS A FACTOR OF 2016 PRESIDENTIAL CAMPAIGN

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P. S. Kanevskiy

2017-01-01

Full Text Available Current article is dedicated to analysis of social cleavages in the American elections and the ways they influenced on presidential election in 2016. Originally developed by S. Rokkan and S.M. Lipset, social cleavages became a classic theme for contemporary political sociology. However, despite the fact that the theory has been developing primarily by Americans, it has been rarely used to analyze electoral system in the USA. Traditionally it’s been aimed at European and developing countries where electoral fragmentation is seen more clearly. But recent changes in the American society and the political system demonstrate the emergence of social cleavages that had not been inherent before. The article shows how American electoral space transformed since the 1980s and how it became more fragmented under the influence of social, economic and ideological factors. Elections in 2016 became a watershed for social cleavages that accumulated through time and aggravated even more considering internal crises in the Democratic and more so in the Republican parties. Donald Trump’s victory is an impersonation of the American party system crisis and of the mainstream politicians’ inability to find proper explanation of the changing electorate. Author shows that American society today is polarized even more than many European countries while group identification determines vectors of political change.

Electrostatic instability of some jellium model lattices of high symmetry to their plane cleavage

International Nuclear Information System (INIS)

Kholopov, Eugene V; Kalashnikova, Vita V

2007-01-01

Jellium model structures composed of regular lattices of equal point charges immersed in a neutralizing uniform background are considered. The symmetric description eliminating the effect of potentials without transverse structural modulation is extended to the events specified by alternating distances between point-charge planes. Based on modulated potentials typical of plane-wise lattice summation, the energy of interaction between two semi-infinite hemi-crystals divided by a plane is obtained for cubic and hexagonal crystals, where all the characteristic orientations of the cleavage plane are taken into account. We found that simple cubic and hexagonal lattices, as well as cubic and hexagonal diamond structures, turn out to be unstable for certain cleavage planes. The most favourable cleavage planes for the bcc, fcc and hcp structures are also emphasized

Next-generation sequencing of multiple individuals per barcoded library by deconvolution of sequenced amplicons using endonuclease fragment analysis

DEFF Research Database (Denmark)

Andersen, Jeppe D; Pereira, Vania; Pietroni, Carlotta

2014-01-01

The simultaneous sequencing of samples from multiple individuals increases the efficiency of next-generation sequencing (NGS) while also reducing costs. Here we describe a novel and simple approach for sequencing DNA from multiple individuals per barcode. Our strategy relies on the endonuclease...... digestion of PCR amplicons prior to library preparation, creating a specific fragment pattern for each individual that can be resolved after sequencing. By using both barcodes and restriction fragment patterns, we demonstrate the ability to sequence the human melanocortin 1 receptor (MC1R) genes from 72...... individuals using only 24 barcoded libraries....

Stimulation of topoisomerase II mediated DNA cleavage at specific sequence elements by the 2-nitroimidazole Ro 15-0216

International Nuclear Information System (INIS)

Sorensen, B.S.; Jensen, P.S.; Andersen, A.H.; Christiansen, K.; Alsner, J.; Thomsen, B.; Westergaard, O.

1990-01-01

The effect of the 2-nitroimidazole Ro 15-0216 upon the interaction between purified topoisomerase II and its DNA substrate was investigated. The cleavage reaction in the presence of this DNA-nonintercalative drug took place with the hallmarks of a regular topoisomerase II mediated cleavage reaction, including covalent linkage of the enzyme to the cleaved DNA. In the presence of Ro 15-0216, topoisomerase II mediated cleavage was extensively stimulated at major cleavage sites of which only one existed in the 4363 base pair pBR322 molecule. The sites stimulated by Ro 15-0216 shared a pronounced sequence homology, indicating that a specific nucleotide sequence is crucial for the action of this drug. The effect of Ro 15-0216 thus differs from that of the clinically important topoisomerase II targeted agents such as mAMSA, VM26, and VP16, which enhance enzyme-mediated cleavage at a multiple number of sites. In contrast to the previous described drugs, Ro 15-0216 did not exert any inhibitory effect on the enzyme's catalytic activity. This observation might be ascribed to the low stability of the cleavage complexes formed in the presence of Ro 15-0216 as compared to the stability of the ones formed in the presence of traditional topoisomerase II targeted drugs

Kinetics of phycocyanobilin cleavage from C-phycocyanin by methanolysis

DEFF Research Database (Denmark)

Malwade, Chandrakant Ramkrishna; Roda Serrat, Maria Cinta; Christensen, Knud Villy

2016-01-01

Phycocyanobilin (PCB) is an important linear tetrapyrrolic molecule for food as well as pharmaceutical industry. It is obtained from blue-green algae, where it is attached covalently to phycobiliproteins (C-PC and APC) present in the light harvesting complexes. In this work, cleavage of PCB from...

cis-Apa: a practical linker for the microwave-assisted preparation of cyclic pseudopeptides via RCM cyclative cleavage.

Science.gov (United States)

Baron, Alice; Verdié, Pascal; Martinez, Jean; Lamaty, Frédéric

2011-02-04

A new linker cis-5-aminopent-3-enoic acid (cis-Apa) was prepared for the synthesis of cyclic pseudopeptides by cyclization-cleavage by using ring-closing methatesis (RCM). We developed a new synthetic pathway for the preparation of the cis-Apa linker that was tested in the cyclization-cleavage process of different RGD peptide sequences. Different macrocyclic peptidomimetics were prepared by using this integrated microwave-assisted method, showing that the readily available cis-Apa amino acid is well adapted as a linker in the cyclization-cleavage process.

30 CFR 819.21 - Auger mining: Protection of underground mining.

Science.gov (United States)

2010-07-01

... 30 Mineral Resources 3 2010-07-01 2010-07-01 false Auger mining: Protection of underground mining. 819.21 Section 819.21 Mineral Resources OFFICE OF SURFACE MINING RECLAMATION AND ENFORCEMENT... STANDARDS-AUGER MINING § 819.21 Auger mining: Protection of underground mining. Auger holes shall not extend...

The Conserved ATM Kinase RAG2-S365 Phosphorylation Site Limits Cleavage Events in Individual Cells Independent of Any Repair Defect

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Susannah L. Hewitt

2017-10-01

Full Text Available Many DNA lesions associated with lymphoid malignancies are linked to off-target cleavage by the RAG1/2 recombinase. However, off-target cleavage has mostly been analyzed in the context of DNA repair defects, confounding any mechanistic understanding of cleavage deregulation. We identified a conserved SQ phosphorylation site on RAG2 365 to 366 that is involved in feedback control of RAG cleavage. Mutation of serine 365 to a non-phosphorylatable alanine permits bi-allelic and bi-locus RAG-mediated breaks in the same cell, leading to reciprocal translocations. This phenomenon is analogous to the phenotype we described for ATM kinase inactivation. Here, we establish deregulated cleavage itself as a driver of chromosomal instability without the associated repair defect. Intriguingly, a RAG2-S365E phosphomimetic rescues the deregulated cleavage of ATM inactivation, reducing the incidence of reciprocal translocations. These data support a model in which feedback control of cleavage and maintenance of genome stability involves ATM-mediated phosphorylation of RAG2.

Contract Mining versus Owner Mining – The Way Forward | Suglo ...

African Journals Online (AJOL)

Ghana Mining Journal ... By contracting out one or more of their mining operations, the mining companies can concentrate on their core businesses. This paper reviews ... The general trends in the mining industry show that contract mining will be the way forward for most mines under various circumstances in the future.

Differential distribution of a SINE element in the Entamoeba histolytica and Entamoeba dispar genomes: Role of the LINE-encoded endonuclease

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Gupta Abhishek K

2011-05-01

Full Text Available Abstract Background Entamoeba histolytica and Entamoeba dispar are closely related protistan parasites but while E. histolytica can be invasive, E. dispar is completely non pathogenic. Transposable elements constitute a significant portion of the genome in these species; there being three families of LINEs and SINEs. These elements can profoundly influence the expression of neighboring genes. Thus their genomic location can have important phenotypic consequences. A genome-wide comparison of the location of these elements in the E. histolytica and E. dispar genomes has not been carried out. It is also not known whether the retrotransposition machinery works similarly in both species. The present study was undertaken to address these issues. Results Here we extracted all genomic occurrences of full-length copies of EhSINE1 in the E. histolytica genome and matched them with the homologous regions in E. dispar, and vice versa, wherever it was possible to establish synteny. We found that only about 20% of syntenic sites were occupied by SINE1 in both species. We checked whether the different genomic location in the two species was due to differences in the activity of the LINE-encoded endonuclease which is required for nicking the target site. We found that the endonucleases of both species were essentially very similar, both in their kinetic properties and in their substrate sequence specificity. Hence the differential distribution of SINEs in these species is not likely to be influenced by the endonuclease. Further we found that the physical properties of the DNA sequences adjoining the insertion sites were similar in both species. Conclusions Our data shows that the basic retrotransposition machinery is conserved in these sibling species. SINEs may indeed have occupied all of the insertion sites in the genome of the common ancestor of E. histolytica and E. dispar but these may have been subsequently lost from some locations. Alternatively, SINE

Coupling fibroblast growth factor 23 production and cleavage: iron deficiency, rickets, and kidney disease.

Science.gov (United States)

Wolf, Myles; White, Kenneth E

2014-07-01

High levels of fibroblast growth factor 23 (FGF23) cause the rare disorders of hypophosphatemic rickets and are a risk factor for cardiovascular disease and death in patients with chronic kidney disease (CKD). Despite major advances in understanding FGF23 biology, fundamental aspects of FGF23 regulation in health and in CKD remain mostly unknown. Autosomal dominant hypophosphatemic rickets (ADHR) is caused by gain-of-function mutations in FGF23 that prevent its proteolytic cleavage, but affected individuals experience a waxing and waning course of phosphate wasting. This led to the discovery that iron deficiency is an environmental trigger that stimulates FGF23 expression and hypophosphatemia in ADHR. Unlike osteocytes in ADHR, normal osteocytes couple increased FGF23 production with commensurately increased FGF23 cleavage to ensure that normal phosphate homeostasis is maintained in the event of iron deficiency. Simultaneous measurement of FGF23 by intact and C-terminal assays supported these breakthroughs by providing minimally invasive insight into FGF23 production and cleavage in bone. These findings also suggest a novel mechanism of FGF23 elevation in patients with CKD, who are often iron deficient and demonstrate increased FGF23 production and decreased FGF23 cleavage, consistent with an acquired state that mimics the molecular pathophysiology of ADHR. Iron deficiency stimulates FGF23 production, but normal osteocytes couple increased FGF23 production with increased cleavage to maintain normal circulating levels of biologically active hormone. These findings uncover a second level of FGF23 regulation within osteocytes, failure of which culminates in elevated levels of biologically active FGF23 in ADHR and perhaps CKD.

Developing a capillary electrophoresis based method for dynamically monitoring enzyme cleavage activity using quantum dots-peptide assembly.

Science.gov (United States)

Wang, Jianhao; Fan, Jie; Liu, Li; Ding, Shumin; Liu, Xiaoqian; Wang, Jianpeng; Gao, Liqian; Chattopadhaya, Souvik; Miao, Peng; Xia, Jiang; Qiu, Lin; Jiang, Pengju

2017-10-01

Herein, a novel assay has been developed for monitoring PreScission protease (His-PSP) mediated enzyme cleavage of ATTO 590 labeled peptide substrate (ATTO-LEV). This novel method is based on combining the use of capillary electrophoresis and fluorescence detection (CE-FL) to dynamically monitor the enzyme cleavage activity. A multivalent peptide substrate was first constructed by immobilizing His-tagged ATTO 590 labeled peptide substrate (ATTO-LEVH6) onto the surface of CdSe/ZnS quantum dots (QDs). Once successfully immobilized, the novel multivalent peptide substrate resulted in the Förster resonance energy transfer (FRET) from QDs to ATTO 590. The ATTO-LEVH6-QD assembly was then incubated with His-PSP to study the proteolytic cleavage of surface bound ATTO-LEVH6 by CE-FL. Our data suggests that PreScission-mediated proteolytic cleavage is enzyme concentration- and incubation time-dependent. By combining capillary electrophoresis, QDs and FRET, our study herein not only provides a new method for the detection and dynamically monitoring of PSP enzyme cleavage activity, but also can be extended to the detection of many other enzymes and proteases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metabolic Engineering to Develop a Pathway for the Selective Cleavage of Carbon-Nitrogen Bonds

Energy Technology Data Exchange (ETDEWEB)

John J. Kilbane II

2005-10-01

The objective of the project is to develop a biochemical pathway for the selective cleavage of C-N bonds in molecules found in petroleum. Specifically a novel biochemical pathway will be developed for the selective cleavage of C-N bonds in carbazole. The cleavage of the first C-N bond in carbazole is accomplished by the enzyme carbazole dioxygenase, that catalyzes the conversion of carbazole to 2-aminobiphenyl-2,3-diol. The genes encoding carbazole dioxygenase were cloned from Sphingomonas sp. GTIN11 and from Pseudomonas resinovorans CA10. The selective cleavage of the second C-N bond has been challenging, and efforts to overcome that challenge have been the focus of recent research in this project. Enrichment culture experiments succeeded in isolating bacterial cultures that can metabolize 2-aminobiphenyl, but no enzyme capable of selectively cleaving the C-N bond in 2-aminobiphenyl has been identified. Aniline is very similar to the structure of 2-aminobiphenyl and aniline dioxygenase catalyzes the conversion of aniline to catechol and ammonia. For the remainder of the project the emphasis of research will be to simultaneously express the genes for carbazole dioxygenase and for aniline dioxygenase in the same bacterial host and then to select for derivative cultures capable of using carbazole as the sole source of nitrogen.

Selection of mining method for No.3 uranium ore body in the independent mining area at a uranium mine

International Nuclear Information System (INIS)

Ding Fulong; Ding Dexin; Ye Yongjun

2010-01-01

Mining operation in the existed mining area at a uranium mine is near completion and it is necessary to mine the No.3 uranium ore body in another mining area at the mine. This paper, based on the geological conditions, used analogical method for analyzing the feasible methods and the low cost and high efficiency mining method was suggested for the No.3 ore body in the independent mining area at the uranium mine. (authors)

Type II restriction endonucleases--a historical perspective and more.

Science.gov (United States)

Pingoud, Alfred; Wilson, Geoffrey G; Wende, Wolfgang

2014-07-01

This article continues the series of Surveys and Summaries on restriction endonucleases (REases) begun this year in Nucleic Acids Research. Here we discuss 'Type II' REases, the kind used for DNA analysis and cloning. We focus on their biochemistry: what they are, what they do, and how they do it. Type II REases are produced by prokaryotes to combat bacteriophages. With extreme accuracy, each recognizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nearby. The discoveries of these enzymes in the 1970s, and of the uses to which they could be put, have since impacted every corner of the life sciences. They became the enabling tools of molecular biology, genetics and biotechnology, and made analysis at the most fundamental levels routine. Hundreds of different REases have been discovered and are available commercially. Their genes have been cloned, sequenced and overexpressed. Most have been characterized to some extent, but few have been studied in depth. Here, we describe the original discoveries in this field, and the properties of the first Type II REases investigated. We discuss the mechanisms of sequence recognition and catalysis, and the varied oligomeric modes in which Type II REases act. We describe the surprising heterogeneity revealed by comparisons of their sequences and structures. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Coupling of the nucleotide incision and 3' {yields} 5' exonuclease activities in Escherichia coli endonuclease IV: Structural and genetic evidences

Energy Technology Data Exchange (ETDEWEB)

Golan, Gali [Hebrew University of Jerusalem, Jerusalem 91904 (Israel); Ishchenko, Alexander A. [Groupe Reparation de l' ADN, CNRS UMR 8126, Univ. Paris-Sud, Institut de Cancerologie Gustave Roussy, 39, rue Camille Desmoulins, F-94805 Villejuif Cedex (France); Khassenov, Bekbolat [National Center for Biotechnology, Astana (Kazakhstan); Shoham, Gil, E-mail: gil2@vms.huji.ac.il [Hebrew University of Jerusalem, Jerusalem 91904 (Israel); Saparbaev, Murat K., E-mail: smurat@igr.fr [Groupe Reparation de l' ADN, CNRS UMR 8126, Univ. Paris-Sud, Institut de Cancerologie Gustave Roussy, 39, rue Camille Desmoulins, F-94805 Villejuif Cedex (France)

2010-03-01

Aerobic respiration generates reactive oxygen species (ROS) as a by-product of cellular metabolism which can damage DNA. The complex nature of oxidative DNA damage requires actions of several repair pathways. Oxidized DNA bases are substrates for two overlapping pathways: base excision repair (BER) and nucleotide incision repair (NIR). In the BER pathway a DNA glycosylase cleaves the N-glycosylic bond between the abnormal base and deoxyribose, leaving either an abasic site or single-stranded DNA break. Alternatively, in the NIR pathway, an apurinic/apyrimidinic (AP) endonuclease incises duplex DNA 5' next to oxidatively damaged nucleotide. The multifunctional Escherichia coli endonuclease IV (Nfo) is involved in both BER and NIR pathways. Nfo incises duplex DNA 5' of a damaged residue but also possesses an intrinsic 3' {yields} 5' exonuclease activity. Herein, we demonstrate that Nfo-catalyzed NIR and exonuclease activities can generate a single-strand gap at the 5' side of 5,6-dihydrouracil residue. Furthermore, we show that Nfo mutants carrying amino acid substitutions H69A and G149D are deficient in both NIR and exonuclease activities, suggesting that these two functions are genetically linked and governed by the same amino acid residues. The crystal structure of Nfo-H69A mutant reveals the loss of one of the active site zinc atoms (Zn1) and rearrangements of the catalytic site, but no gross changes in the overall enzyme conformation. We hypothesize that these minor changes strongly affect the DNA binding of Nfo. Decreased affinity may lead to a different kinking angle of the DNA helix and this in turn thwart nucleotide incision and exonuclease activities of Nfo mutants but to lesser extent of their AP endonuclease function. Based on the biochemical and genetic data we propose a model where nucleotide incision coupled to 3' {yields} 5' exonuclease activity prevents formation of lethal double-strand breaks when repairing bi

Repair of potentially lethal damage by introduction of T4 DNA ligase in eucaryotic cells

International Nuclear Information System (INIS)

Durante, M.; Grossi, G.F.; Napolitano, M.; Gialanella, G.

1991-01-01

The bacterial enzyme PvuII, which generates blunt-ended DNA double-strand breaks, and T4 DNA ligase, which seals adjacent DNA fragments in coupling to ATP cleavage, were introduced in mouse C3H10T1/2 fibroblasts using osmolytic shock of pinocytic vesicles. Cells were then assayed for their clonogenic ability. In agreement with previous studies by others, the authors found that PvuII restriction endonuclease simulates ionizing radiation effects by causing a dose-dependent loss of reproductive capacity. They show that concomitant treatment with DNA ligase considerably increases cell survival. Survival curves were shown to be dependent on ligase enzyme dose and on ATP concentration in the hypertonic medium. They conclude that T4 DNA ligase is able to repair some potentially lethal damage produced by restriction endonucleases in eucaryotic cells. (author)

Treatment of mine-water from decommissioning uranium mines

International Nuclear Information System (INIS)

Fan Quanhui

2002-01-01

Treatment methods for mine-water from decommissioning uranium mines are introduced and classified. The suggestions on optimal treatment methods are presented as a matter of experience with decommissioned Chenzhou Uranium Mine

Influence of the austenitizing temperature in the cleavage facet size of Niocor 2

International Nuclear Information System (INIS)

Darwish, F.A.I.; Teixeira, J.C.G.; Fernandes, R.A.; Juer, S.

1983-01-01

Convetional Charpy specimens of Niocor 2 steel cooled in air from various austenitizing temperatures were fractured at -196 0 C so as to insure failure by cleavage. The cleavage facet size distribution was determined and then correlated with the grain size and other aspects of the microstructure. The results that the average facet size can be increased through a coarsening of the microstructure. For the case where the γ→α transformation products are predominantely acicular, the facet size is shown to depend on substructural aspects primarily the lath packet size. (Author) [pt

[Cleavage of DNA fragments induced by UV nanosecond laser excitation at 193 nm].

Science.gov (United States)

Vtiurina, N N; Grokhovskiĭ, S L; Filimonov, I V; Medvedkov, O I; Nechipurenko, D Iu; Vasil'ev, S A; Nechipurenko, Iu D

2011-01-01

The cleavage of dsDNA fragments in aqueous solution after irradiation with UV laser pulses at 193 nm has been studied. Samples were investigated using polyacrylamide gel electrophoresis. The intensity of damage of particular phosphodiester bond after hot alkali treatment was shown to depend on the base pair sequence. It was established that the probability of cleavage is twice higher for sites of DNA containing two or more successively running guanine residues. A possible mechanism of damage to the DNA molecule connected with the migration of holes along the helix is discussed.

Affect of Presenilin Mutations on APP Cleavage; Insights into the Pathogenesis of FAD

Directory of Open Access Journals (Sweden)

Nuomin eLi

2016-03-01

Full Text Available Alzheimer disease (AD is characterized by progressive memory loss, reduction in cognitive functions, and damage to the brain. The β-amyloid precursor protein can be sequentially cleaved by β- secretase and γ-secretase. Mutations in the presenilin1（PS1） are the most common cause of Familial Alzheimer’s disease ( FAD. PS1 mutations can alter the activity of γ-secretase on the cleavage of the β-amyloid precursor protein, causing increased Aβ production. Previous studies show that the βAPP-C-terminal fragment is first cleaved by β-scretase, primarily generating long fragments of Aβ48 and Aβ49, followed by the stepwise cleavage of every three amino acid residues at the C terminus, resulting in Aβ48-, 45-, 42 line and Aβ49-, 46-, 43-, 40 line. Here, we used LC-MS/MS to analyze unique peptides IAT, VVIA, ITL，TVI，IVI through sequential cleavage, combined with ELISA to test the level of Aβ42 and Aβ40 for validation. The results show that most FAD mutant PS1 can alter the level of Aβ42 and Aβ40 monitored by the Aβ42/Aβ40 ratio. Among them, 6 mutants (I143T, H163P, S170F, Q223R, M233V and G384A affect the Aβ42/40 ratio through both Aβ49-40 and Aβ48-38 lines; L166P through decreasing the Aβ49-40 line, 6 mutants (I143V, M146V, G217A, E280A, L381V and L392V through increasing the Aβ48-42 line. More importantly, we found some mutations can affect the γ-secretase cleavage preference of α-CTF and β-CTF. In conclusion, we found that the FAD PS1 mutations mainly increase the generation of Aβ42 by decreasing the cleavage of Aβ42-Aβ38 and Aβ43-Aβ40.

Micrococcus luteus correndonucleases. II. Mechanism of action of two endonucleases specific for DNA containing pyrimidine dimers

International Nuclear Information System (INIS)

Riazuddin, S.; Grossman, L.

1977-01-01

Py--Py correndonucleases I and II from Micrococcus luteus act exclusively on thymine-thymine, cytosine-cytosine, and thymine-cytosine cyclobutyl dimers in DNA, catalyzing incision 5' to the damage and generating 3'-hydroxyl and 5'-phosphoryl termini. Both enzymes initiate excision of pyrimidine dimers in vitro by correxonucleases and DNA polymerase I. The respective incised DNAs, however, differ in their ability to act as substrate for phage T4 polynucleotide ligase or bacterial alkaline phosphatase, suggesting that each endonuclease is specific for a conformationally unique site. The possibility that their respective action generates termini which represent different degrees of single strandedness is suggested by the unequal protection by Escherichia coli binding protein from the hydrolytic action of exonuclease VII

Studies on the repair of damaged DNA in bacteriophage, bacterial and mammalian systems. Comprehensive report, 1 February 1981-15 September 1983

International Nuclear Information System (INIS)

Friedberg, E.C.

1983-01-01

We have explored the molecular mechanism of the repair of DNA at a number of different levels of biological organization, by investigating bacteriophage, bacterial, yeast and mammalian (including human) cells. We have demonstrated that uv endonuclease of phage T4 not only possesses pyrimidine dimer (PD)-DNA glycosylase activity but also apyrimidinic (AP) endonuclease activity. The demonstration of both activities provided an explanation for the specific endonucleosytic cleavage of DNA at sites of pyrimidine dimers catalyzed by this small protein. A new apurinic/apyrimidinic (AP) endonuclease, specific for sites of of base loss in single stranded DNA has been isolated from E. celi and presumably recognizes these lesions in single stranded regions of duplex DNA. We have partially purified this enzyme and have carried out a preliminary characterization of the activity. We treated xeroderma pigmentosum and normal cells with sodium butyrate in the hope of restoring normal levels of excision repair to the former. Although this result was not obtained, we established that all cells treated with sodium butyrate show enhanced levels of repair synthesis, thus providing a means for increasing the sensitivity of this commonly used technique for measuring DNA repair in mammalian cells in culture

30 CFR 77.1712 - Reopening mines; notification; inspection prior to mining.

Science.gov (United States)

2010-07-01

... to mining. 77.1712 Section 77.1712 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION... prior to mining. Prior to reopening any surface coal mine after it has been abandoned or declared... an authorized representative of the Secretary before any mining operations in such mine are...

Pre-steady-state fluorescence analysis of damaged DNA transfer from human DNA glycosylases to AP endonuclease APE1.

Science.gov (United States)

Kuznetsova, Alexandra A; Kuznetsov, Nikita A; Ishchenko, Alexander A; Saparbaev, Murat K; Fedorova, Olga S

2014-10-01

DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (apurinic/apyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-endonucleases. Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4(cat) and UNG from different structural superfamilies were used. We found that all DNA glycosylases may utilise direct protein-protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. We hypothesize a fast "flip-flop" exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4(cat), AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway. Copyright © 2014 Elsevier B.V. All rights reserved.

High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA.

Science.gov (United States)

Chandrananda, Dineika; Thorne, Natalie P; Bahlo, Melanie

2015-06-17

High-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. However, many biological properties of this extracellular genetic material remain unknown. Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data. In this study, we investigate various features of cell-free DNA in plasma using deep-sequencing data from two pregnant women (>70X, >50X) and compare them with matched cellular DNA. We utilize a descriptive approach to examine how the biological cleavage of cell-free DNA affects different sequence signatures such as fragment lengths, sequence motifs at fragment ends and the distribution of cleavage sites along the genome. We show that the size distributions of these cell-free DNA molecules are dependent on their autosomal and mitochondrial origin as well as the genomic location within chromosomes. DNA mapping to particular microsatellites and alpha repeat elements display unique size signatures. We show how cell-free fragments occur in clusters along the genome, localizing to nucleosomal arrays and are preferentially cleaved at linker regions by correlating the mapping locations of these fragments with ENCODE annotation of chromatin organization. Our work further demonstrates that cell-free autosomal DNA cleavage is sequence dependent. The region spanning up to 10 positions on either side of the DNA cleavage site show a consistent pattern of preference for specific nucleotides. This sequence motif is present in cleavage sites localized to nucleosomal cores and linker regions but is absent in nucleosome-free mitochondrial DNA. These background signals in cell-free DNA sequencing data stem from the non-random biological cleavage of these fragments. This

Embryo apoptosis identification: Oocyte grade or cleavage stage?

Science.gov (United States)

Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

2015-01-01

Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced. PMID:26858565

Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand.

Science.gov (United States)

Teasley, Daniel C; Parajuli, Shankar; Nguyen, Mai; Moore, Hayley R; Alspach, Elise; Lock, Ying Jie; Honaker, Yuchi; Saharia, Abhishek; Piwnica-Worms, Helen; Stewart, Sheila A

2015-06-12

The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

RECQ5 Helicase Cooperates with MUS81 Endonuclease in Processing Stalled Replication Forks at Common Fragile Sites during Mitosis

DEFF Research Database (Denmark)

Di Marco, Stefano; Hasanova, Zdenka; Kanagaraj, Radhakrishnan

2017-01-01

The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent...... on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain...

Dual CRISPR-Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica.

Science.gov (United States)

Gao, Difeng; Smith, Spencer; Spagnuolo, Michael; Rodriguez, Gabriel; Blenner, Mark

2018-05-29

CRISPR-Cas9 technology has been successfully applied in Yarrowia lipolytica for targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest. The targeted regions for excision vary in length from 0.3 kb up to 3.5 kb and contain both non-coding and coding regions. The majority of the gene excisions are repaired by perfect nonhomologous end-joining without indel. Based on this dual cleavage system, two targeted markerless integration methods are developed by providing repair templates. While both strategies are effective, homology mediated end joining (HMEJ) based method are twice as efficient as homology recombination (HR) based method. In both cases, dual cleavage leads to similar or improved gene integration efficiencies compared to gene excision without integration. This dual cleavage strategy will be useful for not only generating more predictable and robust gene knockout, but also for efficient targeted markerless integration, and simultaneous knockout and integration in Y. lipolytica. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acid mine drainage: mining and water pollution issues in British Columbia

Energy Technology Data Exchange (ETDEWEB)

NONE

1998-12-31

The importance of protecting water quality and some of the problems associated with mineral development are described. Negative impacts of mining operations such as sedimentation, water disturbances, and water pollution from waste rock and tailings are considered. Mining wastes, types of water pollution from mining, the legacy of acid mine drainage, predicting acid mine drainage, preventing and mitigating acid mine drainage, examples from the past, and cyanide heap-leaching are discussed. The real costs of mining at the Telkwa open pit coal mine are assessed. British Columbia mines that are known for or are potentially acid generating are shown on a map. 32 refs., 10 figs.

Porcine deltacoronavirus nsp5 inhibits interferon-β production through the cleavage of NEMO.

Science.gov (United States)

Zhu, Xinyu; Fang, Liurong; Wang, Dang; Yang, Yuting; Chen, Jiyao; Ye, Xu; Foda, Mohamed Frahat; Xiao, Shaobo

2017-02-01

Porcine deltacoronavirus (PDCoV) causes acute enteric disease and mortality in seronegative neonatal piglets. Previously we have demonstrated that PDCoV infection suppresses the production of interferon-beta (IFN-β), while the detailed mechanisms are poorly understood. Here, we demonstrate that nonstructural protein 5 (nsp5) of PDCoV, the 3C-like protease, significantly inhibits Sendai virus (SEV)-induced IFN-β production by targeting the NF-κB essential modulator (NEMO), confirmed by the diminished function of NEMO cleaved by PDCoV. The PDCoV nsp5 cleavage site in the NEMO protein was identified as glutamine 231, and was identical to the porcine epidemic diarrhea virus nsp5 cleavage site, revealing the likelihood of a common target in NEMO for coronaviruses. Furthermore, this cleavage impaired the ability of NEMO to activate the IFN response and downstream signaling. Taken together, our findings reveal PDCoV nsp5 to be a newly identified IFN antagonist and enhance the understanding of immune evasion by deltacoronaviruses. Copyright © 2016 Elsevier Inc. All rights reserved.

Responsible Mining: A Human Resources Strategy for Mine Development Project

OpenAIRE

Sampathkumar, Sriram (Ram)

2012-01-01

Mining is a global industry. Most mining companies operate internationally, often in remote, challenging environments and consequently frequently have respond to unusual and demanding Human Resource (HR) requirements. It is my opinion that the strategic imperative behind success in mining industry is responsible mining. The purpose of this paper is to examine how an effective HR strategy can be a competitive advantage that contributes to the success of a mining project in the global mining in...

Extending mine life

International Nuclear Information System (INIS)

Anon.

1984-01-01

Mine layouts, new machines and techniques, research into problem areas of ground control and so on, are highlighted in this report on extending mine life. The main resources taken into account are coal mining, uranium mining, molybdenum and gold mining

Active site mutations change the cleavage specificity of neprilysin.

Directory of Open Access Journals (Sweden)

Travis Sexton

Full Text Available Neprilysin (NEP, a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe(563 and Ser(546. Among the mutants studied in detail we observed changes in their activity towards leucine(5-enkephalin, insulin B chain, and amyloid β(1-40. For example, NEP(F563I displayed an increase in preference towards cleaving leucine(5-enkephalin relative to insulin B chain, while mutant NEP(S546E was less discriminating than neprilysin. Mutants NEP(F563L and NEP(S546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß(1-40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential.

Cleavage of thymine N3-H bonds by low-energy electrons attached to base π* orbitals

International Nuclear Information System (INIS)

Theodore, Magali; Sobczyk, Monika; Simons, Jack

2006-01-01

In this work, we extend our earlier studies on single strand break (SSB) formation in DNA to consider the possibility of cleaving a thymine N 3 -H bond to generate a nitrogen-centered anion and a hydrogen radical which might proceed to induce further bond cleavages. In earlier studies, we considered SSBs induced by low-energy electrons that attach to DNA bases' π* orbitals or to phosphate P=O π* orbitals to cleave sugar-phosphate C-O bonds or base-sugar N 1 -C bonds. We also studied the effects of base π-stacking on the rates of such bond cleavages. To date, our results suggest that sugar-phosphate C-O bonds have the lowest barriers to cleavage, that attachment of electrons with energies below 2 eV most likely occurs at the base π* orbitals, that electrons with energy above 2 eV can also attach to phosphate P=O π* orbitals, and that base π stacking has a modest but slowing effect on the rates of SSB formation. However, we had not yet examined the possibility that base N 3 -H bonds could rupture subsequent to base π* orbital capture. In the present work, the latter possibility is considered and it is found that the barrier to cleavage of the N 3 -H bond in thymine is considerably higher than for cleaving sugar-phosphate C-O bonds, so our prediction that SSB formation is dominated by C-O bond cleavage remains intact

A consistency-based feature selection method allied with linear SVMs for HIV-1 protease cleavage site prediction.

Directory of Open Access Journals (Sweden)

Orkun Oztürk

Full Text Available BACKGROUND: Predicting type-1 Human Immunodeficiency Virus (HIV-1 protease cleavage site in protein molecules and determining its specificity is an important task which has attracted considerable attention in the research community. Achievements in this area are expected to result in effective drug design (especially for HIV-1 protease inhibitors against this life-threatening virus. However, some drawbacks (like the shortage of the available training data and the high dimensionality of the feature space turn this task into a difficult classification problem. Thus, various machine learning techniques, and specifically several classification methods have been proposed in order to increase the accuracy of the classification model. In addition, for several classification problems, which are characterized by having few samples and many features, selecting the most relevant features is a major factor for increasing classification accuracy. RESULTS: We propose for HIV-1 data a consistency-based feature selection approach in conjunction with recursive feature elimination of support vector machines (SVMs. We used various classifiers for evaluating the results obtained from the feature selection process. We further demonstrated the effectiveness of our proposed method by comparing it with a state-of-the-art feature selection method applied on HIV-1 data, and we evaluated the reported results based on attributes which have been selected from different combinations. CONCLUSION: Applying feature selection on training data before realizing the classification task seems to be a reasonable data-mining process when working with types of data similar to HIV-1. On HIV-1 data, some feature selection or extraction operations in conjunction with different classifiers have been tested and noteworthy outcomes have been reported. These facts motivate for the work presented in this paper. SOFTWARE AVAILABILITY: The software is available at http

Effects of Olive Metabolites on DNA Cleavage Mediated by Human Type II Topoisomerases

Science.gov (United States)

2016-01-01

Several naturally occurring dietary polyphenols with chemopreventive or anticancer properties are topoisomerase II poisons. To identify additional phytochemicals that enhance topoisomerase II-mediated DNA cleavage, a library of 341 Mediterranean plant extracts was screened for activity against human topoisomerase IIα. An extract from Phillyrea latifolia L., a member of the olive tree family, displayed high activity against the human enzyme. On the basis of previous metabolomics studies, we identified several polyphenols (hydroxytyrosol, oleuropein, verbascoside, tyrosol, and caffeic acid) as potential candidates for topoisomerase II poisons. Of these, hydroxytyrosol, oleuropein, and verbascoside enhanced topoisomerase II-mediated DNA cleavage. The potency of these olive metabolites increased 10–100-fold in the presence of an oxidant. Hydroxytyrosol, oleuropein, and verbascoside displayed hallmark characteristics of covalent topoisomerase II poisons. (1) The activity of the metabolites was abrogated by a reducing agent. (2) Compounds inhibited topoisomerase II activity when they were incubated with the enzyme prior to the addition of DNA. (3) Compounds were unable to poison a topoisomerase IIα construct that lacked the N-terminal domain. Because hydroxytyrosol, oleuropein, and verbascoside are broadly distributed across the olive family, extracts from the leaves, bark, and fruit of 11 olive tree species were tested for activity against human topoisomerase IIα. Several of the extracts enhanced enzyme-mediated DNA cleavage. Finally, a commercial olive leaf supplement and extra virgin olive oils pressed from a variety of Olea europea subspecies enhanced DNA cleavage mediated by topoisomerase IIα. Thus, olive metabolites appear to act as topoisomerase II poisons in complex formulations intended for human dietary consumption. PMID:26132160

Primary processing of CRISPR RNA by the endonuclease Cas6 in Staphylococcus epidermidis.

Science.gov (United States)

Wakefield, Noelle; Rajan, Rakhi; Sontheimer, Erik J

2015-10-07

In many bacteria and archaea, an adaptive immune system (CRISPR-Cas) provides immunity against foreign genetic elements. This system uses CRISPR RNAs (crRNAs) derived from the CRISPR array, along with CRISPR-associated (Cas) proteins, to target foreign nucleic acids. In most CRISPR systems, endonucleolytic processing of crRNA precursors (pre-crRNAs) is essential for the pathway. Here we study the Cas6 endonuclease responsible for crRNA processing in the Type III-A CRISPR-Cas system from Staphylococcus epidermidis RP62a, a model for Type III-A CRISPR-Cas systems, and define substrate requirements for SeCas6 activity. We find that SeCas6 is necessary and sufficient for full-length crRNA biogenesis in vitro, and that it relies on both sequence and stem-loop structure in the 3' half of the CRISPR repeat for recognition and processing. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Maximizing Selective Cleavages at Aspartic Acid and Proline Residues for the Identification of Intact Proteins

Science.gov (United States)

Foreman, David J.; Dziekonski, Eric T.; McLuckey, Scott A.

2018-04-01

A new approach for the identification of intact proteins has been developed that relies on the generation of relatively few abundant products from specific cleavage sites. This strategy is intended to complement standard approaches that seek to generate many fragments relatively non-selectively. Specifically, this strategy seeks to maximize selective cleavage at aspartic acid and proline residues via collisional activation of precursor ions formed via electrospray ionization (ESI) under denaturing conditions. A statistical analysis of the SWISS-PROT database was used to predict the number of arginine residues for a given intact protein mass and predict a m/z range where the protein carries a similar charge to the number of arginine residues thereby enhancing cleavage at aspartic acid residues by limiting proton mobility. Cleavage at aspartic acid residues is predicted to be most favorable in the m/z range of 1500-2500, a range higher than that normally generated by ESI at low pH. Gas-phase proton transfer ion/ion reactions are therefore used for precursor ion concentration from relatively high charge states followed by ion isolation and subsequent generation of precursor ions within the optimal m/z range via a second proton transfer reaction step. It is shown that the majority of product ion abundance is concentrated into cleavages C-terminal to aspartic acid residues and N-terminal to proline residues for ions generated by this process. Implementation of a scoring system that weights both ion fragment type and ion fragment area demonstrated identification of standard proteins, ranging in mass from 8.5 to 29.0 kDa. [Figure not available: see fulltext.

The Generation of Dehydroalanine Residues in Protonated Polypeptides: Ion/Ion Reactions for Introducing Selective Cleavages

Science.gov (United States)

Peng, Zhou; Bu, Jiexun; McLuckey, Scott A.

2017-09-01

We examine a gas-phase approach for converting a subset of amino acid residues in polypeptide cations to dehydroalanine (Dha). Subsequent activation of the modified polypeptide ions gives rise to specific cleavage N-terminal to the Dha residue. This process allows for the incorporation of selective cleavages in the structural characterization of polypeptide ions. An ion/ion reaction within the mass spectrometer between a multiply protonated polypeptide and the sulfate radical anion introduces a radical site into the multiply protonated polypeptide reactant. Subsequent collisional activation of the polypeptide radical cation gives rise to radical side chain loss from one of several particular amino acid side chains (e.g., leucine, asparagine, lysine, glutamine, and glutamic acid) to yield a Dha residue. The Dha residues facilitate preferential backbone cleavages to produce signature c- and z-ions, demonstrated with cations derived from melittin, mechano growth factor (MGF), and ubiquitin. The efficiencies for radical side chain loss and for subsequent generation of specific c- and z-ions have been examined as functions of precursor ion charge state and activation conditions using cations of ubiquitin as a model for a small protein. It is noted that these efficiencies are not strongly dependent on ion trap collisional activation conditions but are sensitive to precursor ion charge state. Moderate to low charge states show the greatest overall yields for the specific Dha cleavages, whereas small molecule losses (e.g., water/ammonia) dominate at the lowest charge states and proton catalyzed amide bond cleavages that give rise to b- and y-ions tend to dominate at high charge states. [Figure not available: see fulltext.

ATP-Dependent C–F Bond Cleavage Allows the Complete Degradation of 4-Fluoroaromatics without Oxygen

Directory of Open Access Journals (Sweden)

Oliver Tiedt

2016-08-01

Full Text Available Complete biodegradation of the abundant and persistent fluoroaromatics requires enzymatic cleavage of an arylic C–F bond, probably the most stable single bond of a biodegradable organic molecule. While in aerobic microorganisms defluorination of fluoroaromatics is initiated by oxygenases, arylic C–F bond cleavage has never been observed in the absence of oxygen. Here, an oxygen-independent enzymatic aryl fluoride bond cleavage is described during the complete degradation of 4-fluorobenzoate or 4-fluorotoluene to CO2 and HF in the denitrifying Thauera aromatica: the ATP-dependent defluorination of 4-fluorobenzoyl-coenzyme A (4-F-BzCoA to benzoyl-coenzyme A (BzCoA and HF, catalyzed by class I BzCoA reductase (BCR. Adaptation to growth with the fluoroaromatics was accomplished by the downregulation of a promiscuous benzoate-CoA ligase and the concomitant upregulation of 4-F-BzCoA-defluorinating/dearomatizing BCR on the transcriptional level. We propose an unprecedented mechanism for reductive arylic C–F bond cleavage via a Birch reduction-like mechanism resulting in a formal nucleophilic aromatic substitution. In the proposed anionic 4-fluorodienoyl-CoA transition state, fluoride elimination to BzCoA is favored over protonation to a fluorinated cyclic dienoyl-CoA.

MONITORING OF MINING

Directory of Open Access Journals (Sweden)

Berislav Šebečić

1996-12-01

Full Text Available The way mining was monitored in the past depended on knowledge, interest and the existing legal regulations. Documentary evidence about this work can be found in archives, libraries and museums. In particular, there is the rich archival material (papers and books concerning the work of the one-time Imperial and Royal Mining Captaincies in Zagreb, Zadar, Klagenfurt and Split, A minor part of the documentation has not yet been transferred to Croatia. From mining handbooks and books we can also find out about mining in Croatia. In the context of Austro-Hungary. For example, we can find out that the first governorships in Zagreb and Zadar headed the Ban, Count Jelacic and Baron Mamula were also the top mining authorities, though this, probably from political motives, was suppressed in the guides and inventories or the Mining Captaincies. At the end of the 1850s, Croatia produced 92-94% of sea salt, up to 8.5% of sulphur, 19.5% of asphalt and 100% of oil for the Austro-Hungarian empire. From data about mining in the Split Mining Captaincy, prepared for the Philadephia Exhibition, it can be seen that in the exploratory mining operations in which there were 33,372 independent mines declared in 1925 they were looking mainly for bauxite (60,0%, then dark coal (19,0%, asphalts (10.3% and lignites (62%. In 1931, within the area covered by the same captaincy, of 74 declared mines, only 9 were working. There were five coal mines, three bauxite mines and one for asphalt. I suggest that within state institution, the Mining Captaincy or Authority be renewed, or that a Mining and Geological Authority be set ap, which would lead to the more complete affirmation of Croatian mining (the paper is published in Croatian.

Mechanisms for lymphocytic choriomeningitis virus glycoprotein cleavage, transport, and incorporation into virions

International Nuclear Information System (INIS)

Kunz, Stefan; Edelmann, Kurt H.; Torre, Juan-Carlos de la; Gorney, Robert; Oldstone, Michael B.A.

2003-01-01

The glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) serves as virus attachment protein to its receptor on host cells and is a key determinant for cell tropism, pathogenesis, and epidemiology of the virus. The GP of LCMV is posttranslationally cleaved by the subtilase SKI-1/S1P into two subunits, the peripheral GP1, which is implicated in receptor binding, and the transmembrane GP2 that is structurally similar to the fusion active membrane proximal portions of the glycoproteins of other enveloped viruses. The present study shows that cleavage by SKI-1/S1P is not required for cell surface expression of LCMVGP on infected cells but is essential for its incorporation into virions and for the production of infectious virus particles. In absence of SKI-1/S1P cleavage, cell-to-cell propagation of the virus was markedly reduced. Further, proteolytic processing of LCMVGP depends on the presence of a cluster of basic amino acids at the C-terminus of the cytoplasmic domain of GP2, a structural motif that is conserved in Old World arenaviruses. The effect of the truncation of the cytoplasmic tail on cleavage suggests a structural interdependence between the cytoplasmic domain and the ectodomains of LCMVGP

Sequence specific inhibition of DNA restriction enzyme cleavage by PNA

DEFF Research Database (Denmark)

Nielsen, P.E.; Egholm, M.; Berg, R.H.

1993-01-01

Plasmids containing double-stranded 10-mer PNA (peptide nucleic acid chimera) targets proximally flanked by two restriction enzyme sites were challenged with the complementary PNA or PNAs having one or two mismatches, and the effect on the restriction enzyme cleavage of the flanking sites was ass...

Cleavage of desmin by cysteine proteases: Calpains and cathepsin B

DEFF Research Database (Denmark)

Baron, Caroline; Jacobsen, S.; Purslow, P.P.

2004-01-01

The intermediate filament protein, desmin, was purified from pork longissimus dorsi and incubated with either P-calpain, m-calpain or cathepsin B. Proteolysis of desmin was followed using SDS-PAGE and Western blotting. After incubation of desmin with the proteases, cleavage sites on the desmin mo...

Gold-Mining

DEFF Research Database (Denmark)

Raaballe, J.; Grundy, B.D.

2002-01-01

  Based on standard option pricing arguments and assumptions (including no convenience yield and sustainable property rights), we will not observe operating gold mines. We find that asymmetric information on the reserves in the gold mine is a necessary and sufficient condition for the existence...... of operating gold mines. Asymmetric information on the reserves in the mine implies that, at a high enough price of gold, the manager of high type finds the extraction value of the company to be higher than the current market value of the non-operating gold mine. Due to this under valuation the maxim of market...

The mining methods at the Fraisse mine

International Nuclear Information System (INIS)

Heurley, P.; Vervialle, J.P.

1985-01-01

The Fraisse mine is one of the four underground mines of the La Crouzille mining divisions of Cogema. Faced with the necessity to mechanize its workings, this mine also had to satisfy a certain number of stringent demands. This has led to concept of four different mining methods for the four workings at present in active operation at this pit, which nevertheless preserve the basic ideas of the methods of top slicing under concrete slabs (TSS) or horizontal cut-and-fill stopes (CFS). An electric scooptram is utilized. With this type of vehicle the stringent demands for the introduction of means for fire fighting and prevention are reduced to a minimum. Finally, the dimensions of the vehicles and the operation of these methods result in a net-to-gross tonnages of close to 1, i.e. a maximum output, combined with a minimum of contamination [fr

Activation of apurinic/apyrimidinic endonuclease in human cells by reactive oxygen species and its correlation with their adaptive response to genotoxicity of free radicals

Science.gov (United States)

Ramana, Chilakamarti V.; Boldogh, Istvan; Izumi, Tadahide; Mitra, Sankar

1998-01-01

Apurinic/apyrimidinic (AP) endonuclease (APE; EC 4.2.99.18) plays a central role in repair of DNA damage due to reactive oxygen species (ROS) because its DNA 3′-phosphoesterase activity removes 3′ blocking groups in DNA that are generated by DNA glycosylase/AP-lyases during removal of oxidized bases and by direct ROS reaction with DNA. The major human APE (APE-1) gene is activated selectively by sublethal levels of a variety of ROS and ROS generators, including ionizing radiation, but not by other genotoxicants—e.g., UV light and alkylating agents. Increased expression of APE mRNA and protein was observed both in the HeLa S3 tumor line and in WI 38 primary fibroblasts, and it was accompanied by translocation of the endonuclease to the nucleus. ROS-treated cells showed a significant increase in resistance to the cytotoxicity of such ROS generators as H2O2 and bleomycin, but not to UV light. This “adaptive response” appears to result from enhanced repair of cytotoxic DNA lesions due to an increased activity of APE-1, which may be limiting in the base excision repair process for ROS-induced toxic lesions. PMID:9560228

[Endonuclease modified comet assay for oxidative DNA damage induced by detection of genetic toxicants].

Science.gov (United States)

Zhao, Jian; Li, Hongli; Zhai, Qingfeng; Qiu, Yugang; Niu, Yong; Dai, Yufei; Zheng, Yuxin; Duan, Huawei

2014-03-01

The aim of this study was to investigate the use of the lesion-specific endonucleases-modified comet assay for analysis of DNA oxidation in cell lines. DNA breaks and oxidative damage were evaluated by normal alkaline and formamidopyrimidine-DNA-glycosylase (FPG) modified comet assays. Cytotoxicity were assessed by MTT method. The human bronchial epithelial cell (16HBE) were treated with benzo (a) pyrene (B(a)P), methyl methanesulfonate (MMS), colchicine (COL) and vincristine (VCR) respectively, and the dose is 20 µmol/L, 25 mg/ml, 5 mg/L and 0.5 mg/L for 24 h, respectively. Oxidative damage was also detected by levels of reactive oxygen species in treated cells. Four genotoxicants give higher cytotoxicity and no significant changes on parameters of comet assay treated by enzyme buffer. Cell survival rate were (59.69 ± 2.60) %, (54.33 ± 2.81) %, (53.11 ± 4.00) %, (51.43 ± 3.92) % in four groups, respectively. There was the direct DNA damage induced by test genotoxicants presented by tail length, Olive tail moment (TM) and tail DNA (%) in the comet assay. The presence of FPG in the assays increased DNA migration in treated groups when compared to those without it, and the difference was statistically significant which indicated that the clastogen and aneugen could induce oxidative damage in DNA strand. In the three parameters, the Olive TM was changed most obviously after genotoxicants treatment. In the contrast group, the Olive TM of B(a) P,MMS, COL,VCR in the contrast groups were 22.99 ± 17.33, 31.65 ± 18.86, 19.86 ± 9.56 and 17.02 ± 9.39, respectively, after dealing with the FPG, the Olive TM were 34.50 ± 17.29, 43.80 ± 10.06, 33.10 ± 12.38, 28.60 ± 10.53, increased by 58.94%, 38.48%, 66.86% and 68.21%, respectively (t value was 3.91, 3.89, 6.66 and 3.87, respectively, and all P comet assay appears more specific for detecting oxidative DNA damage induced by genotoxicants exposure, and the application of comet assay will be expanded. The endonuclease

The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

Science.gov (United States)

Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H

2003-01-01

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

Mined-out land

International Nuclear Information System (INIS)

Reinsalu, Enno; Toomik, Arvi; Valgma, Ingo

2002-01-01

Estonian mineral resources are deposited in low depth and mining fields are large, therefore vast areas are affected by mining. There are at least 800 deposits with total area of 6,000 km 2 and about the same number of underground mines, surface mines, peat fields, quarries, and sand and gravel pits. The deposits cover more than 10% of Estonian mainland. The total area of operating mine claims exceeds 150 km 2 that makes 0.3 % of Estonian area. The book is written mainly for the people who are living or acting in the area influenced by mining. The observations and research could benefit those who are interested in geography and environment, who follow formation and look of mined-out landscapes. The book contains also warnings for careless people on and under the surface of the mined-out land. Part of the book contains results of the research made in 1968-1993 by the first two authors working at the Estonian branch of A.Skochinsky Institute of Mining. Since 1990, Arvi Toomik continued this study at the Northeastern section of the Institute of Ecology of Tallinn Pedagogical University. Enno Reinsalu studied aftereffects of mining at the Mining Department of Tallinn Technical University from 1998 to 2000. Geographical Information System for Mining was studied by Ingo Valgma within his doctoral dissertation, and this book is one of the applications of his study

Can laccases catalyze bond cleavage in lignin?

DEFF Research Database (Denmark)

Munk, Line; Sitarz, Anna Katarzyna; Kalyani, Dayanand

2015-01-01

illustrations of the putative laccase catalyzed reactions, including the possible reactions of the reactive radical intermediates taking place after the initial oxidation of the phenol-hydroxyl groups, we show that i) Laccase activity is able to catalyze bond cleavage in low molecular weight phenolic lignin......-substituted phenols, benzenethiols, polyphenols, and polyamines, which may be oxidized. In addition, the currently available analytical methods that can be used to detect enzyme catalyzed changes in lignin are summarized, and an improved nomenclature for unequivocal interpretation of the action of laccases on lignin...

Trust Mines

Science.gov (United States)

The United States and the Navajo Nation entered into settlement agreements that provide funds to conduct investigations and any needed cleanup at 16 of the 46 priority mines, including six mines in the Northern Abandoned Uranium Mine Region.

Effects of mutations in the VP2/VP4 cleavage site of Swine vesicular disease virus on RNA encapsidation and viral infectivity

NARCIS (Netherlands)

Rebel, J.M.J.; Leendertse, C.H.; Dekker, A.; Moormann, R.J.M.

2003-01-01

We studied VP0 cleavage of Swine vesicular disease virus (SVDV), a member of the Picornaviridae using a full-length cDNA copy of the Dutch SVDV isolate. The influences of mutations, introduced at the cleavage site of SVDV, on VP0 cleavage, RNA encapsidation and viral infection were studied. Double

Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis.

Science.gov (United States)

Senger, Moritz; Stripp, Sven T; Soboh, Basem

2017-07-14

Metalloenzymes catalyze complex and essential processes, such as photosynthesis, respiration, and nitrogen fixation. For example, bacteria and archaea use [NiFe]-hydrogenases to catalyze the uptake and release of molecular hydrogen (H 2 ). [NiFe]-hydrogenases are redox enzymes composed of a large subunit that harbors a NiFe(CN) 2 CO metallo-center and a small subunit with three iron-sulfur clusters. The large subunit is synthesized with a C-terminal extension, cleaved off by a specific endopeptidase during maturation. The exact role of the C-terminal extension has remained elusive; however, cleavage takes place exclusively after assembly of the [NiFe]-cofactor and before large and small subunits form the catalytically active heterodimer. To unravel the functional role of the C-terminal extension, we used an enzymatic in vitro maturation assay that allows synthesizing functional [NiFe]-hydrogenase-2 of Escherichia coli from purified components. The maturation process included formation and insertion of the NiFe(CN) 2 CO cofactor into the large subunit, endoproteolytic cleavage of the C-terminal extension, and dimerization with the small subunit. Biochemical and spectroscopic analysis indicated that the C-terminal extension of the large subunit is essential for recognition by the maturation machinery. Only upon completion of cofactor insertion was removal of the C-terminal extension observed. Our results indicate that endoproteolytic cleavage is a central checkpoint in the maturation process. Here, cleavage temporally orchestrates cofactor insertion and protein assembly and ensures that only cofactor-containing protein can continue along the assembly line toward functional [NiFe]-hydrogenase. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Web Mining

Science.gov (United States)

Fürnkranz, Johannes

The World-Wide Web provides every internet citizen with access to an abundance of information, but it becomes increasingly difficult to identify the relevant pieces of information. Research in web mining tries to address this problem by applying techniques from data mining and machine learning to Web data and documents. This chapter provides a brief overview of web mining techniques and research areas, most notably hypertext classification, wrapper induction, recommender systems and web usage mining.

Data mining, mining data : energy consumption modelling

Energy Technology Data Exchange (ETDEWEB)

Dessureault, S. [Arizona Univ., Tucson, AZ (United States)

2007-09-15

Most modern mining operations are accumulating large amounts of data on production and business processes. Data, however, provides value only if it can be translated into information that appropriate users can utilize. This paper emphasized that a new technological focus should emerge, notably how to concentrate data into information; analyze information sufficiently to become knowledge; and, act on that knowledge. Researchers at the Mining Information Systems and Operations Management (MISOM) laboratory at the University of Arizona have created a method to transform data into action. The data-to-action approach was exercised in the development of an energy consumption model (ECM), in partnership with a major US-based copper mining company, 2 software companies, and the MISOM laboratory. The approach begins by integrating several key data sources using data warehousing techniques, and increasing the existing level of integration and data cleaning. An online analytical processing (OLAP) cube was also created to investigate the data and identify a subset of several million records. Data mining algorithms were applied using the information that was isolated by the OLAP cube. The data mining results showed that traditional cost drivers of energy consumption are poor predictors. A comparison was made between traditional methods of predicting energy consumption and the prediction formed using data mining. Traditionally, in the mines for which data were available, monthly averages of tons and distance are used to predict diesel fuel consumption. However, this article showed that new information technology can be used to incorporate many more variables into the budgeting process, resulting in more accurate predictions. The ECM helped mine planners improve the prediction of energy use through more data integration, measure development, and workflow analysis. 5 refs., 11 figs.

Mining with communities

International Nuclear Information System (INIS)

Veiga, Marcello M.; Scoble, Malcolm; McAllister, Mary Louise

2001-01-01

To be considered as sustainable, a mining community needs to adhere to the principles of ecological sustainability, economic vitality and social equity. These principles apply over a long time span, covering both the life of the mine and post-mining closure. The legacy left by a mine to the community after its closure is emerging as a significant aspect of its planning. Progress towards sustainability is made when value is added to a community with respect to these principles by the mining operation during its life cycle. This article presents a series of cases to demonstrate the diverse potential challenges to achieving a sustainable mining community. These case studies of both new and old mining communities are drawn mainly from Canada and from locations abroad where Canadian companies are now building mines. The article concludes by considering various approaches that can foster sustainable mining communities and the role of community consultation and capacity building. (author)

On the mechanism of action of ribonucleases: dinucleotide cleavage catalyzed by imidazole and Zn2+.

OpenAIRE

Breslow, R; Huang, D L; Anslyn, E

1989-01-01

Cyclization/cleavage of the 2-(p-nitrophenyl) phosphate ester of propylene glycol is catalyzed by imidazole and, much more effectively, by Zn2+ with imidazole. In the latter case, the mechanism involves simultaneous Lewis acid/base catalysis. Similar Zn2+ and imidazole catalysis of cyclization/cleavage is seen with the dinucleotide 3',5'-UpU (uridylyluridine). Again, the zinc system is much more effective than is catalysis by imidazole alone, and in this case simultaneous Lewis acid/base cata...

Ideate about building green mine of uranium mining and metallurgy

International Nuclear Information System (INIS)

Shi Zuyuan

2012-01-01

Analysing the current situation of uranium mining and metallurgy; Setting up goals for green uranium mining and metallurgy, its fundamental conditions, Contents and measures. Putting forward an idea to combine green uranium mining and metallurgy with the state target for green mining, and keeping its own characteristics. (author)

Social big data mining

CERN Document Server

Ishikawa, Hiroshi

2015-01-01

Social Media. Big Data and Social Data. Hypotheses in the Era of Big Data. Social Big Data Applications. Basic Concepts in Data Mining. Association Rule Mining. Clustering. Classification. Prediction. Web Structure Mining. Web Content Mining. Web Access Log Mining, Information Extraction and Deep Web Mining. Media Mining. Scalability and Outlier Detection.

Force-Induced Calpain Cleavage of Talin Is Critical for Growth, Adhesion Development, and Rigidity Sensing.

Science.gov (United States)

Saxena, Mayur; Changede, Rishita; Hone, James; Wolfenson, Haguy; Sheetz, Michael P

2017-12-13

Cell growth depends upon formation of cell-matrix adhesions, but mechanisms detailing the transmission of signals from adhesions to control proliferation are still lacking. Here, we find that the scaffold protein talin undergoes force-induced cleavage in early adhesions to produce the talin rod fragment that is needed for cell cycle progression. Expression of noncleavable talin blocks cell growth, adhesion maturation, proper mechanosensing, and the related property of EGF activation of motility. Further, the expression of talin rod in the presence of noncleavable full-length talin rescues cell growth and other functions. The cleavage of talin is found in early adhesions where there is also rapid turnover of talin that depends upon calpain and TRPM4 activity as well as the generation of force on talin. Thus, we suggest that an important function of talin is its control over cell cycle progression through its cleavage in early adhesions.

Mining and mining authorities in Saarland 2016. Mining economy, mining technology, occupational safety, environmental protection, statistics, mining authority activities. Annual report; Bergbau und Bergbehoerden im Saarland 2016. Bergwirtschaft, Bergtechnik, Arbeitsschutz, Umweltschutz, Statistiken, Taetigkeiten der Bergbehoerden. Jahresbericht

Energy Technology Data Exchange (ETDEWEB)

NONE

2016-07-01

The annual report of the Saarland Upper Mining Authority provides an insight into the activities of mining authorities. Especially, the development of the black coal mining, safety and technology of mining as well as the correlation between mining and environment are stressed.

A Subset of Membrane-Altering Agents and γ-Secretase Modulators Provoke Nonsubstrate Cleavage by Rhomboid Proteases

Directory of Open Access Journals (Sweden)

Siniša Urban

2014-09-01

Full Text Available Rhomboid proteases are integral membrane enzymes that regulate cell signaling, adhesion, and organelle homeostasis pathways, making substrate specificity a key feature of their function. Interestingly, we found that perturbing the membrane pharmacologically in living cells had little effect on substrate processing but induced inappropriate cleavage of nonsubstrates by rhomboid proteases. A subclass of drugs known to modulate γ-secretase activity acted on the membrane directly and induced nonsubstrate cleavage by rhomboid proteases but left true substrate cleavage sites unaltered. These observations highlight an active role for the membrane in guiding rhomboid selectivity and caution that membrane-targeted drugs should be evaluated for cross-activity against membrane-resident enzymes that are otherwise unrelated to the intended drug target. Furthermore, some γ-secretase-modulating activity or toxicity could partly result from global membrane effects.

75 FR 17529 - High-Voltage Continuous Mining Machine Standard for Underground Coal Mines

Science.gov (United States)

2010-04-06

... High-Voltage Continuous Mining Machine Standard for Underground Coal Mines AGENCY: Mine Safety and... of high-voltage continuous mining machines in underground coal mines. It also revises MSHA's design...-- Underground Coal Mines III. Section-by-Section Analysis A. Part 18--Electric Motor-Driven Mine Equipment and...

Conjugation and Evaluation of Triazole?Linked Single Guide RNA for CRISPR?Cas9 Gene Editing

OpenAIRE

He, Kaizhang; Chou, Eldon T.; Begay, Shawn; Anderson, Emily M.; van?Brabant?Smith, Anja

2016-01-01

Abstract The CRISPR?Cas9 gene editing system requires Cas9 endonuclease and guide RNAs (either the natural dual RNA consisting of crRNA and tracrRNA or a chimeric single guide RNA) that direct site?specific double?stranded DNA cleavage. This communication describes a click ligation approach that uses alkyne?azide cycloaddition to generate a triazole?linked single guide RNA (sgRNA). The conjugated sgRNA shows efficient and comparable genome editing activity to natural dual RNA and unmodified s...

Mine water treatment

Energy Technology Data Exchange (ETDEWEB)

Komissarov, S V

1980-10-01

This article discusses composition of chemical compounds dissolved or suspended in mine waters in various coal basins of the USSR: Moscow basin, Kuzbass, Pechora, Kizelovsk, Karaganda, Donetsk and Chelyabinsk basins. Percentage of suspended materials in water depending on water source (water from water drainage system of dust suppression system) is evaluated. Pollution of mine waters with oils and coli bacteria is also described. Recommendations on construction, capacity of water settling tanks, and methods of mine water treatment are presented. In mines where coal seams 2 m or thicker are mined a system of two settling tanks should be used: in the upper one large grains are settled, in the lower one finer grains. The upper tank should be large enough to store mine water discharged during one month, and the lower one to store water discharged over two months. Salty waters from coal mines mining thin coal seams should be treated in a system of water reservoirs from which water evaporates (if climatic conditions permit). Mine waters from mines with thin coal seams but without high salt content can be treated in a system of long channels with water plants, which increase amount of oxygen in treated water. System of biological treatment of waste waters from mine wash-houses and baths is also described. Influence of temperature, sunshine and season of the year on efficiency of mine water treatment is also assessed. (In Russian)

Uranium mining

International Nuclear Information System (INIS)

2008-01-01

Full text: The economic and environmental sustainability of uranium mining has been analysed by Monash University researcher Dr Gavin Mudd in a paper that challenges the perception that uranium mining is an 'infinite quality source' that provides solutions to the world's demand for energy. Dr Mudd says information on the uranium industry touted by politicians and mining companies is not necessarily inaccurate, but it does not tell the whole story, being often just an average snapshot of the costs of uranium mining today without reflecting the escalating costs associated with the process in years to come. 'From a sustainability perspective, it is critical to evaluate accurately the true lifecycle costs of all forms of electricity production, especially with respect to greenhouse emissions, ' he says. 'For nuclear power, a significant proportion of greenhouse emissions are derived from the fuel supply, including uranium mining, milling, enrichment and fuel manufacture.' Dr Mudd found that financial and environmental costs escalate dramatically as the uranium ore is used. The deeper the mining process required to extract the ore, the higher the cost for mining companies, the greater the impact on the environment and the more resources needed to obtain the product. I t is clear that there is a strong sensitivity of energy and water consumption and greenhouse emissions to ore grade, and that ore grades are likely to continue to decline gradually in the medium to long term. These issues are critical to the current debate over nuclear power and greenhouse emissions, especially with respect to ascribing sustainability to such activities as uranium mining and milling. For example, mining at Roxby Downs is responsible for the emission of over one million tonnes of greenhouse gases per year and this could increase to four million tonnes if the mine is expanded.'

Crystallographic and Modeling Studies of RNase III Suggest a Mechanism for Double-Stranded RNA Cleavage | Center for Cancer Research

Science.gov (United States)

Background: Ribonuclease III belongs to the family of Mg2+-dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1–2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an

DNA cleavage enzymes for treatment of persistent viral infections: Recent advances and the pathway forward

Energy Technology Data Exchange (ETDEWEB)

Weber, Nicholas D., E-mail: nweber@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Department of Laboratory Medicine, University of Washington, Seattle, WA 98195 (United States); Aubert, Martine, E-mail: maubert@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Dang, Chung H., E-mail: cdang@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Stone, Daniel, E-mail: dstone2@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Jerome, Keith R., E-mail: kjerome@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Department of Laboratory Medicine, University of Washington, Seattle, WA 98195 (United States); Department of Microbiology, University of Washington, Seattle, WA 98195 (United States)

2014-04-15

Treatment for most persistent viral infections consists of palliative drug options rather than curative approaches. This is often because long-lasting viral DNA in infected cells is not affected by current antivirals, providing a source for viral persistence and reactivation. Targeting latent viral DNA itself could therefore provide a basis for novel curative strategies. DNA cleavage enzymes can be used to induce targeted mutagenesis of specific genes, including those of exogenous viruses. Although initial in vitro and even in vivo studies have been carried out using DNA cleavage enzymes targeting various viruses, many questions still remain concerning the feasibility of these strategies as they transition into preclinical research. Here, we review the most recent findings on DNA cleavage enzymes for human viral infections, consider the most relevant animal models for several human viral infections, and address issues regarding safety and enzyme delivery. Results from well-designed in vivo studies will ideally provide answers to the most urgent remaining questions, and allow continued progress toward clinical application. - Highlights: • Recent in vitro and in vivo results for DNA cleavage enzymes targeting persistent viral infections. • Analysis of the best animal models for testing enzymes for HBV, HSV, HIV and HPV. • Challenges facing in vivo delivery of therapeutic enzymes for persistent viral infections. • Safety issues to be addressed with proper animal studies.

Hemoglobin cleavage site-specificity of the Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3.

Directory of Open Access Journals (Sweden)

Shoba Subramanian

Full Text Available The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P(1 - P(4 amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P(2 position. Second, with overlapping peptides spanning alpha and beta globin and proteolysis-dependent (18O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P(2 Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents.

Internet technologies in the mining industry. Towards unattended mining systems

Energy Technology Data Exchange (ETDEWEB)

Krzykawski, Michal [FAMUR Group, Katowice (Poland)

2009-08-27

Global suppliers of longwall systems focus mainly on maximising the efficiency of the equipment they manufacture. Given the fact that, since 2004, coal demand on world markets has been constantly on the increase, even during an economic downturn, this endeavour seems fully justified. However, it should be remembered that maximum efficiency must be accompanied by maximum safety of all underground operations. This statement is based on the belief that the mining industry, which exploits increasingly deep and dangerous coal beds, faces the necessity to implement comprehensive IT systems for managing all mining processes and, in the near future, to use unmanned mining systems, fully controllable from the mine surface. The computerisation of mines is an indispensable element of the development of the world mining industry, a belief which has been put into practice with e-mine, developed by the FAMUR Group. (orig.)

Glutamic Acid Selective Chemical Cleavage of Peptide Bonds.

Science.gov (United States)

Nalbone, Joseph M; Lahankar, Neelam; Buissereth, Lyssa; Raj, Monika

2016-03-04

Site-specific hydrolysis of peptide bonds at glutamic acid under neutral aqueous conditions is reported. The method relies on the activation of the backbone amide chain at glutamic acid by the formation of a pyroglutamyl (pGlu) imide moiety. This activation increases the susceptibility of a peptide bond toward hydrolysis. The method is highly specific and demonstrates broad substrate scope including cleavage of various bioactive peptides with unnatural amino acid residues, which are unsuitable substrates for enzymatic hydrolysis.

The Role of G-Protein-Coupled Receptor Proteolysis Site Cleavage of Polycystin-1 in Renal Physiology and Polycystic Kidney Disease

Directory of Open Access Journals (Sweden)

Marie Trudel

2016-01-01

Full Text Available Polycystin-1 (PC1 plays an essential role in renal tubular morphogenesis, and PC1 dysfunction causes human autosomal dominant polycystic kidney disease. A fundamental characteristic of PC1 is post-translational modification via cleavage at the juxtamembrane GPCR proteolysis site (GPS motif that is part of the larger GAIN domain. Given the considerable biochemical complexity of PC1 molecules generated in vivo by this process, GPS cleavage has several profound implications on the intracellular trafficking and localization in association with their particular function. The critical nature of GPS cleavage is further emphasized by the increasing numbers of PKD1 mutations that significantly affect this cleavage process. The GAIN domain with the GPS motif therefore represents the key structural element with fundamental importance for PC1 and might be polycystic kidney disease’s (PKD Achilles’ heel in a large spectrum of PKD1 missense mutations. We highlight the central roles of PC1 cleavage for the regulation of its biogenesis, intracellular trafficking and function, as well as its significance in polycystic kidney disease.

Hold your horSSEs: controlling structure-selective endonucleases MUS81 and Yen1/GEN1.

Science.gov (United States)

Blanco, Miguel G; Matos, Joao

2015-01-01

Repair of DNA lesions through homologous recombination promotes the establishment of stable chromosomal interactions. Multiple helicases, topoisomerases and structure-selective endonucleases (SSEs) act upon recombining joint molecules (JMs) to disengage chromosomal connections and safeguard chromosome segregation. Recent studies on two conserved SSEs - MUS81 and Yen1/GEN1- uncovered multiple layers of regulation that operate to carefully tailor JM-processing according to specific cellular needs. Temporal restriction of SSE function imposes a hierarchy in pathway usage that ensures efficient JM-processing while minimizing reciprocal exchanges between the recombining DNAs. Whereas a conserved strategy of fine-tuning SSE functions exists in different model systems, the precise molecular mechanisms to implement it appear to be significantly different. Here, we summarize the current knowledge on the cellular switches that are in place to control MUS81 and Yen1/GEN1 functions.

Requirements and opportunities for mining engineers in the mining industry abroad

Energy Technology Data Exchange (ETDEWEB)

Albrecht, E

1987-04-09

The decline of the German mining industry and the increasing industrialization of mining is forcing ever greater numbers of young German mining graduates to build their careers abroad. The requirements for this - apart from the technical qualifications are a good knowledge of foreign languages and a readiness to leave Germany for a long time, even for ever. If the young mining graduate accepts these conditions, numerous professional opportunities will open up for him, both with German mining companies with interests abroad, in mining supply companies and consultancy firms and with foreign companies. 6 references.

Mining

Directory of Open Access Journals (Sweden)

Khairullah Khan

2014-09-01

Full Text Available Opinion mining is an interesting area of research because of its applications in various fields. Collecting opinions of people about products and about social and political events and problems through the Web is becoming increasingly popular every day. The opinions of users are helpful for the public and for stakeholders when making certain decisions. Opinion mining is a way to retrieve information through search engines, Web blogs and social networks. Because of the huge number of reviews in the form of unstructured text, it is impossible to summarize the information manually. Accordingly, efficient computational methods are needed for mining and summarizing the reviews from corpuses and Web documents. This study presents a systematic literature survey regarding the computational techniques, models and algorithms for mining opinion components from unstructured reviews.

PREVENTION OF ACID MINE DRAINAGE GENERATION FROM OPEN-PIT MINE HIGHWALLS

Science.gov (United States)

Exposed, open pit mine highwalls contribute significantly to the production of acid mine drainage (AMD) thus causing environmental concerns upon closure of an operating mine. Available information on the generation of AMD from open-pit mine highwalls is very limit...

Cell elongation is an adaptive response for clearing long chromatid arms from the cleavage plane

Science.gov (United States)

Kotadia, Shaila; Montembault, Emilie; Sullivan, William

2012-01-01

Chromosome segregation must be coordinated with cell cleavage to ensure correct transmission of the genome to daughter cells. Here we identify a novel mechanism by which Drosophila melanogaster neuronal stem cells coordinate sister chromatid segregation with cleavage furrow ingression. Cells adapted to a dramatic increase in chromatid arm length by transiently elongating during anaphase/telophase. The degree of cell elongation correlated with the length of the trailing chromatid arms and was concomitant with a slight increase in spindle length and an enlargement of the zone of cortical myosin distribution. Rho guanine-nucleotide exchange factor (Pebble)–depleted cells failed to elongate during segregation of long chromatids. As a result, Pebble-depleted adult flies exhibited morphological defects likely caused by cell death during development. These studies reveal a novel pathway linking trailing chromatid arms and cortical myosin that ensures the clearance of chromatids from the cleavage plane at the appropriate time during cytokinesis, thus preserving genome integrity. PMID:23185030

Metabolic cleavage of cell-penetrating peptides in contact with epithelial models

DEFF Research Database (Denmark)

Tréhin, Rachel; Nielsen, Hanne Mørck; Jahnke, Heinz-Georg

2004-01-01

We assessed the metabolic degradation kinetics and cleavage patterns of some selected CPP (cell-penetrating peptides) after incubation with confluent epithelial models. Synthesis of N-terminal CF [5(6)-carboxyfluorescein]-labelled CPP, namely hCT (human calcitonin)-derived sequences, Tat(47-57) a...

A direction of developing a mining method and mining complexes

Energy Technology Data Exchange (ETDEWEB)

Gabov, V.V.; Efimov, I.A. [St. Petersburg State Mining Institute, St. Petersburg (Russian Federation). Vorkuta Branch

1996-12-31

The analyses of a mining method as a main factor determining the development stages of mining units is presented. The paper suggests a perspective mining method which differs from the known ones by following peculiarities: the direction selectivity of cuts with regard to coal seams structure; the cutting speed, thickness and succession of dusts. This method may be done by modulate complexes (a shield carrying a cutting head for coal mining), their mining devices being supplied with hydraulic drive. An experimental model of the module complex has been developed. 2 refs.

Carotenoid Cleavage Oxygenases from Microbes and Photosynthetic Organisms: Features and Functions

Directory of Open Access Journals (Sweden)

Oussama Ahrazem

2016-10-01

Full Text Available Apocarotenoids are carotenoid-derived compounds widespread in all major taxonomic groups, where they play important roles in different physiological processes. In addition, apocarotenoids include compounds with high economic value in food and cosmetics industries. Apocarotenoid biosynthesis starts with the action of carotenoid cleavage dioxygenases (CCDs, a family of non-heme iron enzymes that catalyze the oxidative cleavage of carbon–carbon double bonds in carotenoid backbones through a similar molecular mechanism, generating aldehyde or ketone groups in the cleaving ends. From the identification of the first CCD enzyme in plants, an increasing number of CCDs have been identified in many other species, including microorganisms, proving to be a ubiquitously distributed and evolutionarily conserved enzymatic family. This review focuses on CCDs from plants, algae, fungi, and bacteria, describing recent progress in their functions and regulatory mechanisms in relation to the different roles played by the apocarotenoids in these organisms.

Base substitutions at scissile bond sites are sufficient to alter RNA-binding and cleavage activity of RNase III.

Science.gov (United States)

Kim, Kyungsub; Sim, Se-Hoon; Jeon, Che Ok; Lee, Younghoon; Lee, Kangseok

2011-02-01

RNase III, a double-stranded RNA-specific endoribonuclease, degrades bdm mRNA via cleavage at specific sites. To better understand the mechanism of cleavage site selection by RNase III, we performed a genetic screen for sequences containing mutations at the bdm RNA cleavage sites that resulted in altered mRNA stability using a transcriptional bdm'-'cat fusion construct. While most of the isolated mutants showed the increased bdm'-'cat mRNA stability that resulted from the inability of RNase III to cleave the mutated sequences, one mutant sequence (wt-L) displayed in vivo RNA stability similar to that of the wild-type sequence. In vivo and in vitro analyses of the wt-L RNA substrate showed that it was cut only once on the RNA strand to the 5'-terminus by RNase III, while the binding constant of RNase III to this mutant substrate was moderately increased. A base substitution at the uncleaved RNase III cleavage site in wt-L mutant RNA found in another mutant lowered the RNA-binding affinity by 11-fold and abolished the hydrolysis of scissile bonds by RNase III. Our results show that base substitutions at sites forming the scissile bonds are sufficient to alter RNA cleavage as well as the binding activity of RNase III. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Snake venom serine proteinases specificity mapping by proteomic identification of cleavage sites.

Science.gov (United States)

Zelanis, André; Huesgen, Pitter F; Oliveira, Ana Karina; Tashima, Alexandre K; Serrano, Solange M T; Overall, Christopher M

2015-01-15

Many snake venom toxins are serine proteases but their specific in vivo targets are mostly unknown. Various act on components of the coagulation cascade, and fibrinolytic and kallikrein-kinin systems to trigger various pathological effects observed in the envenomation. Despite showing high similarity in terms of primary structure snake venom serine proteinases (SVSPs) show exquisite specificity towards macromolecular substrates. Therefore, the characterization of their peptide bond specificity is important for understanding the active site preference associated with effective proteolysis as well as for the design of peptide substrates and inhibitors. Bothrops jararaca contains various SVSPs among which Bothrops protease A is a specific fibrinogenolytic agent and PA-BJ is a platelet-activating enzyme. In this study we used proteome derived peptide libraries in the Proteomic Identification of protease Cleavage Sites (PICS) approach to explore the peptide bond specificity of Bothrops protease A and PA-BJ in order to determine their individual peptide cleavage sequences. A total of 371 cleavage sites (208 for Bothrops protease A and 163 for PA-BJ) were detected and both proteinases displayed a clear preference for arginine at the P1 position. Moreover, the analysis of the specificity profiles of Bothrops protease A and PA-BJ revealed subtle differences in the preferences along P6-P6', despite a common yet unusual preference for Pro at P2. Taken together, these results map the subsite specificity of both SVSPs and shed light in the functional differences between these proteinases. Proteolysis is key to various pathological effects observed upon envenomation by viperid snakes. The use of the Proteomic Identification of protease Cleavage Sites (PICS) approach for the easy mapping of proteinase subsite preferences at both the prime- and non-prime sides concurrently gives rise to a fresh understanding of the interaction of the snake venom serine proteinases with peptide and

Unconjugated Bilirubin Inhibits Proteolytic Cleavage of von Willebrand Factor by ADAMTS13 Protease

Science.gov (United States)

Lu, Rui-Nan; Yang, Shangbin; Wu, Haifeng M.; Zheng, X. Long

2015-01-01

Summary Background Bilirubin is a yellow breakdown product of heme catabolism. Increased serum levels of unconjugated bilirubin are conditions commonly seen in premature neonates and adults with acute hemolysis including thrombotic microangiopathy. Previous studies have shown that unconjugated bilirubin lowers plasma ADAMTS13 activity, but the mechanism is not fully understood. Objectives The study is to determine whether unconjugated bilirubin directly inhibits the cleavage of von Willebrand factor (VWF) and its analogs by ADAMTS13. Methods Fluorogenic, SELDI-TOF mass spectrometric assay, and Western blotting analyses were employed to address this question. Results Unconjugated bilirubin inhibits the cleavage of F485-rVWF73-H, D633-rVWF73-H, and GST-rVWF71-11K by ADAMTS13 in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of ~13 μM, ~70 μM, and ~17 μM, respectively. Unconjugated bilirubin also dose-dependently inhibits the cleavage of multimeric VWF by ADAMTS13 under denaturing conditions. The inhibitory activity of bilirubin on the cleavage of D633-rVWF73-H and multimeric VWF, but not F485-rVWF73-H, was eliminated after incubation with bilirubin oxidase that converts bilirubin to biliverdin. Furthermore, plasma ADAMTS13 activity in patients with hyperbilirubinemia is lower prior to than after treatment with bilirubin oxidase. Conclusions unconjugated bilirubin directly inhibits ADAMTS13’s ability to cleave both peptidyl and native VWF substrates in addition to its interference with certain fluorogenic assays. Our findings may help proper interpretation of ADAMTS13 results under pathological conditions. Whether elevated serum unconjugated bilirubin has an adverse effect in vivo remains to be determined in our future study. PMID:25782102

30 CFR 780.27 - Reclamation plan: Surface mining near underground mining.

Science.gov (United States)

2010-07-01

... 30 Mineral Resources 3 2010-07-01 2010-07-01 false Reclamation plan: Surface mining near underground mining. 780.27 Section 780.27 Mineral Resources OFFICE OF SURFACE MINING RECLAMATION AND ENFORCEMENT, DEPARTMENT OF THE INTERIOR SURFACE COAL MINING AND RECLAMATION OPERATIONS PERMITS AND COAL...

Minimizing the Impact of Mining Activities for Sustainable Mined-Out ...

African Journals Online (AJOL)

Minimizing the Impact of Mining Activities for Sustainable Mined-Out Area ... sensing and Geographical Information System (GIS) in assessing environmental impact of ... Keywords: Solid mineral, Impact assessment, Mined-out area utilization, ...

Archveyor{trademark} automated mining system - implementation at the Conant mine

Energy Technology Data Exchange (ETDEWEB)

Hofmann, W.J. [Arch of Illinois, Percy, IL (United States)

1997-12-01

Arch Mineral Corporation, through the Arch Technology Department, has developed an automated continuous haulage mining system called the `Archveyor{trademark}`. The original technology came from a Russian patent. Kloeckner-Becorit (K-B) further developed the system and called it the `Mobile Conveyor`. This system was utilized in both coal and trona mines in the United States and Canada. Consolidation Coal designed their version of this continuous haulage system, called the `Tramveyor`. The Tramveyor is presently operating in their Dilworth Mine, in Pennsylvania. This system has no computer guidance system related to the continuous miner or the Tramveyor. Arch Mineral Corporation has further developed this continuous haulage mining system. Their system is a programmable, logic-controlled (PLC) automated mining system. A highwall version of the Archveyor{trademark} is being operated at Arch of Wyoming near Hanna, Wyoming. This paper introduces the first underground version of Archveyor{trademark} to be implemented at Conant Mine in southern Illinois. During the development process, the Archveyor{trademark} mining system consists of a continuous miner, a bolter car, the Archveyor{trademark} (itself), a stageloader, and an operator`s cab. During the secondary mining process the bolter car is taken out of the system.

Monoclonal antibodies to the reactive centre loop (RCL) of human corticosteroid-binding globulin (CBG) can protect against proteolytic cleavage.

Science.gov (United States)

Lewis, John G; Elder, Peter A

2017-07-01

Corticosteroid-binding globulin (CBG) binds most of the cortisol in circulation and is a non-functional member of the family of serine protease inhibitors (serpins) with an exposed elastase sensitive reactive centre loop (RCL). The RCL can be cleaved by human neutrophil elastase, released from activated neutrophils, and can also be cleaved at nearby site(s) by elastase released by Pseudomonas aeruginosa, and at two further sites, also within the RCL, by bovine chymotrypsin. Cleavage of the RCL results in a conformational change accompanied by a marked decrease in affinity for cortisol and hence its release at the site of proteolysis. These cleavages are irreversible and the similar half-lives of cleaved and intact CBG could mean that there may be some advantage in slowing the rate of CBG cleavage in acute inflammation thereby increasing the proportion of intact CBG in circulation. Here we show, for the first time, that pre-incubation of tethered human CBG with two monoclonal antibodies to the RCL of CBG protects against cleavage by all three enzymes. Furthermore, in plasma, pre-incubation with both RCL monoclonal antibodies delays neutrophil elastase cleavage of the RCL and one of these RCL monoclonal antibodies also delays bovine chymotrypsin cleavage of the RCL. These findings may provide a basis and rationale for the concept of the use of RCL antibodies as therapeutic agents to effectively increase the proportion of intact CBG in circulation which may be of benefit in acute inflammation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Photoenhanced Oxidative DNA Cleavage with Non-Heme Iron(II) Complexes

NARCIS (Netherlands)

Li, Qian; Browne, Wesley R.; Roelfes, Gerard

2010-01-01

The DNA cleavage activity of iron(II) complexes of a series of monotopic pentadentate N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine (N4Py)-derived ligands (1-5) was investigated under laser irradiation at 473, 400.8, and 355 nm in the absence of a reducing agent and compared to that under

Management of mining-related damages in abandoned underground coal mine areas using GIS

International Nuclear Information System (INIS)

Lee, U.J.; Kim, J.A.; Kim, S.S.; Kim, W.K.; Yoon, S.H.; Choi, J.K.

2005-01-01

The mining-related damages such as ground subsidence, acid mine drainage (AMD), and deforestation in the abandoned underground coal mine areas become an object of public concern. Therefore, the system to manage the mining-related damages is needed for the effective drive of rehabilitation activities. The management system for Abandoned Underground Coal Mine using GIS includes the database about mining record and information associated with the mining-related damages and application programs to support mine damage prevention business. Also, this system would support decision-making policy for rehabilitation and provide basic geological data for regional construction works in abandoned underground coal mine areas. (authors)

Text Mining.

Science.gov (United States)

Trybula, Walter J.

1999-01-01

Reviews the state of research in text mining, focusing on newer developments. The intent is to describe the disparate investigations currently included under the term text mining and provide a cohesive structure for these efforts. A summary of research identifies key organizations responsible for pushing the development of text mining. A section…

Surface mining

Science.gov (United States)

Robert Leopold; Bruce Rowland; Reed Stalder

1979-01-01

The surface mining process consists of four phases: (1) exploration; (2) development; (3) production; and (4) reclamation. A variety of surface mining methods has been developed, including strip mining, auger, area strip, open pit, dredging, and hydraulic. Sound planning and design techniques are essential to implement alternatives to meet the myriad of laws,...

Uranium mining

International Nuclear Information System (INIS)

Lange, G.

1975-01-01

The winning of uranium ore is the first stage of the fuel cycle. The whole complex of questions to be considered when evaluating the profitability of an ore mine is shortly outlined, and the possible mining techniques are described. Some data on uranium mining in the western world are also given. (RB) [de

Determination of polyphenolic content, HPLC analyses and DNA cleavage activity of Malaysian Averrhoa carambola L. fruit extracts

Directory of Open Access Journals (Sweden)

Zakia Khanam

2015-10-01

Full Text Available In developing countries, the increasing gap between population growth and food supply has created renewed interest in finding reliable and cheap natural resources of nutraceutical value and health promoting properties. Therefore, the present study deals with the phytochemical analyses and DNA cleavage activity of Averrhoa carambola L. fruit (starfruit extracts. The phytochemical studies involve colour tests and quantification of phenolics and flavonoids of the prepared ethanolic and aqueous extracts. Identification of phenolic acids and flavonoids present in the extracts were conducted by high performance liquid chromatography (HPLC equipped with diode array detector (DAD. DNA cleavage activity of the extracts was evaluated through gel electrophoresis against plasmid Escherichia coli DNA at different concentrations (0.125–0.60 μg/μl. The results of the study exhibited that the starfruit is a rich source of polyphenols and all the extracts exhibited a dose dependent DNA cleavage activity, whereas ethanolic extract induced more cleavage as compared to the aqueous extract. In conclusion, the present study provides preliminary evidence with regard to nutraceutical value of the fruit. So, further extensive study is a prerequisite to exploit DNA cleaving properties of the fruit extracts for therapeutic application.

Supplementary data for the mechanism for cleavage of three typical glucosidic bonds induced by hydroxyl free radical

Directory of Open Access Journals (Sweden)

Yujie Dai

2017-12-01

Full Text Available The data presented in this article are related to the research article entitled “The mechanism for cleavage of three typical glucosidic bonds induced by hydroxyl free radical” (Dai et al., 2017 [1]. This article includes the structures of three kinds of disaccharides such as maltose, fructose and cellobiose, the diagrammatic sketch of the hydrogen abstraction reaction of the disaccharides by hydroxyl radical, the structure of the transition states for pyran ring opening of moiety A and cleavage of α(1→2 glycosidic bond starting from the hydrogen abstraction of C6–H in moiety A of sucrose, the transition state structure for cleavage of α(1→2 glycosidic bond starting from the hydrogen abstraction of C1′-H in moiety B of sucrose, the transition state structure, sketch for the reaction process and relative energy change of the reaction pathway for direct cleavage of α(1→4 glycosidic bond starting from hydrogen abstraction of C6′–H of moiety B of maltose.

Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

Science.gov (United States)

Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

2015-01-01

Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

Two-dimensional NMR evidence for cleavage of lignin and xylan substituents in wheat straw through hydrothermal pretreatment and enzymatic hydrolysis

DEFF Research Database (Denmark)

Yelle, Daniel J.; Kaparaju, Laxmi-Narasimha Prasad; Hunt, Christopher G.

2013-01-01

correlation spectroscopy, via an heteronuclear single quantum coherence experiment, revealed substantial lignin β-aryl ether cleavage, deacetylation via cleavage of the natural acetates at the 2-O- and 3-O-positions of xylan, and uronic acid depletion via cleavage of the (1 → 2)-linked 4-O....... g., further deacylation revealed by the depletion in ferulate and p-coumarate structures). Supplementary chemical analyses showed that the hydrothermal pretreatment increased the cellulose and lignin concentration with partial removal of extractives and hemicelluloses. The subsequent enzymatic...

Realizatinon of “zero emission” of mining water effluents from Sasa mine

OpenAIRE

Mirakovski, Dejan; Doneva, Nikolinka; Hadzi-Nikolova, Marija; Gocevski, Borce

2015-01-01

Sasa mine continuously takes actions to minimize the environmental impact of mining activities, in order to fulfill the national legislation in the field of environmental protection which comply with European legislation. This paper shows the drainage system of the horizon 830, which is performed in order to prevent free leakage of mining groundwater, as a part of these actions. This system provides a zero emission of mining water in the environment from Sasa mine. Key words: mining water...

Sustainable rehabilitation of mining waste and acid mine drainage using geochemistry, mine type, mineralogy, texture, ore extraction and climate knowledge.

Science.gov (United States)

Anawar, Hossain Md

2015-08-01

The oxidative dissolution of sulfidic minerals releases the extremely acidic leachate, sulfate and potentially toxic elements e.g., As, Ag, Cd, Cr, Cu, Hg, Ni, Pb, Sb, Th, U, Zn, etc. from different mine tailings and waste dumps. For the sustainable rehabilitation and disposal of mining waste, the sources and mechanisms of contaminant generation, fate and transport of contaminants should be clearly understood. Therefore, this study has provided a critical review on (1) recent insights in mechanisms of oxidation of sulfidic minerals, (2) environmental contamination by mining waste, and (3) remediation and rehabilitation techniques, and (4) then developed the GEMTEC conceptual model/guide [(bio)-geochemistry-mine type-mineralogy- geological texture-ore extraction process-climatic knowledge)] to provide the new scientific approach and knowledge for remediation of mining wastes and acid mine drainage. This study has suggested the pre-mining geological, geochemical, mineralogical and microtextural characterization of different mineral deposits, and post-mining studies of ore extraction processes, physical, geochemical, mineralogical and microbial reactions, natural attenuation and effect of climate change for sustainable rehabilitation of mining waste. All components of this model should be considered for effective and integrated management of mining waste and acid mine drainage. Copyright © 2015 Elsevier Ltd. All rights reserved.

Philippine Mining Capitalism: The Changing Terrains of Struggle in the Neoliberal Mining Regime

Directory of Open Access Journals (Sweden)

Alvin A. Camba

2016-06-01

Full Text Available This article analyzes how the mining sector and anti-mining groups compete for mining outcomes in the Philippines. I argue that the transition to a neoliberal mineral regime has empowered the mining sector and weakened the mining groups by shifting the terrains of struggle onto the domains of state agencies and scientific networks. Since the neoliberal era, the mining sector has come up with two strategies. First, technologies of subjection elevate various public institutions to elect and select the processes aimed at making mining accountable and sensitive to the demands of local communities. However, they often refuse or lack the capacity to intervene effectively. Second, technologies of subjectivities allow a selective group of industry experts to single-handedly determine the environmental viability of mining projects. Mining consultants, specialists, and scientists chosen by mining companies determine the potential environmental damage on water bodies, air pollution, and soil erosion. Because of the mining capital’s access to economic and legal resources, anti-mining communities across the Philippines have been forced to compete on an unequal terrain for a meaningful social dialogue and mining outcomes.

Copper-catalyzed transformation of ketones to amides via C(CO)-C(alkyl) bond cleavage directed by picolinamide.

Science.gov (United States)

Ma, Haojie; Zhou, Xiaoqiang; Zhan, Zhenzhen; Wei, Daidong; Shi, Chong; Liu, Xingxing; Huang, Guosheng

2017-09-13

Copper catalyzed chemoselective cleavage of the C(CO)-C(alkyl) bond leading to C-N bond formation with chelation assistance of N-containing directing groups is described. Inexpensive Cu(ii)-acetate serves as a convenient catalyst for this transformation. This method highlights the emerging strategy to transform unactivated alkyl ketones into amides in organic synthesis and provides a new strategy for C-C bond cleavage.

Small molecule inhibitors uncover synthetic genetic interactions of human flap endonuclease 1 (FEN1 with DNA damage response genes.

Directory of Open Access Journals (Sweden)

Thomas A Ward

Full Text Available Flap endonuclease 1 (FEN1 is a structure selective endonuclease required for proficient DNA replication and the repair of DNA damage. Cellularly active inhibitors of this enzyme have previously been shown to induce a DNA damage response and, ultimately, cell death. High-throughput screens of human cancer cell-lines identify colorectal and gastric cell-lines with microsatellite instability (MSI as enriched for cellular sensitivity to N-hydroxyurea series inhibitors of FEN1, but not the PARP inhibitor olaparib or other inhibitors of the DNA damage response. This sensitivity is due to a synthetic lethal interaction between FEN1 and MRE11A, which is often mutated in MSI cancers through instabilities at a poly(T microsatellite repeat. Disruption of ATM is similarly synthetic lethal with FEN1 inhibition, suggesting that disruption of FEN1 function leads to the accumulation of DNA double-strand breaks. These are likely a result of the accumulation of aberrant replication forks, that accumulate as a consequence of a failure in Okazaki fragment maturation, as inhibition of FEN1 is toxic in cells disrupted for the Fanconi anemia pathway and post-replication repair. Furthermore, RAD51 foci accumulate as a consequence of FEN1 inhibition and the toxicity of FEN1 inhibitors increases in cells disrupted for the homologous recombination pathway, suggesting a role for homologous recombination in the resolution of damage induced by FEN1 inhibition. Finally, FEN1 appears to be required for the repair of damage induced by olaparib and cisplatin within the Fanconi anemia pathway, and may play a role in the repair of damage associated with its own disruption.

Technological highwall mining

Energy Technology Data Exchange (ETDEWEB)

Davison, I. [Highwall Systems (United States)

2006-09-15

The paper explores the issues facing highwall mining. Based in Chilhowie, Virginia, American Highwall Systems has developed a highwall mining system that will allow the mining of coal seams from 26 in to 10 ft in thickness. The first production model, AH51, began mining in August 2006. Technologies incorporated into the company's mining machines to improve the performance, enhance the efficiency, and improve the reliability of the highwall mining equipment incorporate technologies from many disciplines. Technology as applied to design engineering, manufacturing and fabrication engineering, control and monitoring computer hardware and software has played an important role in the evolution of the American Highwall Systems design concept. 5 photos.

ChloroP, a neural network-based method for predicting chloroplast transitpeptides and their cleavage sites

DEFF Research Database (Denmark)

Emanuelsson, O.; Nielsen, Henrik; von Heijne, Gunnar

1999-01-01

the cleavage sites given in SWISS-PROT. An analysis of 715 Arabidopsis thaliana sequences from SWISS-PROT suggests that the ChloroP method should be useful for the identification of putative transit peptides in genome-wide sequence data. The ChloroP predictor is available as a web-server at http......We present a neural network based method (ChloroP) for identifying chloroplast transit peptides and their cleavage sites. Using cross-validation, 88% of the sequences in our homology reduced training set were correctly classified as transit peptides or nontransit peptides. This performance level...

Kiruna research mine

Energy Technology Data Exchange (ETDEWEB)

Oestensen, A

1983-12-01

The research mine at Kiruna is the first large-scale mining research project sponsored by the Swedish government. Under the leadership of the Swedish Mining Research Foundation, a five-year project involving development of new mining systems and machinery will be carried out in cooperation with the Lulea Institute of Technology and a number of Swedish industrial companies.

Fast cleavage of phycocyanobilin from phycocyanin for use in food colouring.

Science.gov (United States)

Roda-Serrat, Maria Cinta; Christensen, Knud Villy; El-Houri, Rime Bahij; Fretté, Xavier; Christensen, Lars Porskjær

2018-02-01

Phycocyanins from cyanobacteria are possible sources for new natural blue colourants. Their chromophore, phycocyanobilin (PCB), was cleaved from the apoprotein by solvolysis in alcohols and alcoholic aqueous solutions. In all cases two PCB isomers were obtained, while different solvent adducts were formed upon the use of different reagents. The reaction is believed to take place via two competing pathways, a concerted E2 elimination and a S N 2 nucleophilic substitution. Three cleavage methods were compared in terms of yield and purity: conventional reflux, sealed vessel heated in an oil bath, and microwave assisted reaction. The sealed vessel method is a new approach for fast cleavage of PCB from phycocyanin and gave at 120°C the same yield within 30min compared to 16h by the conventional reflux method (P<0.05). In addition the sealed vessel method resulted in improved purity compared to the other methods. Microwave irradiation increased product degradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

TRAIL-induced cleavage and inactivation of SPAK sensitizes cells to apoptosis

International Nuclear Information System (INIS)

Polek, Tara C.; Talpaz, Moshe; Spivak-Kroizman, Taly R.

2006-01-01

Ste20-related proline-alanine-rich kinase (SPAK) has been linked to various cellular processes, including proliferation, differentiation, and ion transport regulation. Recently, we showed that SPAK mediates signaling by the TNF receptor, RELT. The presence of a caspase cleavage site in SPAK prompted us to study its involvement in apoptotic signaling induced by another TNF member, TRAIL. We show that TRAIL stimulated caspase 3-like proteases that cleaved SPAK at two distinct sites. Cleavage had little effect on the activity of SPAK but removed its substrate-binding domain. In addition, TRAIL reduced the activity of SPAK in HeLa cells in a caspase-independent manner. Thus, TRAIL inhibited SPAK by two mechanisms: activation of caspases, which removed its substrate-binding domain, and caspase-independent down-regulation of SPAK activity. Furthermore, reducing the amount of SPAK by siRNA increased the sensitivity of HeLa cells to TRAIL-induced apoptosis. Thus, TRAIL down-regulation of SPAK is an important event that enhances its apoptotic effects

Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2

Energy Technology Data Exchange (ETDEWEB)

Hamilton, Brian S.; Chung, Changik; Cyphers, Soreen Y.; Rinaldi, Vera D.; Marcano, Valerie C.; Whittaker, Gary R., E-mail: grw7@cornell.edu

2014-07-25

Highlights: • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza HA cleavage activation. • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza virus infection. • Comparative analysis of HAI-2 for vesicular stomatitis virus and human parainfluenza virus type-1. • Analysis of the activity of HAI-2 in a mouse model of influenza. - Abstract: Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant to the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza.

Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2

International Nuclear Information System (INIS)

Hamilton, Brian S.; Chung, Changik; Cyphers, Soreen Y.; Rinaldi, Vera D.; Marcano, Valerie C.; Whittaker, Gary R.

2014-01-01

Highlights: • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza HA cleavage activation. • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza virus infection. • Comparative analysis of HAI-2 for vesicular stomatitis virus and human parainfluenza virus type-1. • Analysis of the activity of HAI-2 in a mouse model of influenza. - Abstract: Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant to the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza

Overview of mine drainage geochemistry at historical mines, Humboldt River basin and adjacent mining areas, Nevada. Chapter E.

Science.gov (United States)

Nash, J. Thomas; Stillings, Lisa L.

2004-01-01

Reconnaissance hydrogeochemical studies of the Humboldt River basin and adjacent areas of northern Nevada have identified local sources of acidic waters generated by historical mine workings and mine waste. The mine-related acidic waters are rare and generally flow less than a kilometer before being neutralized by natural processes. Where waters have a pH of less than about 3, particularly in the presence of sulfide minerals, the waters take on high to extremely high concentrations of many potentially toxic metals. The processes that create these acidic, metal-rich waters in Nevada are the same as for other parts of the world, but the scale of transport and the fate of metals are much more localized because of the ubiquitous presence of caliche soils. Acid mine drainage is rare in historical mining districts of northern Nevada, and the volume of drainage rarely exceeds about 20 gpm. My findings are in close agreement with those of Price and others (1995) who estimated that less than 0.05 percent of inactive and abandoned mines in Nevada are likely to be a concern for acid mine drainage. Most historical mining districts have no draining mines. Only in two districts (Hilltop and National) does water affected by mining flow into streams of significant size and length (more than 8 km). Water quality in even the worst cases is naturally attenuated to meet water-quality standards within about 1 km of the source. Only a few historical mines release acidic water with elevated metal concentrations to small streams that reach the Humboldt River, and these contaminants and are not detectable in the Humboldt. These reconnaissance studies offer encouraging evidence that abandoned mines in Nevada create only minimal and local water-quality problems. Natural attenuation processes are sufficient to compensate for these relatively small sources of contamination. These results may provide useful analogs for future mining in the Humboldt River basin, but attention must be given to

Present and future mine effluents management at Zirovski Vrh uranium mine

International Nuclear Information System (INIS)

Logar, Z.; Likar, B.; Gantar, I.

2002-01-01

Zirovski Vrh uranium mine and its facilities are situated on the northeastern slopes of the Zirovski Vrh ridge (960 m) and on the southern slopes of Crna gora (611 m) respectively. Mine elevation is from 430 m (bottom of the valley) to 580 m (P-1 adit). All effluents from the mine and mill objects flow into the Brebovscica river (with average yearly flow of 0.74 m 3 /s): run off mine water; mine waste pile Jazbec outflow; mill tailings Borst outflows; effluents from mine temporary mine waste piles P-1, P-9, P-36 are of minor significance. The first three effluents and the recipient surface water flows (the Todrascica brook and the Brebovscica river) are monitored extensively. The impact of radioactive polluted outflows on named waters is proved, but far under the maximal permitted limit values. The authorised maximal limits values for mine effluents were obtained in 1996. Detail design will ensure that this values will not be exceeded in the future. The long term planes are to minimise the uranium concentrations in the run off mine water by target underground drilling. The mine waste pile and the mill tailings will be covered by engineered cover system to avoid clean water contamination by weathering and ablution as well. The existing effluents from the mill tailings will diminish after the remediation and consolidation of the tailing. The Government of Slovenia funds the remediation of the uranium production site Zirovski Vrh. Estimated needed funds for remediation of the main objects are shown in the table below. The total investment includes also the costs for effluents control. Area Mio US$ Underground mine remediation 19.00 Mine waste pile remediation 6.50 Mill tailings remediation 2.24 Total investment costs 27.74 Above figures do not include operation costs of the Zirovski Vrh Mine, approximately US$ 2.2 Mio per year nowadays. The last implementation schedule foresights the end of remediation works in year 2005. After that starts trial monitoring of 5 years

Hydrogeochemical assessment of mine-impacted water and sediment of iron ore mining

Science.gov (United States)

Nur Atirah Affandi, Fatin; Kusin, Faradiella Mohd; Aqilah Sulong, Nur; Madzin, Zafira

2018-04-01

This study was carried out to evaluate the hydrogeochemical behaviour of mine-impacted water and sediment of a former iron ore mining area. Sampling of mine water and sediment were carried out at selected locations within the mine including the former mining ponds, mine tailings and the nearby stream. The water samples were analysed for their hydrochemical facies, major and trace elements including heavy metals. The water in the mining ponds and the mine tailings was characterised as highly acidic (pH 2.54-3.07), but has near-neutral pH in the nearby stream. Results indicated that Fe and Mn in water have exceeded the recommended guidelines values and was also supported by the results of geochemical modelling. The results also indicated that sediments in the mining area were contaminated with Cd and As as shown by the potential ecological risk index values. The total risk index of heavy metals in the sediment were ranked in the order of Cd>As>Pb>Cu>Zn>Cr. Overall, the extent of potential ecological risks of the mining area were categorised as having low to moderate ecological risk.

Collaborative Data Mining

Science.gov (United States)

Moyle, Steve

Collaborative Data Mining is a setting where the Data Mining effort is distributed to multiple collaborating agents - human or software. The objective of the collaborative Data Mining effort is to produce solutions to the tackled Data Mining problem which are considered better by some metric, with respect to those solutions that would have been achieved by individual, non-collaborating agents. The solutions require evaluation, comparison, and approaches for combination. Collaboration requires communication, and implies some form of community. The human form of collaboration is a social task. Organizing communities in an effective manner is non-trivial and often requires well defined roles and processes. Data Mining, too, benefits from a standard process. This chapter explores the standard Data Mining process CRISP-DM utilized in a collaborative setting.

SKI2 mediates degradation of RISC 5'-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis.

Science.gov (United States)

Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

2015-12-15

Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20-24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5', but not 3'-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5' to the cleavage site, but several examples of 3'-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5'-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5'-cleavage fragments. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

76 FR 63238 - Proximity Detection Systems for Continuous Mining Machines in Underground Coal Mines

Science.gov (United States)

2011-10-12

... Detection Systems for Continuous Mining Machines in Underground Coal Mines AGENCY: Mine Safety and Health... Agency's proposed rule addressing Proximity Detection Systems for Continuous Mining Machines in... proposed rule for Proximity Detection Systems on Continuous Mining Machines in Underground Coal Mines. Due...

76 FR 70075 - Proximity Detection Systems for Continuous Mining Machines in Underground Coal Mines

Science.gov (United States)

2011-11-10

... Detection Systems for Continuous Mining Machines in Underground Coal Mines AGENCY: Mine Safety and Health... proposed rule addressing Proximity Detection Systems for Continuous Mining Machines in Underground Coal... Detection Systems for Continuous Mining Machines in Underground Coal Mines. MSHA conducted hearings on...

Determine Appropriate Post Mining Land Use in Indonesia Coal Mining Using Land Suitability Evaluation

OpenAIRE

Maryati, Sri; Shimada, Hideki; Hamanaka, Akihiro; Sasaoka, Takashi; Matsui, Kikuo

2012-01-01

Coal mining industry gives many benefits for Indonesia including contribution in total Indonesian GDP. Most of coal mines in Indonesia are open pit mining method which disturbs large area of land. One of open pit mining impact is damage land and related to soil erosion occurrences it will degrade land by top soil loses. Indonesia Government has issued mine closure regulation to encourage mining industry provide post mining land use. Determination of post mining land use should be considering ...

A guide to mines and mining houses of the Republic of South Africa

Energy Technology Data Exchange (ETDEWEB)

1989-02-01

This guide lists the mining houses of the Republic of South Africa and the mines under their respective control. It is intended as a guide for British exporters wishing to develop business with the South African mining industry and hence includes buying offices and senior buying personnel in South Africa (and in the UK where relevant). Amongst the 16 mining houses included are the Chamber of Mines of South Africa, De Beers Consolidated Mines Ltd., Rand Mines Ltd., Sasol Ltd. and the South African Iron and Steel Industrial Corporation Ltd.

Numerical Study on 4-1 Coal Seam of Xiaoming Mine in Ascending Mining

Science.gov (United States)

Tianwei, Lan; Hongwei, Zhang; Sheng, Li; Weihua, Song; Batugin, A. C.; Guoshui, Tang

2015-01-01

Coal seams ascending mining technology is very significant, since it influences the safety production and the liberation of dull coal, speeds up the construction of energy, improves the stability of stope, and reduces or avoids deep hard rock mining induced mine disaster. Combined with the Xiaoming ascending mining mine 4-1, by numerical calculation, the paper analyses ascending mining 4-1 factors, determines the feasibility of ascending mining 4-1 coalbed, and proposes roadway layout program about working face, which has broad economic and social benefits. PMID:25866840

Ghana Mining Journal

African Journals Online (AJOL)

... in the Ghana mining journal: Geology and Mineral Exploration, Mining, Quarrying, Geomechanics, Groundwater Studies, Hydrocarbon Development, Mineral Processing, Metallurgy, Material Science, Mineral Management Policies, Mineral Economics, Environmental Aspects, Computer Applications and Mining Education.

Relationship between synthesis and cleavage of poliovirus-specific proteins.

OpenAIRE

Thomas, A A; Voorma, H O; Boeye, A

1983-01-01

Poliovirus proteinase was studied in vitro in lysates from poliovirus-infected HeLa cells. Preincubation of these lysates caused (i) a reduction in poliovirus proteinase activity and (ii) a partial dependence on exogenous mRNA for optimal translation. Proteins translated from endogenous poliovirus RNA in preincubated extracts from virus-infected HeLa cells are poorly cleaved. This cleavage deficiency is alleviated by adding fresh poliovirus RNA to the translation system, thus, allowing re-ini...

Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein–staphylococcal nuclease hybrid

International Nuclear Information System (INIS)

Mori, Tomoaki; Nakamura, Kento; Masaoka, Keisuke; Fujita, Yusuke; Morisada, Ryosuke; Mori, Koichi; Tobimatsu, Takamasa; Sera, Takashi

2016-01-01

Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to construct an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms. - Highlights: • A novel RNA restriction enzyme using SNase was developed tor cleave viral RNA. • Our enzyme cleaved influenza RNA with rates >120-fold higher rates a PIN-fusion one. • Our artificial enzyme with the L5 linker showed the highest RNA cleavage rate. • Our artificial enzyme site-selectively cleaved influenza RNA in vitro.

Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity

International Nuclear Information System (INIS)

Du, Lanying; Kao, Richard Y.; Zhou, Yusen; He, Yuxian; Zhao, Guangyu; Wong, Charlotte; Jiang, Shibo; Yuen, Kwok-Yung; Jin, Dong-Yan; Zheng, Bo-Jian

2007-01-01

The spike (S) protein of SARS coronavirus (SARS-CoV) has been known to recognize and bind to host receptors, whose conformational changes then facilitate fusion between the viral envelope and host cell membrane, leading to viral entry into target cells. However, other functions of SARS-CoV S protein such as proteolytic cleavage and its implications to viral infection are incompletely understood. In this study, we demonstrated that the infection of SARS-CoV and a pseudovirus bearing the S protein of SARS-CoV was inhibited by a protease inhibitor Ben-HCl. Also, the protease Factor Xa, a target of Ben-HCl abundantly expressed in infected cells, was able to cleave the recombinant and pseudoviral S protein into S1 and S2 subunits, and the cleavage was inhibited by Ben-HCl. Furthermore, this cleavage correlated with the infectivity of the pseudovirus. Taken together, our study suggests a plausible mechanism by which SARS-CoV cleaves its S protein to facilitate viral infection

Legal Policy Of Peoples Rights In Around Mining Corporate Post-Mining Activities

Directory of Open Access Journals (Sweden)

Teng Berlianty

2015-08-01

Full Text Available This study aims to gain an understanding of the essence of the rights of communities around post-mining corporate responsibility towards the fulfillment of the rights of communities around post-mining as well as government policies to protect the sustainability of the post-mining communities around the mining business. This type of research is a normative legal research methods using primary legal materials secondary and tertiary. With the approach of sociolegal through down the field in Gebe to get data concrete. Data were analyzed with qualitative analysis. The results showed that the essence of the rights of communities around mining operations after the mine in the form of the right to a decent life welfare the right to social security in the form of employment the guarantee of free education and healthcare for the local population as well as the right to a good environment and healthy as a guarantee of the continuity of human existence and future generations. These rights have not been fully realized post-mining. Corporate responsibility in accordance with Article 74 of Law No. 40 of 2007 on the fulfillment of the rights of communities around mining operations after the mine in the form of welfare responsibilities clothing food and shelter especially electricity and water have not been met then the social responsibility to empower communities around the mine as stakeholders as well as environmental responsibility. Legal policy such as the empowerment of communities around the mine in order to be self-sufficient after the post-mining public service policies in education and health as a form of existence of government using existing programs nationally and subordinate to the PT. Antam. as well as environmental protection policies in the form of post-mining reclamation formulated in the companys liabilities.

Cleavage/alteration of interleukin-8 by matrix metalloproteinase-9 in the female lower genital tract.

Science.gov (United States)

Zariffard, M Reza; Anastos, Kathryn; French, Audrey L; Munyazesa, Elisaphane; Cohen, Mardge; Landay, Alan L; Spear, Gregory T

2015-01-01

Interleukin-8 (IL-8, CXCL8) plays important roles in immune responses at mucosal sites including in the lower genital tract. Since several types of bacteria produce proteases that cleave IL-8 and many types of bacteria can be present in lower genital tract microbiota, we assessed genital fluids for IL-8 cleavage/alteration. Genital fluids collected by lavage from 200 women (23 HIV-seronegative and 177 HIV-seropositive) were tested for IL-8 cleavage/alteration by ELISA. IL-8 cleaving/altering activity was observed in fluids from both HIV-positive (28%) and HIV-negative women (35%). There was no clear relationship between the activity and the types of bacteria present in the lower genital tract as determined by high-throughput sequencing of the 16S rRNA gene. Protease inhibitors specific for matrix metalloproteinases (MMPs) reduced the activity and a multiplex assay that detects both inactive and active MMPs showed the presence of multiple MMPs, including MMP-1, -3, -7, -8, -9, -10 and -12 in genital secretions from many of the women. The IL-8-cleaving/altering activity significantly correlated with active MMP-9 as well as with cleavage of a substrate that is acted on by several active MMPs. These studies show that multiple MMPs are present in the genital tract of women and strongly suggest that MMP-9 in genital secretions can cleave IL-8 at this mucosal site. These studies suggest that MMP-mediated cleavage of IL-8 can modulate inflammatory responses in the lower genital tract.

Geochemical Characterization of Mine Waste, Mine Drainage, and Stream Sediments at the Pike Hill Copper Mine Superfund Site, Orange County, Vermont

Science.gov (United States)

Piatak, Nadine M.; Seal, Robert R.; Hammarstrom, Jane M.; Kiah, Richard G.; Deacon, Jeffrey R.; Adams, Monique; Anthony, Michael W.; Briggs, Paul H.; Jackson, John C.

2006-01-01

The Pike Hill Copper Mine Superfund Site in the Vermont copper belt consists of the abandoned Smith, Eureka, and Union mines, all of which exploited Besshi-type massive sulfide deposits. The site was listed on the U.S. Environmental Protection Agency (USEPA) National Priorities List in 2004 due to aquatic ecosystem impacts. This study was intended to be a precursor to a formal remedial investigation by the USEPA, and it focused on the characterization of mine waste, mine drainage, and stream sediments. A related study investigated the effects of the mine drainage on downstream surface waters. The potential for mine waste and drainage to have an adverse impact on aquatic ecosystems, on drinking- water supplies, and to human health was assessed on the basis of mineralogy, chemical concentrations, acid generation, and potential for metals to be leached from mine waste and soils. The results were compared to those from analyses of other Vermont copper belt Superfund sites, the Elizabeth Mine and Ely Copper Mine, to evaluate if the waste material at the Pike Hill Copper Mine was sufficiently similar to that of the other mine sites that USEPA can streamline the evaluation of remediation technologies. Mine-waste samples consisted of oxidized and unoxidized sulfidic ore and waste rock, and flotation-mill tailings. These samples contained as much as 16 weight percent sulfides that included chalcopyrite, pyrite, pyrrhotite, and sphalerite. During oxidation, sulfides weather and may release potentially toxic trace elements and may produce acid. In addition, soluble efflorescent sulfate salts were identified at the mines; during rain events, the dissolution of these salts contributes acid and metals to receiving waters. Mine waste contained concentrations of cadmium, copper, and iron that exceeded USEPA Preliminary Remediation Goals. The concentrations of selenium in mine waste were higher than the average composition of eastern United States soils. Most mine waste was

Mine drainage treatment

OpenAIRE

Golomeova, Mirjana; Zendelska, Afrodita; Krstev, Boris; Golomeov, Blagoj; Krstev, Aleksandar

2012-01-01

Water flowing from underground and surface mines and contains high concentrations of dissolved metals is called mine drainage. Mine drainage can be categorized into several basic types by their alkalinity or acidity. Sulfide rich and carbonate poor materials are expected to produce acidic drainage, and alkaline rich materials, even with significant sulfide concentrations, often produce net alkaline water. Mine drainages are dangerous because pollutants may decompose in the environment. In...

Mining dictionary: underground mining; open-cast mining; preparation and beneficiation; geology of mineral deposits

Energy Technology Data Exchange (ETDEWEB)

Goergen, H; Stoll, R D; Vriesen, R D; Welzenberg, B

1981-01-01

The dictionary reflects the latest technical developments in the vocabulary of mining methods and the mining industry. Volume I of the dictionary is English to German, Volume II German to English. 36,000 entries are included.

Mining Views : database views for data mining

NARCIS (Netherlands)

Blockeel, H.; Calders, T.; Fromont, É.; Goethals, B.; Prado, A.; Nijssen, S.; De Raedt, L.

2007-01-01

We propose a relational database model towards the integration of data mining into relational database systems, based on the so called virtual mining views. We show that several types of patterns and models over the data, such as itemsets, association rules, decision trees and clusterings, can be

Coastal mining

Science.gov (United States)

Bell, Peter M.

The Exclusive Economic Zone (EEZ) declared by President Reagan in March 1983 has met with a mixed response from those who would benefit from a guaranteed, 200-nautical-mile (370-km) protected underwater mining zone off the coasts of the United States and its possessions. On the one hand, the U.S. Department of the Interior is looking ahead and has been very successful in safeguarding important natural resources that will be needed in the coming decades. On the other hand, the mining industry is faced with a depressed metals and mining market.A report of the Exclusive Economic Zone Symposium held in November 1983 by the U.S. Geological Survey, the Mineral Management Service, and the Bureau of Mines described the mixed response as: “ … The Department of Interior … raring to go into promotion of deep-seal mining but industrial consortia being very pessimistic about the program, at least for the next 30 or so years.” (Chemical & Engineering News, February 5, 1983).

Two tandem RNase III cleavage sites determine betT mRNA stability in response to osmotic stress in Escherichia coli.

Directory of Open Access Journals (Sweden)

Minji Sim

Full Text Available While identifying genes regulated by ribonuclease III (RNase III in Escherichia coli, we observed that steady-state levels of betT mRNA, which encodes a transporter mediating the influx of choline, are dependent on cellular concentrations of RNase III. In the present study, we also observed that steady-state levels of betT mRNA are dependent on RNase III activity upon exposure to osmotic stress, indicating the presence of cis-acting elements controlled by RNase III in betT mRNA. Primer extension analyses of betT mRNA revealed two tandem RNase III cleavage sites in its stem-loop region, which were biochemically confirmed via in vitro cleavage assays. Analyses of cleavage sites suggested the stochastic selection of cleavage sites by RNase III, and mutational analyses indicated that RNase III cleavage at either site individually is insufficient for efficient betT mRNA degradation. In addition, both the half-life and abundance of betT mRNA were significantly increased in association with decreased RNase III activity under hyper-osmotic stress conditions. Our findings demonstrate that betT mRNA stability is controlled by RNase III at the post-transcriptional level under conditions of osmotic stress.

Specialized mining GIS system MineGIS SMZ Jelšava

Directory of Open Access Journals (Sweden)

Peter Sasvári

2005-12-01

Full Text Available Following, the real needs for new mining information system requested by SMZ Jelšava, the Department of Mineral Deposits and Applied Geology (KLaAG at the Technical University of Košice (TUKE has prepared a specification for the specialized mining geographic information system called MineGIS SMZ Jelšava. The main roles of the new system have been defined as follows of reserves: the administration, analyse and the visualization of all mining geo-data related to the estimation.

A camel-derived MERS-CoV with a variant spike protein cleavage site and distinct fusion activation properties

Science.gov (United States)

Millet, Jean Kaoru; Goldstein, Monty E; Labitt, Rachael N; Hsu, Hung-Lun; Daniel, Susan; Whittaker, Gary R

2016-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) continues to circulate in both humans and camels, and the origin and evolution of the virus remain unclear. Here we characterize the spike protein of a camel-derived MERS-CoV (NRCE-HKU205) identified in 2013, early in the MERS outbreak. NRCE-HKU205 spike protein has a variant cleavage motif with regard to the S2′ fusion activation site—notably, a novel substitution of isoleucine for the otherwise invariant serine at the critical P1′ cleavage site position. The substitutions resulted in a loss of furin-mediated cleavage, as shown by fluorogenic peptide cleavage and western blot assays. Cell–cell fusion and pseudotyped virus infectivity assays demonstrated that the S2′ substitutions decreased spike-mediated fusion and viral entry. However, cathepsin and trypsin-like protease activation were retained, albeit with much reduced efficiency compared with the prototypical EMC/2012 human strain. We show that NRCE-HKU205 has more limited fusion activation properties possibly resulting in more restricted viral tropism and may represent an intermediate in the complex pattern of MERS-CoV ecology and evolution. PMID:27999426

AID to overcome the limitations of genomic information by introducing somatic DNA alterations.

Science.gov (United States)

Honjo, Tasuku; Muramatsu, Masamichi; Nagaoka, Hitoshi; Kinoshita, Kazuo; Shinkura, Reiko

2006-05-01

The immune system has adopted somatic DNA alterations to overcome the limitations of the genomic information. Activation induced cytidine deaminase (AID) is an essential enzyme to regulate class switch recombination (CSR), somatic hypermutation (SHM) and gene conversion (GC) of the immunoglobulin gene. AID is known to be required for DNA cleavage of S regions in CSR and V regions in SHM. However, its molecular mechanism is a focus of extensive debate. RNA editing hypothesis postulates that AID edits yet unknown mRNA, to generate specific endonucleases for CSR and SHM. By contrast, DNA deamination hypothesis assumes that AID deaminates cytosine in DNA, followed by DNA cleavage by base excision repair enzymes. We summarize the basic knowledge for molecular mechanisms for CSR and SHM and then discuss the importance of AID not only in the immune regulation but also in the genome instability.

South African mine valuation

Energy Technology Data Exchange (ETDEWEB)

Storrar, C D

1977-01-01

This article sets out the basic concepts of mine valuation, with gold mining receiving more space than base minerals and coal. Sampling practice is given special attention. Chapter headings are methods of investigation, sampling, underground sampling, averaging of underground sampling, diamond-drill sampling, mass and mineral content of ore, organization of a sample office, working costs, mining pay limits, ore reserves, ore accounting, maintenance of grade, forecasting operations and life of mine, statistical mine valuation, state's share of profits and taxation, and financial valuation of mining ventures.

Microbes from mined sites: Harnessing their potential for reclamation of derelict mine sites

International Nuclear Information System (INIS)

Thavamani, Palanisami; Samkumar, R. Amos; Satheesh, Viswanathan; Subashchandrabose, Suresh R.; Ramadass, Kavitha; Naidu, Ravi; Venkateswarlu, Kadiyala; Megharaj, Mallavarapu

2017-01-01

Derelict mines pose potential risks to environmental health. Several factors such as soil structure, organic matter, and nutrient content are the greatly affected qualities in mined soils. Soil microbial communities are an important element for successful reclamation because of their major role in nutrient cycling, plant establishment, geochemical transformations, and soil formation. Yet, microorganisms generally remain an undervalued asset in mined sites. The microbial diversity in derelict mine sites consists of diverse species belonging to four key phyla: Proteobacteria, Acidobacteria, Firmicutes, and Bacteroidetes. The activity of plant symbiotic microorganisms including root-colonizing rhizobacteria and ectomycorrhizal fungi of existing vegetation in the mined sites is very high since most of these microbes are extremophiles. This review outlines the importance of microorganisms to soil health and the rehabilitation of derelict mines and how microbial activity and diversity can be exploited to better plan the soil rehabilitation. Besides highlighting the major breakthroughs in the application of microorganisms for mined site reclamation, we provide a critical view on plant−microbiome interactions to improve revegetation at the mined sites. Also, the need has been emphasized for deciphering the molecular mechanisms of adaptation and resistance of rhizosphere and non-rhizosphere microbes in abandoned mine sites, understanding their role in remediation, and subsequent harnessing of their potential to pave the way in future rehabilitation strategies for mined sites. - Highlights: • Abandoned mines pose potential risks to human and environmental health. • Re-establishment of a self-sustaining vegetative cover at derelict mines is a major challenge. • Soil microbial communities are very important for successful reclamation of mined sites. • Role of microorganisms in soil function in derelict mines needs to be understood.

Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication.

Directory of Open Access Journals (Sweden)

Eveline Kindler

2017-02-01

Full Text Available Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I. This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU activity is key to prevent early induction of double-stranded RNA (dsRNA host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses.

Mining Views : database views for data mining

NARCIS (Netherlands)

Blockeel, H.; Calders, T.; Fromont, É.; Goethals, B.; Prado, A.

2008-01-01

We present a system towards the integration of data mining into relational databases. To this end, a relational database model is proposed, based on the so called virtual mining views. We show that several types of patterns and models over the data, such as itemsets, association rules and decision

Discrete population balance models of random agglomeration and cleavage in polymer pyrolysis

Directory of Open Access Journals (Sweden)

John E. J. Staggs

2017-05-01

Full Text Available The processes of random agglomeration and cleavage (both of which are important for the development of new models of polymer combustion, but are also applicable in a wide range of fields including atmospheric physics, radiation modelling and astrophysics are analysed using population balance methods. The evolution of a discrete distribution of particles is considered within this framework, resulting in a set of ordinary differential equations for the individual particle concentrations. Exact solutions for these equations are derived, together with moment generating functions. Application of the discrete Laplace transform (analogous to the Z-transform is found to be effective in these problems, providing both exact solutions for particle concentrations and moment generating functions. The combined agglomeration-cleavage problem is also considered. Unfortunately, it has been impossible to find an exact solution for the full problem, but a stable steady state has been identified and computed.

High utility-itemset mining and privacy-preserving utility mining

Directory of Open Access Journals (Sweden)

Jerry Chun-Wei Lin

2016-03-01

Full Text Available In recent decades, high-utility itemset mining (HUIM has emerging a critical research topic since the quantity and profit factors are both concerned to mine the high-utility itemsets (HUIs. Generally, data mining is commonly used to discover interesting and useful knowledge from massive data. It may, however, lead to privacy threats if private or secure information (e.g., HUIs are published in the public place or misused. In this paper, we focus on the issues of HUIM and privacy-preserving utility mining (PPUM, and present two evolutionary algorithms to respectively mine HUIs and hide the sensitive high-utility itemsets in PPUM. Extensive experiments showed that the two proposed models for the applications of HUIM and PPUM can not only generate the high quality profitable itemsets according to the user-specified minimum utility threshold, but also enable the capability of privacy preserving for private or secure information (e.g., HUIs in real-word applications.

Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism.

Directory of Open Access Journals (Sweden)

Annie Nguyen

Full Text Available Cystatin C (Cys C is a small, potent, cysteine protease inhibitor. An Ala25Thr (A25T polymorphism in Cys C has been associated with both macular degeneration and late-onset Alzheimer's disease. Previously, studies have suggested that this polymorphism may compromise the secretion of Cys C. Interestingly, we found that untagged A25T, A25T tagged C-terminally with FLAG, or A25T FLAG followed by green fluorescent protein (GFP, were all secreted as efficiently from immortalized human cells as their wild-type (WT counterparts (e.g., 112%, 100%, and 88% of WT levels from HEK-293T cells, respectively. Supporting these observations, WT and A25T Cys C variants also showed similar intracellular steady state levels. Furthermore, A25T Cys C did not activate the unfolded protein response and followed the same canonical endoplasmic reticulum (ER-Golgi trafficking pathway as WT Cys C. WT Cys C has been shown to undergo signal sequence cleavage between residues Gly26 and Ser27. While the A25T polymorphism did not affect Cys C secretion, we hypothesized that it may alter where the Cys C signal sequence is preferentially cleaved. Under normal conditions, WT and A25T Cys C have the same signal sequence cleavage site after Gly26 (referred to as 'site 2' cleavage. However, in particular circumstances when the residues around site 2 are modified (such as by the presence of an N-terminal FLAG tag immediately after Gly26, or by a Gly26Lys (G26K mutation, A25T has a significantly higher likelihood than WT Cys C of alternative signal sequence cleavage after Ala20 ('site 1' or even earlier in the Cys C sequence. Overall, our results indicate that the A25T polymorphism does not cause a significant reduction in Cys C secretion, but instead predisposes the protein to be cleaved at an alternative signal sequence cleavage site if site 2 is hindered. Additional N-terminal amino acids resulting from alternative signal sequence cleavage may, in turn, affect the protease

Haplotype-based case-control study on human apurinic/apyrimidinic endonuclease 1/redox effector factor-1 gene and essential hypertension.

Science.gov (United States)

Naganuma, Takahiro; Nakayama, Tomohiro; Sato, Naoyuki; Fu, Zhenyan; Soma, Masayoshi; Yamaguchi, Mai; Shimodaira, Masanori; Aoi, Noriko; Usami, Ron

2010-02-01

Oxidative DNA damage is involved in the pathophysiology of essential hypertension (EH), which is a multifactorial disorder. Apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/REF-1) is an essential endonuclease in the base excision repair pathway of oxidatively damaged DNA, in addition to having reducing properties that promote the binding of redox-sensitive transcription factors. Blood pressure in APE1/REF-1-knockout mice is reported to be significantly higher than in wild-type mice. The aim of this study was to investigate the relationship between EH and the human APE1/REF-1 gene through a haplotype-based case-control study using single-nucleotide polymorphisms (SNPs). We selected five SNPs in the human APE1/REF-1 gene (rs1760944, rs3136814, rs17111967, rs3136817, and rs1130409), and performed case-control studies in 265 EH patients and 266 age-matched normotensive (NT) subjects. rs17111967 was found to show nonheterogeneity among Japanese subjects. There were no significant differences in the overall distribution of genotypes or alleles for each SNP between EH and NT groups. In the overall distribution of the haplotype-based case-control study constructed based on rs1760944, rs3136817, and rs1130409, the frequency of the G-T-T haplotype was significantly higher in the EH group than in the NT group (2.1% vs. 0.0%, P = 0.001). Multiple logistic regression analysis also revealed significant differences for the G-T-T haplotype, even after adjustment for confounding factors (OR = 8.600, 95% CI: 1.073-68.951, P = 0.043). Based on the present results, the G-T-T haplotype appears to be a genetic marker of EH, and the APE1/REF-1 gene appears to be a susceptibility gene for EH.

76 FR 54163 - Proximity Detection Systems for Continuous Mining Machines in Underground Coal Mines

Science.gov (United States)

2011-08-31

... Detection Systems for Continuous Mining Machines in Underground Coal Mines AGENCY: Mine Safety and Health... (except full-face continuous mining machines) with proximity detection systems. Miners working near..., each underground coal mine operator would be required to install proximity detection systems on...

Uranium mine ventilation

International Nuclear Information System (INIS)

Katam, K.; Sudarsono

1982-01-01

Uranium mine ventilation system aimed basically to control and decreasing the air radioactivity in mine caused by the radon emanating from uranium ore. The control and decreasing the air ''age'' in mine, with adding the air consumption volume, increasing the air rate consumption, closing the mine-out area; using closed drainage system. Air consumption should be 60m 3 /minute for each 9m 2 uranium ore surfaces with ventilation rate of 15m/minute. (author)

Appalachian mine soil morphology and properties: Effects of weathering and mining method

Energy Technology Data Exchange (ETDEWEB)

Haering, K.C.; Daniels, W.L.; Galbraith, J.M. [Virginia Polytechnic Institute & State University, Blacksburg, VA (United States)

2004-08-01

Surface coal mining and reclamation methods in the Appalachians have changed dramatically since the passage of the Surface Mining Control and Reclamation Act (SMCRA) of 1977 and subsequent improvements in mining and reclamation technology. In this study, 30 pre-SMCRA mine soil profiles (4-20 yr old) were examined and sampled in 1980 and compared with 20 mine soil profiles (8-13 yr old) described in the same area in 2002 after it had been completely remined by modern deep cut methods. Mine soils in both sampling years had high rock fragment content (42-81%), relatively well-developed A horizons, and generally exhibited A-C or A-AC-C horizonation. Although six Bw horizons were described in 1980, only two met all requirements for cambic horizons. The 1980 mine soils developed in overburden dominated by oxidized, preweathered material due to relatively shallow mining cuts. The 1980 mine soils had lower rock fragment content, finer textures, lower pH, and tended to be more heterogeneous in horizonation, morphology, and texture than soils observed in 2002, which had formed primarily in unweathered overburden from deeper cuts. Half the pedons sampled in both years had densic materials within 70 cm of the surface. Four poorly to very poorly drained soil profiles were described in each sampling year containing distinct hydric soil indicators in surface horizons. While older pre-SMCRA mine soils do have many properties in common with newer mine soils, their properties are highly influenced by the fact that they generally have formed in more weathered overburden from higher in the geologic column. Overall, Appalachian mine soils are much more complex in subsoil morphology than commonly assumed, and differential compaction greatly complicates their internal drainage and limits their overall productivity potential.

First Mining workshop of Mining and metallurgical of MERCOSUR

International Nuclear Information System (INIS)

1994-01-01

In the city of Montevideo, capital of the Oriental Republic of Uruguay, at 23 days of September 1994, under the First Meeting of Mercosur Mining Metallurgical, meet representatives of the mining sector in the countries signed the Treaty of Asuncion , attended as observers, authorities of the Republic of Bolivia and Ecuador and representatives of the productive labor, legislative and research. The primary objective is to integrate the mining sectors of those countries, taking into account the specificity of the mining, given by the resource it uses, the need for high-risk investment with slow recoveries of capital and infrastructure problems, taking into account leverage and its remarkable impact on the development of regional economies.

SKI2 mediates degradation of RISC 5′-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis

Science.gov (United States)

Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

2015-01-01

Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20–24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5′, but not 3′-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5′ to the cleavage site, but several examples of 3′-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5′-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5′-cleavage fragments. PMID:26464441

On 3D Geo-visualization of a Mine Surface Plant and Mine Roadway

Institute of Scientific and Technical Information of China (English)

WANG Yunjia; FU Yongming; FU Erjiang

2007-01-01

Constructing the 3D virtual scene of a coal mine is the objective requirement for modernizing and processing information on coal mining production. It is also the key technology to establish a "digital mine". By exploring current worldwide research, software and hardware tools and application demands, combined with the case study site (the Dazhuang mine of Pingdingshan coal group), an approach for 3D geo-visualization of a mine surface plant and mine roadway is deeply discussed. In this study, the rapid modeling method for a large range virtual scene based on Arc/Info and SiteBuilder3D is studied, and automatic generation of a 3D scene from a 2D scene is realized. Such an automatic method which can convert mine roadway systems from 2D to 3D is realized for the Dazhuang mine. Some relevant application questions are studied, including attribute query, coordinate query, distance measure, collision detection and the dynamic interaction between 2D and 3D virtual scenes in the virtual scene of a mine surface plant and mine roadway. A prototype system is designed and developed.

Survey of nine surface mines in North America. [Nine different mines in USA and Canada

Energy Technology Data Exchange (ETDEWEB)

Hayes, L.G.; Brackett, R.D.; Floyd, F.D.

1981-01-01

This report presents the information gathered by three mining engineers in a 1980 survey of nine surface mines in the United States and Canada. The mines visited included seven coal mines, one copper mine, and one tar sands mine selected as representative of present state of the art in open pit, strip, and terrace pit mining. The purpose of the survey was to investigate mining methods, equipment requirements, operating costs, reclamation procedures and costs, and other aspects of current surface mining practices in order to acquire basic data for a study comparing conventional and terrace pit mining methods, particularly in deeper overburdens. The survey was conducted as part of a project under DOE Contract No. DE-AC01-79ET10023 titled The Development of Optimal Terrace Pit Coal Mining Systems.

Mining planing introduction

International Nuclear Information System (INIS)

Toledo, R.D.

1985-01-01

Basic concepts concerning mining parameters, plan establishment and typical procedure methods applied throughout the physical execution of mining operations are here determined, analyzed and discussed. Technological and economic aspects of the exploration phase are presented as well as general mathematical and statistical methods for estimating, analyzing and representing mineral deposits which are virtually essential for good mining project execution. The characterization of important mineral substances and the basic parameters of mining works are emphasized in conjunction with long, medium and short term mining planning. Finally, geological modelling, ore reserves calculations and final economic evaluations are considered using a hypothetical example in order to consolidate the main elaborated ideas. (D.J.M.) [pt

African mining '91

International Nuclear Information System (INIS)

1991-01-01

The second 'African Mining' conference was held in June 1991, and followed the first event held in May 1987. That full four-year period was characterized by substantial changes in the political and economic climate of many countries in both hemispheres. The results of many of these changing facets of our industry are described in the papers in African Mining'91. Many of the papers deal with advances in technology, which is the main reason for the meeting. There are 37 papers under the headings general, mining, metallurgy and geology and exploration. Most papers are concerned with gold, copper and mineral mining. One paper concerning uranium mining operations in Namibia is indexed separately. (author)

The remediation of abandoned workings of a mining area in Ningxiang uranium mine

International Nuclear Information System (INIS)

Liu Yaochi; Zhou Xinghuo; Liu Bing

2004-01-01

The typical mining under buildings and river was used in a mining area of Ningxiang uranium mine. After the mining ended, 32.1% of the 2.68 m 3 abandoned workings did not fill because of limitation of the cut-and-fill mining method at that time. To remedy this, the mine used new filling methods. After the remedial action, the filling coefficient of pits reached 100%, and that of tunnels reached 86%. It can be proved by the monitoring data that the subsiding of surface has been effectively controlled at the abandoned workings

Identification of Cleavage Sites Recognized by the 3C-Like Cysteine Protease within the Two Polyproteins of Strawberry Mottle Virus

Directory of Open Access Journals (Sweden)

Hélène Sanfaçon

2017-04-01

Full Text Available Strawberry mottle virus (SMoV, family Secoviridae, order Picornavirales is one of several viruses found in association with strawberry decline disease in Eastern Canada. The SMoV genome consists of two positive-sense single-stranded RNAs, each encoding one large polyprotein. The RNA1 polyprotein (P1 includes the domains for a putative helicase, a VPg, a 3C-like cysteine protease and an RNA-dependent RNA polymerase at its C-terminus, and one or two protein domains at its N-terminus. The RNA2 polyprotein (P2 is predicted to contain the domains for a movement protein (MP and one or several coat proteins at its N-terminus, and one or more additional domains for proteins of unknown function at its C-terminus. The RNA1-encoded 3C-like protease is presumed to cleave the two polyproteins in cis (P1 and in trans (P2. Using in vitro processing assays, we systematically scanned the two polyproteins for cleavage sites recognized by this protease. We identified five cis-cleavage sites in P1, with cleavage between the putative helicase and VPg domains being the most efficient. The presence of six protein domains in the SMoV P1, including two upstream of the putative helicase domain, is a feature shared with nepoviruses but not with comoviruses. Results from trans-cleavage assays indicate that the RNA1-encoded 3C-like protease recognized a single cleavage site, which was between the predicted MP and coat protein domains in the P2 polyprotein. The cleavage site consensus sequence for the SMoV 3C-like protease is AxE (E or Q/(G or S.

The levels of disclosure relating to mine closure obligations by platinum mining companies

Directory of Open Access Journals (Sweden)

Joline Sturdy

2017-06-01

Aim: The aim of this study is to establish the extent to which platinum mines listed on the Johannesburg Stock Exchange (JSE comply with a recommended disclosure framework. Setting: South Africa is the largest producer of platinum in the world. The study covers all platinum mines listed on the JSE. Methods: Using a framework, a census of the annual financial statements, integrated annual reports and sustainability reports or websites was conducted to determine the level of compliance of disclosure relating to mine closure obligations to the recommended disclosure framework. Results: The results show disclosure relating to mine closure obligations of platinum mines listed on the JSE is inconsistent and not sufficient for stakeholders to understand the scope, key assumptions, parameters or reliability of the assessment and calculation of mine closure obligations. Conclusion: The assumptions used to determine mine closure obligations are specialised and multi-disciplinary. The accuracy and reliability of mine closure obligations will improve dramatically through greater transparency and access to information. It is recommended that the JSE listings for mining companies should require a competent person’s report to provide disclosure on assumptions, key values and processes applied to determine the mine closure obligations. Furthermore, it is recommended that the Department of Mineral Resources implements a mechanism of independent assessment of mine closure obligations by experts on an ongoing basis.

Host Factors Influencing the Retrohoming Pathway of Group II Intron RmInt1, Which Has an Intron-Encoded Protein Naturally Devoid of Endonuclease Activity.

Directory of Open Access Journals (Sweden)

Rafael Nisa-Martínez

Full Text Available Bacterial group II introns are self-splicing catalytic RNAs and mobile retroelements that have an open reading frame encoding an intron-encoded protein (IEP with reverse transcriptase (RT and RNA splicing or maturase activity. Some IEPs carry a DNA endonuclease (En domain, which is required to cleave the bottom strand downstream from the intron-insertion site for target DNA-primed reverse transcription (TPRT of the inserted intron RNA. Host factors complete the insertion of the intron. By contrast, the major retrohoming pathway of introns with IEPs naturally lacking endonuclease activity, like the Sinorhizobium meliloti intron RmInt1, is thought to involve insertion of the intron RNA into the template for lagging strand DNA synthesis ahead of the replication fork, with possible use of the nascent strand to prime reverse transcription of the intron RNA. The host factors influencing the retrohoming pathway of such introns have not yet been described. Here, we identify key candidates likely to be involved in early and late steps of RmInt1 retrohoming. Some of these host factors are common to En+ group II intron retrohoming, but some have different functions. Our results also suggest that the retrohoming process of RmInt1 may be less dependent on the intracellular free Mg2+ concentration than those of other group II introns.

Economics of mine water treatment

OpenAIRE

Dvořáček, Jaroslav; Vidlář, Jiří; Štěrba, Jiří; Heviánková, Silvie; Vaněk, Michal; Barták, Pavel

2012-01-01

Mine water poses a significant problem in lignite coal mining. The drainage of mine water is the fundamental prerequisite of mining operations. Under the legislation of the Czech Republic, mine water that discharges into surface watercourse is subject to the permission of the state administration body in the water management sector. The permission also stipulates the limits for mine water pollution. Therefore, mine water has to be purified prior to discharge. Although all...

Mine waste management

International Nuclear Information System (INIS)

Hutchinson, I.P.G.; Ellison, R.D.

1992-01-01

This book reports on mine waste management. Topics covered include: Performance review of modern mine waste management units; Mine waste management requirements; Prediction of acid generation potential; Attenuation of chemical constituents; Climatic considerations; Liner system design; Closure requirements; Heap leaching; Ground water monitoring; and Economic impact evaluation

Post-mining in France

International Nuclear Information System (INIS)

2007-01-01

This plentifully illustrated book aims at showing how new equilibria are building up during the transition between mining activity and post-mining, and at stressing on the necessity to keep up the cultural elements, the competencies and knowledge of mining works. The first chapter - mine and men - shows the importance of mineral substances in the objects of the everyday life, illustrates the importance of the mining tradition in France and describes the technical and administrative organisation of the end of the mining activity (works, rehabilitation, regulation, monitoring..). Chapter two - exploitation methods - presents the surface and underground facilities and their impact on the environment (extraction machines, workshops, ore processing plants, decantation ponds..). The third chapter deals with the rehabilitation and monitoring aspects: impact of mining activity stoppage on underground and surface waters, land stability, soils cleansing.. The last chapter summarizes the history of French mining region by region: Nord-Pas-de-Calais, Lorraine-Alsace, Massif central, Bretagne-Normandie, Provence-Alpes-Cote d'Azur and Pyrenees

Treating mine water

Energy Technology Data Exchange (ETDEWEB)

Matlak, E S; Kochegarova, L V; Zaslavskaya, I Yu

1980-10-01

Taking into account the negative influence of mine waters with suspended matter on the natural environment on the surface, the maximum treatment of mine water underground, is proposed. It is noted that full treatment of mine water, using conventional filtration methods, would be rather expensive, but a limited treatment of mine water is possible. Such treated mine water can be used in dust suppression and fire fighting systems. Mine water treated underground should be free of any odor, with pH level ranging from 6 to 9.5, with suspended matter content not exceeding 50 mg/l and coli-titre not less than 300 cm$SUP$3. It is suggested that water treatment to produce water characterized by these parameters is possible and economical. Recommendations on construction of underground sedimentation tanks and channels, and a hydraulic system of cleaning sedimentation tanks are proposed. The settling would be stored underground in abandoned workings. (2 refs.) (In Russian)

MONITORING METAL POLLUTION LEVELS IN MINE WASTES AROUND A COAL MINE SITE USING GIS

Directory of Open Access Journals (Sweden)

D. Sanliyuksel Yucel

2017-11-01

Full Text Available In this case study, metal pollution levels in mine wastes at a coal mine site in Etili coal mine (Can coal basin, NW Turkey are evaluated using geographical information system (GIS tools. Etili coal mine was operated since the 1980s as an open pit. Acid mine drainage is the main environmental problem around the coal mine. The main environmental contamination source is mine wastes stored around the mine site. Mine wastes were dumped over an extensive area along the riverbeds, and are now abandoned. Mine waste samples were homogenously taken at 10 locations within the sampling area of 102.33 ha. The paste pH and electrical conductivity values of mine wastes ranged from 2.87 to 4.17 and 432 to 2430 μS/cm, respectively. Maximum Al, Fe, Mn, Pb, Zn and Ni concentrations of wastes were measured as 109300, 70600, 309.86, 115.2, 38 and 5.3 mg/kg, respectively. The Al, Fe and Pb concentrations of mine wastes are higher than world surface rock average values. The geochemical analysis results from the study area were presented in the form of maps. The GIS based environmental database will serve as a reference study for our future work.

Monitoring Metal Pollution Levels in Mine Wastes around a Coal Mine Site Using GIS