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Sample records for endonuclease ecoo109i studied

  1. Recognition of an expanded genetic alphabet by type-II restriction endonucleases and their application to analyze polymerase fidelity.

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    Chen, Fei; Yang, Zunyi; Yan, Maocai; Alvarado, J Brian; Wang, Ganggang; Benner, Steven A

    2011-05-01

    To explore the possibility of using restriction enzymes in a synthetic biology based on artificially expanded genetic information systems (AEGIS), 24 type-II restriction endonucleases (REases) were challenged to digest DNA duplexes containing recognition sites where individual Cs and Gs were replaced by the AEGIS nucleotides Z and P [respectively, 6-amino-5-nitro-3-(1'-β-D-2'-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one]. These AEGIS nucleotides implement complementary hydrogen bond donor-donor-acceptor and acceptor-acceptor-donor patterns. Results allowed us to classify type-II REases into five groups based on their performance, and to infer some specifics of their interactions with functional groups in the major and minor grooves of the target DNA. For three enzymes among these 24 where crystal structures are available (BcnI, EcoO109I and NotI), these interactions were modeled. Further, we applied a type-II REase to quantitate the fidelity polymerases challenged to maintain in a DNA duplex C:G, T:A and Z:P pairs through repetitive PCR cycles. This work thus adds tools that are able to manipulate this expanded genetic alphabet in vitro, provides some structural insights into the working of restriction enzymes, and offers some preliminary data needed to take the next step in synthetic biology to use an artificial genetic system inside of living bacterial cells. © The Author(s) 2011. Published by Oxford University Press.

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

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    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.

  3. 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

  4. A comparative study of cold- and warm-adapted Endonucleases A using sequence analyses and molecular dynamics simulations.

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    Davide Michetti

    Full Text Available The psychrophilic and mesophilic endonucleases A (EndA from Aliivibrio salmonicida (VsEndA and Vibrio cholera (VcEndA have been studied experimentally in terms of the biophysical properties related to thermal adaptation. The analyses of their static X-ray structures was no sufficient to rationalize the determinants of their adaptive traits at the molecular level. Thus, we used Molecular Dynamics (MD simulations to compare the two proteins and unveil their structural and dynamical differences. Our simulations did not show a substantial increase in flexibility in the cold-adapted variant on the nanosecond time scale. The only exception is a more rigid C-terminal region in VcEndA, which is ascribable to a cluster of electrostatic interactions and hydrogen bonds, as also supported by MD simulations of the VsEndA mutant variant where the cluster of interactions was introduced. Moreover, we identified three additional amino acidic substitutions through multiple sequence alignment and the analyses of MD-based protein structure networks. In particular, T120V occurs in the proximity of the catalytic residue H80 and alters the interaction with the residue Y43, which belongs to the second coordination sphere of the Mg2+ ion. This makes T120V an amenable candidate for future experimental mutagenesis.

  5. Crystallographic and bioinformatic studies on restriction endonucleases: inference of evolutionary relationships in the "midnight zone" of homology.

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    Bujnicki, Janusz M

    2003-10-01

    Type II restriction endonucleases (ENases) cleave DNA with remarkable sequence specificity. Their discovery in 1970 and studies on molecular genetics and biochemistry carried out over the past four decades laid foundations for recombinant DNA techniques. Today, restriction enzymes are indispensable tools in molecular biology and molecular medicine and a paradigm for proteins that specifically interact with DNA as well as a challenging target for protein engineering. The sequence-structure-function relationships for these proteins are therefore of central interest in biotechnology. However, among numerous ENase sequences, only a few exhibit statistically significant similarity in pairwise comparisons, which was initially interpreted as evidence for the lack of common origin. Nevertheless, X-ray crystallographic studies of seemingly dissimilar type II ENases demonstrated that they share a common structural core and metal-binding/catalytic site, arguing for extreme divergence rather than independent evolution. A similar nuclease domain has been also identified in various enzymes implicated in DNA repair and recombination. Ironically, following the series of crystallographic studies suggesting homology of all type II ENases, bioinformatic studies provided evidence that some restriction enzymes are in fact diverged members of unrelated nuclease superfamilies: Nuc, HNH and GIY-YIG. Hence, the restriction enzymes as a whole, represent a group of functionally similar proteins, which evolved on multiple occasions and subsequently diverged into the "midnight zone" of homology, where common origins within particular groups can be inferred only from structure-guided comparisons. The structure-guided approaches used for this purpose include: identification of functionally important residues using superposition of atomic coordinates, alignment of sequence profiles enhanced by secondary structures, fold recognition, and homology modeling. This review covers recent results of

  6. Thermodynamic DNA Looping by a Two-Site Restriction Endonuclease Studied using Optical Tweezers

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    Gemmen, Gregory J.

    2005-03-01

    Many enzyme-DNA interactions involve multimeric protein complexes that bind at two distant sites such that the DNA is looped. An example is the type IIe restriction enzyme Sau3AI, which requires two recognition sites to cleave the DNA. Here we study this process at the single DNA level using force measuring optical tweezers. We characterize cleavage rates of single DNA molecules in the presence of Sau3AI as a function of enzyme concentration, incubation time, and the fractional extension of the DNA molecule. Activity is completely inhibited by tensions of a few picoNewtons. By replacing Mg^2+ with Ca^2+, the Sau3AI dimers form but do not cleave the DNA, thus trapping DNA loops. We are able to pull apart these loops, measuring the force needed and the length of DNA released for each. We also characterize the number and length distributions of these loops as a function of incubation time and DNA fractional extension. The results of these studies are discussed in the context of a Brownian dynamics model of DNA looping.

  7. Catalytic domain of restriction endonuclease BmrI as a cleavage module for engineering endonucleases with novel substrate specificities.

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    Chan, Siu-hong; Bao, Yongming; Ciszak, Ewa; Laget, Sophie; Xu, Shuang-yong

    2007-01-01

    Creating endonucleases with novel sequence specificities provides more possibilities to manipulate DNA. We have created a chimeric endonuclease (CH-endonuclease) consisting of the DNA cleavage domain of BmrI restriction endonuclease and C.BclI, a controller protein of the BclI restriction-modification system. The purified chimeric endonuclease, BmrI198-C.BclI, cleaves DNA at specific sites in the vicinity of the recognition sequence of C.BclI. Double-strand (ds) breaks were observed at two sites: 8 bp upstream and 18 bp within the C-box sequence. Using DNA substrates with deletions of C-box sequence, we show that the chimeric endonuclease requires the 5' half of the C box only for specific cleavage. A schematic model is proposed for the mode of protein-DNA binding and DNA cleavage. The present study demonstrates that the BmrI cleavage domain can be used to create combinatorial endonucleases that cleave DNA at specific sequences dictated by the DNA-binding partner. The resulting endonucleases will be useful in vitro and in vivo to create ds breaks at specific sites and generate deletions.

  8. 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)

  9. Haplotype-based case-control study on human apurinic/apyrimidinic endonuclease 1/redox effector factor-1 gene and essential hypertension.

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    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.

  10. In silico analysis of evolutionary patterns in restriction endonucleases.

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    Singh, Tiratha Raj; Pardasani, Kamal Raj

    2009-01-01

    Restriction endonucleases represent one of the best studied examples of DNA binding proteins. Type II restriction endonucleases recognize short sequences of foreign DNA and cleave the target on both strands with remarkable sequence specificity. Type II restriction endonucleases are part of restriction modification systems. Restriction modification systems occur ubiquitously among bacteria and archaea. Restriction endonucleases are indispensable tools in molecular biology and biotechnology. They are important model system for specific protein-nucleic acid interactions and also serve as good example for investigating structural, functional and evolutionary relationships among various biomolecules. The interaction between restriction endonucleases and their recognition sequences plays a crucial role in biochemical activities like catalytic site/metal binding, DNA repair and recombination etc. We study various patterns in restriction endonucleases type II and analyzed their structural, functional and evolutionary role. Our studies support X-ray crystallographic studies, arguing for divergence and molecular evolution. Conservation patterns of the nuclease superfamily have also been analyzed by estimating site-specific evolutionary rates for the analyzed structures related to respective chains in this study.

  11. Study on detection of mutation DNA fragment in gastric cancer by restriction endonuclease fingerprinting with capillary electrophoresis.

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    Wang, Rong; Xie, Hua; Xu, Yue-Bing; Jia, Zheng-Ping; Meng, Xian-Dong; Zhang, Juan-Hong; Ma, Jun; Wang, Juan; Wang, Xian-Hua

    2012-03-01

    The DNA fragment detection focusing technique has further enhanced the sensitivity and information of DNA targets. The DNA fragment detection method was established by capillary electrophoresis with laser-induced fluorescence detection and restriction endonuclease chromatographic fingerprinting (CE-LIF-REF) in our experiment. The silica capillary column was coated with short linear polyarclarylamide (SLPA) using nongel sieving technology. The excision product of various restricted enzymes of DNA fragments was obtained by REF with the molecular biology software Primer Premier 5. The PBR322/BsuRI DNA marker was used to establish the optimization method. The markers were focused electrophoretically and detected by CE-LIF. The results demonstrate that the CE-LIF-REF with SLPA can improve separation, sensitivity and speed of analysis. This technique may be applied to analysis of the excision product of various restricted enzymes of prokaryotic plasmid (pIRES2), eukaryote plasmid (pcDNA3.1) and the PCR product of codon 248 region of gastric cancer tissue. The results suggest that this method could very sensitively separate the excision products of various restricted enzymes at a much better resolution than the traditional agarose electrophoresis. Copyright © 2011 John Wiley & Sons, Ltd.

  12. Towards the Structural Characterization of Intrinsically Disordered Proteins by SAXS and MD Simulation

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    Oroguchi, Tomotaka; Ikeguchi, Mitsunori; Sato, Mamoru

    2011-01-01

    Dynamical structures of intrinsically disordered proteins (IDPs) and multi-domain proteins that include large ID regions between the domains are unable to be determined by such conventional methods as X-ray crystallography and electron microscopy. Small-angle X-ray scattering (SAXS) is suitable to determine low-resolution structures of proteins and protein complexes in solution, but the structural data on protein dynamics are averaged over the structural ensemble in protein solution. To overcome this problem, we have developed a novel method, named MD-SAXS, of the combined use of SAXS and molecular dynamics (MD) simulation to analyze protein dynamics in solution of multi-subunit protein complexes and multi-domain proteins toward the structural characterization of IDPs. Here we show validity of the method through the structural characterization of restriction Endonuclease EcoO109I.

  13. Crystal structures of Lymphocytic choriomeningitis virus endonuclease domain complexed with diketo-acid ligands

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    Magali Saez-Ayala

    2018-03-01

    Full Text Available The Arenaviridae family, together with the Bunyaviridae and Orthomyxoviridae families, is one of the three negative-stranded RNA viral families that encode an endonuclease in their genome. The endonuclease domain is at the N-terminus of the L protein, a multifunctional protein that includes the RNA-dependent RNA polymerase. The synthesis of mRNA in arenaviruses is a process that is primed by capped nucleotides that are `stolen' from the cellular mRNA by the endonuclease domain in cooperation with other domains of the L protein. This molecular mechanism has been demonstrated previously for the endonuclease of the prototype Lymphocytic choriomeningitis virus (LCMV. However, the mode of action of this enzyme is not fully understood as the original structure did not contain catalytic metal ions. The pivotal role played by the cap-snatching process in the life cycle of the virus and the highly conserved nature of the endonuclease domain make it a target of choice for the development of novel antiviral therapies. Here, the binding affinities of two diketo-acid (DKA compounds (DPBA and L-742,001 for the endonuclease domain of LCMV were evaluated using biophysical methods. X-ray structures of the LCMV endonuclease domain with catalytic ions in complex with these two compounds were determined, and their efficacies were assessed in an in vitro endonuclease-activity assay. Based on these data and computational simulation, two new DKAs were synthesized. The LCMV endonuclease domain exhibits a good affinity for these DKAs, making them a good starting point for the design of arenavirus endonuclease inhibitors. In addition to providing the first example of an X-ray structure of an arenavirus endonuclease incorporating a ligand, this study provides a proof of concept that the design of optimized inhibitors against the arenavirus endonuclease is possible.

  14. Biochemical characterization of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2.

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    Zhang, Likui; Huang, Yanchao; Xu, Dandan; Yang, Lixiang; Qian, Kaicheng; Chang, Guozhu; Gong, Yong; Zhou, Xiaojian; Ma, Kesen

    2016-09-01

    His-Asn-His (HNH) proteins are a very common family of small nucleic acid-binding proteins that are generally associated with endonuclease activity and are found in all kingdoms of life. Although HNH endonucleases from mesophiles have been widely investigated, the biochemical functions of HNH endonucleases from thermophilic bacteriophages remain unknown. Here, we characterized the biochemical properties of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2. The recombinant GVE2 HNH endonuclease exhibited non-specific cleavage activity at high temperature. The optimal temperature of the GVE2 HNH endonuclease for cleaving DNA was 60-65 °C, and the enzyme retained its DNA cleavage activity even after heating at 100 °C for 30 min, suggesting the enzyme is a thermostable endonuclease. The GVE2 HNH endonuclease cleaved DNA over a wide pH spectrum, ranging from 5.5 to 9.0, and the optimal pH for the enzyme activity was 8.0-9.0. Furthermore, the GVE2 HNH endonuclease activity was dependent on a divalent metal ion. While the enzyme is inactive in the presence of Cu(2+), the GVE2 HNH endonuclease displayed cleavage activity of varied efficiency with Mn(2+), Mg(2+), Ca(2+), Fe(2+), Co(2+), Zn(2+), and Ni(2+). The GVE2 HNH endonuclease activity was inhibited by NaCl. This study provides the basis for determining the role of this endonuclease in life cycle of the bacteriophage GVE2 and suggests the potential application of the enzyme in molecular biology and biotechnology.

  15. Haplotype-based case-control study between human apurinic/apyrimidinic endonuclease 1/redox effector factor-1 gene and cerebral infarction.

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    Naganuma, Takahiro; Nakayama, Tomohiro; Sato, Naoyuki; Fu, Zhenyan; Yamaguchi, Mai; Soma, Masayoshi; Aoi, Noriko; Usami, Ron; Doba, Nobutaka; Hinohara, Shigeaki

    2009-10-01

    The aim of this study was to investigate the relationship between cerebral infarction (CI) and the human apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/REF-1) gene using single-nucleotide polymorphisms (SNPs) and a haplotype-based case-control study. We selected 5 SNPs in the human APE1/REF1 gene (rs1760944, rs3136814, rs17111967, rs3136817 and rs1130409), and performed case-control studies in 177 CI patients and 309 control subjects. rs17111967 was found to have no heterogeneity in Japanese. The overall distribution of the haplotype-based case-control study constructed by rs1760944, rs3136814 and rs1130409 showed a significant difference. The frequency of the G-C-T haplotype was significantly higher in the CI group than in the control group (2.5% vs. 0.0%, p>0.001). Based on the results of the haplotype-based case-control-study, the G-C-T haplotype may be a genetic marker of CI, and the APE1/REF-1 gene may be a CI susceptibility gene.

  16. Structural comparison of AP endonucleases from the exonuclease III family reveals new amino acid residues in human AP endonuclease 1 that are involved in incision of damaged DNA.

    Science.gov (United States)

    Redrejo-Rodríguez, Modesto; Vigouroux, Armelle; Mursalimov, Aibek; Grin, Inga; Alili, Doria; Koshenov, Zhanat; Akishev, Zhiger; Maksimenko, Andrei; Bissenbaev, Amangeldy K; Matkarimov, Bakhyt T; Saparbaev, Murat; Ishchenko, Alexander A; Moréra, Solange

    2016-01-01

    Oxidatively damaged DNA bases are substrates for two overlapping repair pathways: DNA glycosylase-initiated base excision repair (BER) and apurinic/apyrimidinic (AP) endonuclease-initiated nucleotide incision repair (NIR). In the BER pathway, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases, whereas in the NIR pathway, the same AP endonuclease incises DNA 5' to an oxidized base. The majority of characterized AP endonucleases possess classic BER activities, and approximately a half of them can also have a NIR activity. At present, the molecular mechanism underlying DNA substrate specificity of AP endonucleases remains unclear mainly due to the absence of a published structure of the enzyme in complex with a damaged base. To identify critical residues involved in the NIR function, we performed biochemical and structural characterization of Bacillus subtilis AP endonuclease ExoA and compared its crystal structure with the structures of other AP endonucleases: Escherichia coli exonuclease III (Xth), human APE1, and archaeal Mth212. We found conserved amino acid residues in the NIR-specific enzymes APE1, Mth212, and ExoA. Four of these positions were studied by means of point mutations in APE1: we applied substitution with the corresponding residue found in NIR-deficient E. coli Xth (Y128H, N174Q, G231S, and T268D). The APE1-T268D mutant showed a drastically decreased NIR activity and an inverted Mg(2+) dependence of the AP site cleavage activity, which is in line with the presence of an aspartic residue at the equivalent position among other known NIR-deficient AP endonucleases. Taken together, these data show that NIR is an evolutionarily conserved function in the Xth family of AP endonucleases. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Inteins, introns, and homing endonucleases: recent revelations about the life cycle of parasitic genetic elements

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    Hilario Elena

    2006-11-01

    Full Text Available Abstract Self splicing introns and inteins that rely on a homing endonuclease for propagation are parasitic genetic elements. Their life-cycle and evolutionary fate has been described through the homing cycle. According to this model the homing endonuclease is selected for function only during the spreading phase of the parasite. This phase ends when the parasitic element is fixed in the population. Upon fixation the homing endonuclease is no longer under selection, and its activity is lost through random processes. Recent analyses of these parasitic elements with functional homing endonucleases suggest that this model in its most simple form is not always applicable. Apparently, functioning homing endonuclease can persist over long evolutionary times in populations and species that are thought to be asexual or nearly asexual. Here we review these recent findings and discuss their implications. Reasons for the long-term persistence of a functional homing endonuclease include: More recombination (sexual and as a result of gene transfer than previously assumed for these organisms; complex population structures that prevent the element from being fixed; a balance between active spreading of the homing endonuclease and a decrease in fitness caused by the parasite in the host organism; or a function of the homing endonuclease that increases the fitness of the host organism and results in purifying selection for the homing endonuclease activity, even after fixation in a local population. In the future, more detailed studies of the population dynamics of the activity and regulation of homing endonucleases are needed to decide between these possibilities, and to determine their relative contributions to the long term survival of parasitic genes within a population. Two outstanding publications on the amoeba Naegleria group I intron (Wikmark et al. BMC Evol Biol 2006, 6:39 and the PRP8 inteins in ascomycetes (Butler et al.BMC Evol Biol 2006, 6:42 provide

  18. Identification of Campylobacter pyloridis isolates by restriction endonuclease DNA analysis

    NARCIS (Netherlands)

    Langenberg, W.; Rauws, E. A.; Widjojokusumo, A.; Tytgat, G. N.; Zanen, H. C.

    1986-01-01

    Campylobacter pyloridis isolates recovered from gastric biopsy specimens of 16 patients were examined by restriction endonuclease DNA analysis with HindIII. For 8 of these 16 patients two different isolates were compared to study the persistence of the colonizing strains and the stability of their

  19. Problem-Solving Test: Restriction Endonuclease Mapping

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    Szeberenyi, Jozsef

    2011-01-01

    The term "restriction endonuclease mapping" covers a number of related techniques used to identify specific restriction enzyme recognition sites on small DNA molecules. A method for restriction endonuclease mapping of a 1,000-basepair (bp)-long DNA molecule is described in the fictitious experiment of this test. The most important fact needed to…

  20. Isothermal detection of RNA with restriction endonucleases.

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    Yan, Lei; Nakayama, Shizuka; Yitbarek, Saron; Greenfield, Isabel; Sintim, Herman O

    2011-01-07

    Herein, we demonstrate how to detect nucleic acids that do not contain restriction endonuclease recognition sites with restriction endonucleases. We show that the topology of DNA probes used in this detection strategy remarkably affects the efficiency of RNA/DNA detection.

  1. Endonucleases induced TRAIL-insensitive apoptosis in ovarian carcinoma cells

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    Geel, Tessa M. [Department of Pathology and Medical Biology, Groningen University Institute for Drug Exploration (GUIDE), University Medical Center Groningen (UMCG), Hanzeplein 1, 9713 GZ, Groningen (Netherlands); Meiss, Gregor [Institute of Biochemistry, Justus-Liebig-University Giessen, D-35392 Giessen (Germany); Gun, Bernardina T. van der; Kroesen, Bart Jan; Leij, Lou F. de [Department of Pathology and Medical Biology, Groningen University Institute for Drug Exploration (GUIDE), University Medical Center Groningen (UMCG), Hanzeplein 1, 9713 GZ, Groningen (Netherlands); Zaremba, Mindaugas; Silanskas, Arunas [Institute of Biotechnology, Vilnius LT-02241 (Lithuania); Kokkinidis, Michael [IMBB/FORTH and University of Crete/Department of Biology, GR-71409 Heraklion/Crete (Greece); Pingoud, Alfred [Institute of Biochemistry, Justus-Liebig-University Giessen, D-35392 Giessen (Germany); Ruiters, Marcel H. [Department of Pathology and Medical Biology, Groningen University Institute for Drug Exploration (GUIDE), University Medical Center Groningen (UMCG), Hanzeplein 1, 9713 GZ, Groningen (Netherlands); Synvolux therapeutics, Groningen (Netherlands); McLaughlin, Pamela M. [Department of Pathology and Medical Biology, Groningen University Institute for Drug Exploration (GUIDE), University Medical Center Groningen (UMCG), Hanzeplein 1, 9713 GZ, Groningen (Netherlands); Rots, Marianne G., E-mail: m.g.rots@med.umcg.nl [Department of Pathology and Medical Biology, Groningen University Institute for Drug Exploration (GUIDE), University Medical Center Groningen (UMCG), Hanzeplein 1, 9713 GZ, Groningen (Netherlands)

    2009-09-10

    TRAIL induced apoptosis of tumor cells is currently entering phase II clinical settings, despite the fact that not all tumor types are sensitive to TRAIL. TRAIL resistance in ovarian carcinomas can be caused by a blockade upstream of the caspase 3 signaling cascade. We explored the ability of restriction endonucleases to directly digest DNA in vivo, thereby circumventing the caspase cascade. For this purpose, we delivered enzymatically active endonucleases via the cationic amphiphilic lipid SAINT-18{sup Registered-Sign }:DOPE to both TRAIL-sensitive and insensitive ovarian carcinoma cells (OVCAR and SKOV-3, respectively). Functional nuclear localization after delivery of various endonucleases (BfiI, PvuII and NucA) was indicated by confocal microscopy and genomic cleavage analysis. For PvuII, analysis of mitochondrial damage demonstrated extensive apoptosis both in SKOV-3 and OVCAR. This study clearly demonstrates that cellular delivery of restriction endonucleases holds promise to serve as a novel therapeutic tool for the treatment of resistant ovarian carcinomas.

  2. Catalytic activity control of restriction endonuclease--triplex forming oligonucleotide conjugates.

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    Silanskas, Arunas; Zaremba, Mindaugas; Sasnauskas, Giedrius; Siksnys, Virginijus

    2012-02-15

    Targeting of individual genes in complex genomes requires endonucleases of extremely high specificity. To direct cleavage at the unique site(s) in the genome, both naturally occurring and artificial enzymes have been developed. These include homing endonucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and restriction or chemical nucleases coupled to a triple-helix forming oligonucleotide (TFO). The desired cleavage has been demonstrated both in vivo and in vitro for several model systems. However, to limit cleavage strictly to unique sites and avoid undesired reactions, endonucleases with controlled activity are highly desirable. In this study we present a proof-of-concept demonstration of two strategies to generate restriction endonuclease-TFO conjugates with controllable activity. First, we combined the restriction endonuclease caging and TFO coupling procedures to produce a caged MunI-TFO conjugate, which can be activated by UV-light upon formation of a triple helix. Second, we coupled TFO to a subunit interface mutant of restriction endonuclease Bse634I which shows no activity due to impaired dimerization but is assembled into an active dimer when two Bse634I monomers are brought into close proximity by triple helix formation at the targeted site. Our results push the restriction endonuclease-TFO conjugate technology one step closer to potential in vivo applications.

  3. Selection of restriction endonucleases using artificial cells.

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    Zheng, Yu; Roberts, Richard J

    2007-01-01

    We describe in this article an in vitro system for the selection of restriction endonucleases using artificial cells. The artificial cells are generated in the form of a water-in-oil emulsion by in vitro compartmentalization. Each aqueous compartment contains a reconstituted transcription/translation mix along with the dispersed DNA templates. In the compartments containing endonuclease genes, an endonuclease expressed in vitro cleaves its own DNA template adjacent to the gene, leaving a sticky end. The pooled DNA templates are then ligated to an adaptor with a compatible end. The endonuclease genes are then enriched by adaptor-specific PCR on the ligation mix. We demonstrate that the system can achieve at least 100-fold enrichment in a single round of selection. It is sensitive enough to enrich an active endonuclease gene from a 1:10(5) model library in 2-3 rounds of selection. Finally, we describe experiments where we selected endonuclease genes directly from a bacterial genomic DNA source in three rounds of selections: the known PstI gene from Providencia stuartii and the new TspMI gene from Thermus sp. manalii. This method provides a unique tool for cloning restriction endonuclease genes and has many other potential applications.

  4. The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit.

    Science.gov (United States)

    Dias, Alexandre; Bouvier, Denis; Crépin, Thibaut; McCarthy, Andrew A; Hart, Darren J; Baudin, Florence; Cusack, Stephen; Ruigrok, Rob W H

    2009-04-16

    The influenza virus polymerase, a heterotrimer composed of three subunits, PA, PB1 and PB2, is responsible for replication and transcription of the eight separate segments of the viral RNA genome in the nuclei of infected cells. The polymerase synthesizes viral messenger RNAs using short capped primers derived from cellular transcripts by a unique 'cap-snatching' mechanism. The PB2 subunit binds the 5' cap of host pre-mRNAs, which are subsequently cleaved after 10-13 nucleotides by the viral endonuclease, hitherto thought to reside in the PB2 (ref. 5) or PB1 (ref. 2) subunits. Here we describe biochemical and structural studies showing that the amino-terminal 209 residues of the PA subunit contain the endonuclease active site. We show that this domain has intrinsic RNA and DNA endonuclease activity that is strongly activated by manganese ions, matching observations reported for the endonuclease activity of the intact trimeric polymerase. Furthermore, this activity is inhibited by 2,4-dioxo-4-phenylbutanoic acid, a known inhibitor of the influenza endonuclease. The crystal structure of the domain reveals a structural core closely resembling resolvases and type II restriction endonucleases. The active site comprises a histidine and a cluster of three acidic residues, conserved in all influenza viruses, which bind two manganese ions in a configuration similar to other two-metal-dependent endonucleases. Two active site residues have previously been shown to specifically eliminate the polymerase endonuclease activity when mutated. These results will facilitate the optimisation of endonuclease inhibitors as potential new anti-influenza drugs.

  5. Massively parallel characterization of restriction endonucleases.

    Science.gov (United States)

    Kamps-Hughes, Nick; Quimby, Aine; Zhu, Zhenyu; Johnson, Eric A

    2013-06-01

    Restriction endonucleases are highly specific in recognizing the particular DNA sequence they act on. However, their activity is affected by sequence context, enzyme concentration and buffer composition. Changes in these factors may lead to either ineffective cleavage at the cognate restriction site or relaxed specificity allowing cleavage of degenerate 'star' sites. Additionally, uncharacterized restriction endonucleases and engineered variants present novel activities. Traditionally, restriction endonuclease activity is assayed on simple substrates such as plasmids and synthesized oligonucleotides. We present and use high-throughput Illumina sequencing-based strategies to assay the sequence specificity and flanking sequence preference of restriction endonucleases. The techniques use fragmented DNA from sequenced genomes to quantify restriction endonuclease cleavage on a complex genomic DNA substrate in a single reaction. By mapping millions of restriction site-flanking reads back to the Escherichia coli and Drosophila melanogaster genomes we were able to quantitatively characterize the cognate and star site activity of EcoRI and MfeI and demonstrate genome-wide decreases in star activity with engineered high-fidelity variants EcoRI-HF and MfeI-HF, as well as quantify the influence on MfeI cleavage conferred by flanking nucleotides. The methods presented are readily applicable to all type II restriction endonucleases that cleave both strands of double-stranded DNA.

  6. Dependence of DNA-protein cross-linking via guanine oxidation upon local DNA sequence as studied by restriction endonuclease inhibition.

    Science.gov (United States)

    Madison, Amanda L; Perez, Zitadel A; To, Phuong; Maisonet, Tiffany; Rios, Eunice V; Trejo, Yuri; Ochoa-Paniagua, Carmen; Reno, Anita; Stemp, Eric D A

    2012-01-10

    Oxidative damage plays a causative role in many diseases, and DNA-protein cross-linking is one important consequence of such damage. It is known that GG and GGG sites are particularly prone to one-electron oxidation, and here we examined how the local DNA sequence influences the formation of DNA-protein cross-links induced by guanine oxidation. Oxidative DNA-protein cross-linking was induced between DNA and histone protein via the flash quench technique, a photochemical method that selectively oxidizes the guanine base in double-stranded DNA. An assay based on restriction enzyme cleavage was developed to detect the cross-linking in plasmid DNA. Following oxidation of pBR322 DNA by flash quench, several restriction enzymes (PpuMI, BamHI, EcoRI) were then used to probe the plasmid surface for the expected damage at guanine sites. These three endonucleases were strongly inhibited by DNA-protein cross-linking, whereas the AT-recognizing enzyme AseI was unaffected in its cleavage. These experiments also reveal the susceptibility of different guanine sites toward oxidative cross-linking. The percent inhibition observed for the endonucleases, and their pBR322 cleavage sites, decreased in the order: PpuMI (5'-GGGTCCT-3' and 5'-AGGACCC-3') > BamHI (5'-GGATCC-3') > EcoRI (5'-GAATTC-3'), a trend consistent with the observed and predicted tendencies for guanine to undergo one-electron oxidation: 5'-GGG-3' > 5'-GG-3' > 5'-GA-3'. Thus, it appears that in mixed DNA sequences the guanine sites most vulnerable to oxidative cross-linking are those that are easiest to oxidize. These results further indicate that equilibration of the electron hole in the plasmid DNA occurs on a time scale faster than that of cross-linking.

  7. 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.

  8. 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.

  9. RNA aptamer inhibitors of a restriction endonuclease.

    Science.gov (United States)

    Mondragón, Estefanía; Maher, L James

    2015-09-03

    Restriction endonucleases (REases) recognize and cleave short palindromic DNA sequences, protecting bacterial cells against bacteriophage infection by attacking foreign DNA. We are interested in the potential of folded RNA to mimic DNA, a concept that might be applied to inhibition of DNA-binding proteins. As a model system, we sought RNA aptamers against the REases BamHI, PacI and KpnI using systematic evolution of ligands by exponential enrichment (SELEX). After 20 rounds of selection under different stringent conditions, we identified the 10 most enriched RNA aptamers for each REase. Aptamers were screened for binding and specificity, and assayed for REase inhibition. We obtained eight high-affinity (Kd ∼12-30 nM) selective competitive inhibitors (IC50 ∼20-150 nM) for KpnI. Predicted RNA secondary structures were confirmed by in-line attack assay and a 38-nt derivative of the best anti-KpnI aptamer was sufficient for inhibition. These competitive inhibitors presumably act as KpnI binding site analogs, but lack the primary consensus KpnI cleavage sequence and are not cleaved by KpnI, making their potential mode of DNA mimicry fascinating. Anti-REase RNA aptamers could have value in studies of REase mechanism and may give clues to a code for designing RNAs that competitively inhibit DNA binding proteins including transcription factors. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. 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

  11. Restriction endonucleases: classification, properties, and applications.

    Science.gov (United States)

    Williams, Raymond J

    2003-03-01

    Restriction endonucleases have become a fundamental tool of molecular biology with many commercial vendors and extensive product lines. While a significant amount has been learned about restriction enzyme diversity, genomic organization, and mechanism, these continue to be active areas of research and assist in classification efforts. More recently, one focus has been their exquisite specificity for the proper recognition sequence and the lack of homology among enzymes recognizing the same DNA sequence. Some questions also remain regarding in vivo function. Site-directed mutagenesis and fusion proteins based on known endonucleases show promise for custom-designed cleavage. An understanding of the enzymes and their properties can improve their productive application by maintaining critical digest parameters and enhancing or avoiding alternative activities.

  12. Substrate generation for endonucleases of CRISPR/cas systems.

    Science.gov (United States)

    Zoephel, Judith; Dwarakanath, Srivatsa; Richter, Hagen; Plagens, André; Randau, Lennart

    2012-09-08

    The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) (1-3). Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems (4). The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster (5). The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs (6-8). These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence (9). A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity(10). Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA (11,12) . These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases (4). Here, we present methods to generate crRNAs and precursor-cRNAs for

  13. Simple and sensitive fluorescence assay of restriction endonuclease on graphene oxide

    Energy Technology Data Exchange (ETDEWEB)

    Gang, Jong Back [Dept. of Nano Chemistry, Gachon University, Incheon (Korea, Republic of)

    2015-09-15

    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.

  14. Identification of Egyptian Fasciola species by PCR and restriction endonucleases digestion of the nuclear small subunit ribosomal RNA gene.

    Science.gov (United States)

    El-Gozamy, Bothina R; Shoukry, Nahla M

    2009-08-01

    Fascioliasis is one of the familiar zoonotic health problems of worldwide distribution including Egypt. In this study, a simple and rapid polymerase chain reaction/restriction fragment length polymorphisms (PCR/RFLPs) assay, using the common restriction endonucleases Aval, EcoRI, Eael, Sac11 and Avail was applied to differentiate between both Fasciola gigantica and F. hepatica. The five restriction endonucleases were used to differentiate between the two species of Fasciola based on -1950 bp long sequence of the 18S nuclear small subunit ribosomal RNA gene. Aval and EcoRI restriction endonucleases failed to differentiate between the two Fasciola species when each restriction enzyme gave the same restriction patterns in both of them. However, F. gigantica and F. hepatica were well-differentiated when their small subunit ribosomal DNA were digested with Eael and Sac 11 restriction endonucleases.

  15. [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.

  16. Depurinized milk downregulates rat thymus MyD88/Akt/p38 function, NF-κB-mediated inflammation, caspase-1 activity but not the endonuclease pathway: in vitro/in vivo study

    Science.gov (United States)

    Kocic, Gordana; Veljkovic, Andrej; Kocic, Hristina; Colic, Miodrag; Mihajlovic, Dusan; Tomovic, Katarina; Stojanovic, Svetlana; Smelcerovic, Andrija

    2017-01-01

    The aim of this study was the evaluation of 15 days dietary regimen of depurinized (DP) milk (obtained using our patented technological procedures) or 1.5% fat UHT milk instead of standard chow diet, on rat thymus and bone marrow MyD88/Akt/p38, NF-κB, caspase-1 and endonuclease pathways, in relation to peripheral blood cell composition. To determine whether the reduced mass of the thymus is a consequence of the direct effect of DP/UHT milk on apoptosis of thymocytes, in vitro Annexin-V-FITC/PI assay was performed. Significant decreases in the thymus wet weight, thymocyte MyD88, Akt-1/phospho-Akt-1 kinase, p38/phospho-p38, NF-κB, caspase-1 activity and CD4+/CD8+ antigen expression were obtained, especially in the DP milk group. The activity of thymocyte alkaline and acid DNase increased in the DP but not in the UHT milk group. The level of IL-6 significantly decreased in DP milk treated group, while the level of total TGF-β and IL-6 increased in UHT milk group. Significant differences in hematological parameters were obtained in commercial milk fed group. Observed results about prevention of experimental diabetes in DP pretreated groups may suggest that purine compounds, uric acid and other volatile toxic compounds of commercial milk may suppress oral tolerance, probably via IL-6 and TGF-β cytokine effects. PMID:28176796

  17. A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site

    Science.gov (United States)

    Karpova, E. A.; Kubareva, E. A.; Shabarova, Z. A.

    1999-01-01

    To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.

  18. New restriction endonucleases from Acetobacter aceti and Bacillus aneurinolyticus.

    Science.gov (United States)

    Sugisaki, H; Maekawa, Y; Kanazawa, S; Takanami, M

    1982-10-11

    Two restriction endonucleases with new sequence specificities have been isolated from Acetobacter aceti IFO 3281 and Bacillus aneurinolyticus IAM 1077 and named AatII and BanII, respectively. Based on analysis of the sequences around the restriction sites, the recognition sequences and cleavage sites of these endonucleases were deduced as below: (formula; see text)

  19. New restriction endonucleases from Acetobacter aceti and Bacillus aneurinolyticus.

    OpenAIRE

    Sugisaki, H; Maekawa, Y; Kanazawa, S; Takanami, M

    1982-01-01

    Two restriction endonucleases with new sequence specificities have been isolated from Acetobacter aceti IFO 3281 and Bacillus aneurinolyticus IAM 1077 and named AatII and BanII, respectively. Based on analysis of the sequences around the restriction sites, the recognition sequences and cleavage sites of these endonucleases were deduced as below: (formula; see text)

  20. 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.

  1. Antibiotic resistance and restriction endonucleases in fecal enterococci of chamois (Rupicapra rupicapra Linnaeus, 1758).

    Science.gov (United States)

    Vandžurová, A; Hrašková, I; Júdová, J; Javorský, P; Pristaš, P

    2012-07-01

    Two hundred eighty-four isolates of enterococci from feces of wild living chamois from alpine environments were tested for sensitivity to three antibiotics. Low frequency of resistance was observed in studied enterococcal populations (about 5 % for tetracycline and erythromycin and 0 % for ampicillin). In six animals, the population of enterococci lacked any detectable resistance. Our data indicated that enterococcal population in feces of the majority of studied animals did not encounter mobile genetic elements encoding antibiotic resistance probably due to spatial separation and/or due to low exposure to the antibiotics. Based on resistance profiles observed, three populations were analyzed for the presence of restriction endonucleases. The restriction enzymes from two isolates-31K and 1K-were further purified and characterized. Restriction endonuclease Efa1KI recognizes CCWGG sequence and is an isoschizomer of BstNI. Endonuclease Efc31KI, a BsmAI isoschizomer, recognizes the sequence GTCTC and it is a first restriction endonuclease identified in Enterococcus faecium. Our data indicate that restriction-modification (R-M) systems do not represent an efficient barrier for antibiotic resistance spreading; enterococcal populations colonized by antibiotics resistance genes were also colonized by the R-M systems.

  2. Endonuclease activities of MutLα and its homologs in DNA mismatch repair.

    Science.gov (United States)

    Kadyrova, Lyudmila Y; Kadyrov, Farid A

    2016-02-01

    MutLα is a key component of the DNA mismatch repair system in eukaryotes. The DNA mismatch repair system has several genetic stabilization functions. Of these functions, DNA mismatch repair is the major one. The loss of MutLα abolishes DNA mismatch repair, thereby predisposing humans to cancer. MutLα has an endonuclease activity that is required for DNA mismatch repair. The endonuclease activity of MutLα depends on the DQHA(X)2E(X)4E motif which is a part of the active site of the nuclease. This motif is also present in many bacterial MutL and eukaryotic MutLγ proteins, DNA mismatch repair system factors that are homologous to MutLα. Recent studies have shown that yeast MutLγ and several MutL proteins containing the DQHA(X)2E(X)4E motif possess endonuclease activities. Here, we review the endonuclease activities of MutLα and its homologs in the context of DNA mismatch repair. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Assaying multiple restriction endonucleases functionalities and inhibitions on DNA microarray with multifunctional gold nanoparticle probes.

    Science.gov (United States)

    Ma, Lan; Zhu, Zhijun; Li, Tao; Wang, Zhenxin

    2014-02-15

    Herein, a double-stranded (ds) DNA microarray-based resonance light scattering (RLS) assay with multifunctional gold nanoparticle (GNP) probes has been developed for studying restriction endonuclease functionality and inhibition. Because of decreasing significantly melting temperature, the enzyme-cleaved dsDNAs easily unwind to form single-stranded (ss) DNAs. The ssDNAs are hybridized with multiplex complementary ssDNAs functionalized GNP probes followed by silver enhancement and RLS detection. Three restriction endonucleases (EcoRI, BamHI and EcoRV) and three potential inhibitors (doxorubicin hydrochloride (DOX), ethidium bromide (EB) and an EcoRI-derived helical peptide (α4)) were selected to demonstrate capability of the assay. Enzyme activities of restriction endonucleases are detected simultaneously with high specificity down to the limits of 2.0 × 10(-2)U/mL for EcoRI, 1.1 × 10(-2)U/mL for BamHI and 1.6 × 10(-2)U/mL for EcoRV, respectively. More importantly, the inhibitory potencies of three inhibitors are showed quantitatively, indicating that our approach has great promise for high-throughput screening of restriction endonuclease inhibitors. © 2013 Elsevier B.V. All rights reserved.

  4. Sequence-dependent cleavage of mismatched DNA by Ban I restriction endonuclease.

    Science.gov (United States)

    Gao, Weimin; Zhu, Dan; Keohavong, Phouthone

    2017-10-01

    Restriction enzymes have previously shown the ability to cleave DNA substrates with mismatched base(s) in recognition sequences; in this study, Ban I endonuclease demonstrated this same ability. Single base substitutions were introduced, and fragments containing various types of unpaired base(s) (heteroduplex fragments) within the Ban I endonuclease recognition sequence, 5'-G|GPyPuCC-3', were generated. Each of the heteroduplex fragments was treated with Ban I endonuclease and analyzed by denaturing gradient gel electrophoresis. Our results showed that heteroduplex fragments containing mismatched bases at either the first or third position of the Ban I recognition sequence or, because of the symmetrical structure of the sequence, the sixth or fourth position on the opposite strand were cleaved by the enzyme. Furthermore, these cleaved fragments contained at least one strand corresponding to the original Ban I recognition sequence. Fragments with mismatches formed by an A (noncanonical, nc) opposite a purine (canonical, ca) or a T (nc) opposite a pyrimidine (ca) were cleaved more efficiently than other types of mismatched bases. These results may help elucidate the mechanisms by which DNA and protein interact during the process of DNA cleavage by Ban I endonuclease. Copyright © 2017 John Wiley & Sons, Ltd.

  5. A Flap Endonuclease (TcFEN1) Is Involved in Trypanosoma cruzi Cell Proliferation, DNA Repair, and Parasite Survival.

    Science.gov (United States)

    Ponce, Ivan; Aldunate, Carmen; Valenzuela, Lucia; Sepúlveda, Sofia; Garrido, Gilda; Kemmerling, Ulrike; Cabrera, Gonzalo; Galanti, Norbel

    2017-07-01

    FLAP endonucleases (FEN) are involved both in DNA replication and repair by processing DNA intermediaries presenting a nucleotide flap using its phosphodiesterase activity. In spite of these important functions in DNA metabolism, this enzyme was not yet studied in Trypanosomatids. Trypanosoma cruzi, the ethiological agent of Chagas disease, presents two dividing cellular forms (epimastigote and amastigote) and one non-proliferative, infective form (trypomastigote). The parasite survives DNA damage produced by reactive species generated in its hosts. The activity of a T. cruzi FLAP endonuclease (TcFEN1) was determined in the three cellular forms of the parasite using a DNA substrate generated by annealing three different oligonucleotides to form a double-stranded DNA with a 5' flap in the middle. This activity showed optimal pH and temperature similar to other known FENs. The substrate cut by the flap endonuclease activity could be ligated by the parasite generating a repaired DNA product. A DNA flap endonuclease coding sequence found in the T. cruzi genome (TcFEN1) was cloned, inserted in parasite expression vectors and transfected to epimastigotes. The purified native recombinant protein showed DNA flap endonuclease activity. This endonuclease was found located in the parasite nucleus of transfected epimastigotes and its over-expression increased both parasite proliferation and survival to H 2 O 2 . The presence of a flap endonuclease activity in T. cruzi and its nuclear location are indicative of the participation of this enzyme in DNA processing of flap fragments during DNA replication and repair in this parasite of ancient evolutive origin. J. Cell. Biochem. 118: 1722-1732, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  6. 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....... This third metal ion has a crucial role, triggering the consecutive hydrolysis of the targeted phosphodiester bonds in the DNA strands and leaving its position once the DSB is generated. The multiple structures show the orchestrated conformational changes in the protein residues, nucleotides and metals......-ion cleavage mechanism. We captured intermediates of the different catalytic steps, and this allowed us to watch the reaction by 'freezing' multiple states. We observed the successive entry of two metals involved in the reaction and the arrival of a third cation in a central position of the active site...

  7. 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.

  8. Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system

    Science.gov (United States)

    Morozova, Natalia; Sabantsev, Anton; Bogdanova, Ekaterina; Fedorova, Yana; Maikova, Anna; Vedyaykin, Alexey; Rodic, Andjela; Djordjevic, Marko; Khodorkovskii, Mikhail; Severinov, Konstantin

    2016-01-01

    Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level. PMID:26687717

  9. Enzymatic cleavage of type II restriction endonucleases on the 2'-O-methyl nucleotide and phosphorothioate substituted DNA.

    Directory of Open Access Journals (Sweden)

    Guojie Zhao

    Full Text Available The effects of nucleotide analogue substitution on the cleavage efficiencies of type II restriction endonucleases have been investigated. Six restriction endonucleases (EcoRV, SpeI, XbaI, XhoI, PstI and SphI were investigated respectively regarding their cleavage when substrates were substituted by 2'-O-methyl nucleotide (2'-OMeN and phosphorothioate (PS. Substitutions were made in the recognition sequence and the two nucleotides flanking the recognition sequence for each endonuclease. The endonuclease cleavage efficiencies were determined using FRET-based assay. Results demonstrated a position-dependent inhibitory effect of substitution on the cleavage efficiency for all the six endonucleases. In general, the 2'-OMeN substitutions had greater impact than the PS substitutions on the enzymatic activities. Nucleotides of optimal substitutions for protection against RE cleavage were identified. Experimental results and conclusions in this study facilitate our insight into the DNA-protein interactions and the enzymatic cleavage mechanism, particularly for those whose detailed structure information is not available. In addition, the information could benefit the development of bioengineering and synthetic biology.

  10. Enzymatic cleavage of type II restriction endonucleases on the 2'-O-methyl nucleotide and phosphorothioate substituted DNA.

    Science.gov (United States)

    Zhao, Guojie; Li, Jun; Tong, Zhaoxue; Zhao, Bin; Mu, Runqing; Guan, Yifu

    2013-01-01

    The effects of nucleotide analogue substitution on the cleavage efficiencies of type II restriction endonucleases have been investigated. Six restriction endonucleases (EcoRV, SpeI, XbaI, XhoI, PstI and SphI) were investigated respectively regarding their cleavage when substrates were substituted by 2'-O-methyl nucleotide (2'-OMeN) and phosphorothioate (PS). Substitutions were made in the recognition sequence and the two nucleotides flanking the recognition sequence for each endonuclease. The endonuclease cleavage efficiencies were determined using FRET-based assay. Results demonstrated a position-dependent inhibitory effect of substitution on the cleavage efficiency for all the six endonucleases. In general, the 2'-OMeN substitutions had greater impact than the PS substitutions on the enzymatic activities. Nucleotides of optimal substitutions for protection against RE cleavage were identified. Experimental results and conclusions in this study facilitate our insight into the DNA-protein interactions and the enzymatic cleavage mechanism, particularly for those whose detailed structure information is not available. In addition, the information could benefit the development of bioengineering and synthetic biology.

  11. Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system.

    Science.gov (United States)

    Morozova, Natalia; Sabantsev, Anton; Bogdanova, Ekaterina; Fedorova, Yana; Maikova, Anna; Vedyaykin, Alexey; Rodic, Andjela; Djordjevic, Marko; Khodorkovskii, Mikhail; Severinov, Konstantin

    2016-01-29

    Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Molecular diversity of Pasteurella multocida isolated from cattle and buffaloes in East Azerbaijan province based on restriction endonuclease analysis

    Directory of Open Access Journals (Sweden)

    jalal shayegh

    2014-05-01

    Full Text Available In order to increase information about the molecular diversity of Pasteurella multocida isolated from cattle and buffalo, 2 buffalo and 8 cattle isolates were investigated by Restriction Endonuclease Analysis (REA. REA was performed with Hha-I Endonuclease which established 2 distinct profiles: I and II.  Cattle and buffalo isolates fell into both REA profiles. Contrary to previous studies, the genetic diversity of the isolates was negligible. Considering the similarity of cattle and buffalo isolates is the present study, further studies witch larger samples should be carried out to investigate the possibility of inter-species transmission.

  13. Solitary restriction endonucleases in prokaryotic genomes.

    Science.gov (United States)

    Ershova, Anna S; Karyagina, Anna S; Vasiliev, Mikhail O; Lyashchuk, Alexander M; Lunin, Vladimir G; Spirin, Sergey A; Alexeevski, Andrei V

    2012-11-01

    Prokaryotic restriction-modification (R-M) systems defend the host cell from the invasion of a foreign DNA. They comprise two enzymatic activities: specific DNA cleavage activity and DNA methylation activity preventing cleavage. Typically, these activities are provided by two separate enzymes: a DNA methyltransferase (MTase) and a restriction endonuclease (RE). In the absence of a corresponding MTase, an RE of Type II R-M system is highly toxic for the cell. Genes of the R-M system are linked in the genome in the vast majority of annotated cases. There are only a few reported cases in which the genes of MTase and RE from one R-M system are not linked. Nevertheless, a few hundreds solitary RE genes are present in the Restriction Enzyme Database (http://rebase.neb.com) annotations. Using the comparative genomic approach, we analysed 272 solitary RE genes. For 57 solitary RE genes we predicted corresponding MTase genes located distantly in a genome. Of the 272 solitary RE genes, 99 are likely to be fragments of RE genes. Various explanations for the existence of the remaining 116 solitary RE genes are also discussed.

  14. UVI31+ is a DNA endonuclease that dynamically localizes to chloroplast pyrenoids in C. reinhardtii.

    Directory of Open Access Journals (Sweden)

    Manish Shukla

    Full Text Available UVI31+ is an evolutionarily conserved BolA family protein. In this study we examine the presence, localization and possible functions of this protein in the context of a unicellular alga, Chlamydomonas reinhardtii. UVI31+ in C. reinhardtii exhibits DNA endonuclease activity and is induced upon UV stress. Further, UVI31+ that normally localizes to the cell wall and pyrenoid regions gets redistributed into punctate foci within the whole chloroplast, away from the pyrenoid, upon UV stress. The observed induction upon UV-stress as well as the endonuclease activity suggests plausible role of this protein in DNA repair. We have also observed that UV31+ is induced in C. reinhardtii grown in dark conditions, whereby the protein localization is enhanced in the pyrenoid. Biomolecular interaction between the purified pyrenoids and UVI31+ studied by NMR demonstrates the involvement of the disordered loop domain of the protein in its interaction.

  15. 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.

  16. Restriction endonucleases: natural and directed evolution.

    Science.gov (United States)

    Gupta, Richa; Capalash, Neena; Sharma, Prince

    2012-05-01

    Type II restriction endonucleases (REs) are highly sequence-specific compared with other classes of nucleases. PD-(D/E)XK nucleases, initially represented by only type II REs, now comprise a large and extremely diverse superfamily of proteins and, although sharing a structurally conserved core, typically display little or no detectable sequence similarity except for the active site motifs. Sequence similarity can only be observed in methylases and few isoschizomers. As a consequence, REs are classified according to combinations of functional properties rather than on the basis of genetic relatedness. New alignment matrices and classification systems based on structural core connectivity and cleavage mechanisms have been developed to characterize new REs and related proteins. REs recognizing more than 300 distinct specificities have been identified in RE database (REBASE: http://rebase.neb.com/cgi-bin/statlist ) but still the need for newer specificities is increasing due to the advancement in molecular biology and applications. The enzymes have undergone constant evolution through structural changes in protein scaffolds which include random mutations, homologous recombinations, insertions, and deletions of coding DNA sequences but rational mutagenesis or directed evolution delivers protein variants with new functions in accordance with defined biochemical or environmental pressures. Redesigning through random mutation, addition or deletion of amino acids, methylation-based selection, synthetic molecules, combining recognition and cleavage domains from different enzymes, or combination with domains of additional functions change the cleavage specificity or substrate preference and stability. There is a growing number of patents awarded for the creation of engineered REs with new and enhanced properties.

  17. Investigation of the salicylaldehyde thiosemicarbazone scaffold for inhibition of influenza virus PA endonuclease.

    Science.gov (United States)

    Rogolino, Dominga; Bacchi, Alessia; De Luca, Laura; Rispoli, Gabriele; Sechi, Mario; Stevaert, Annelies; Naesens, Lieve; Carcelli, Mauro

    2015-10-01

    The influenza virus PA endonuclease is an attractive target for the development of novel anti-influenza virus therapeutics, which are urgently needed because of the emergence of drug-resistant viral strains. Reported PA inhibitors are assumed to chelate the divalent metal ion(s) (Mg²⁺ or Mn²⁺) in the enzyme's catalytic site, which is located in the N-terminal part of PA (PA-Nter). In the present work, a series of salicylaldehyde thiosemicarbazone derivatives have been synthesized and evaluated for their ability to inhibit the PA-Nter catalytic activity. Compounds 1-6 have been evaluated against influenza virus, both in enzymatic assays with influenza virus PA-Nter and in virus yield assays in MDCK cells. In order to establish a structure-activity relationship, the hydrazone analogue of the most active thiosemicarbazone has also been evaluated. Since chelation may represent a mode of action of such class of molecules, we studied the interaction of two of them, one with and one without biological activity versus the PA enzyme, towards Mg²⁺, the ion that is probably involved in the endonuclease activity of the heterotrimeric influenza polymerase complex. The crystal structure of the magnesium complex of the o-vanillin thiosemicarbazone ligand 1 is also described. Moreover, docking studies of PA endonuclease with compounds 1 and 2 were performed, to further analyse the possible mechanism of action of this class of inhibitors.

  18. 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

  19. Simple and cost-effective restriction endonuclease analysis of human adenoviruses.

    Science.gov (United States)

    Adhikary, Arun Kumar; Hanaoka, Nozomu; Fujimoto, Tsuguto

    2014-01-01

    Restriction endonuclease analyses (REAs) constitute the only inexpensive molecular approach capable of typing and characterizing human adenovirus (HAdV) strains based on the entire genome. However, the application of this method is limited by the need for time-consuming and labor-intensive procedures. We herein developed a simple and cost-effective REA for assessing HAdV. The method consists of (1) simple and cost-effective DNA extraction, (2) fast restriction endonuclease (RE) digestion, and (3) speedy mini agarose gel electrophoresis. In this study, DNA was isolated according to the kit-based method and 21.0 to 28.0  μg of viral DNA was extracted from prototypes (HAdV-1, HAdV-3, HAdV-4, and HAdV-37) in each flask. The amount of DNA ranged from 11.4 to 57.0  μg among the HAdV-3 (n=73) isolates. The obtained viral DNA was found to be applicable to more than 10 types of REAs. Fast-cut restriction endonucleases (REs) were able to digest the DNA within 15 minutes, and restriction fragments were easily separated via horizontal mini agarose gel electrophoresis. The whole procedure for 10 samples can be completed within approximately six hours (the conventional method requires at least two days). These results show that our REA is potentially applicable in many laboratories in which HAdVs are isolated.

  20. Peculiarities of Crystallization of the Restriction Endonuclease EcoRII

    Science.gov (United States)

    Karpove, Elizaveta; Pusey, M.arc L.

    1998-01-01

    Nucleases interfere with most standard molecular biology procedures. We have purified and crystallized the restriction endonuclease EcoRII, which belongs to the type II of restriction- modification enzyme, to study the protein crystallization process using a "non standard" macromolecule. A procedure for the purification of EcoRII was developed and 99% pure protein as determined by SDS PAGE electrophoresis obtained. Light scattering experiments were performed to assist in screening protein suitable crystallization conditions. The second virial coefficient was determined as a function of precipitating salt concentration, using sodium chloride, ammonium sulfate, and sodium sulfate. Small (maximum size approximately 0.2 mm) well shaped crystals have been obtained. Larger poorly formed crystals (ca 0.5 mm) have also been obtained, but we have been unable to mount them for diff-raction analysis due to their extreme fragility. Crystallization experiments with PEG have shown that using this precipitant, the best crystals are obtained from slightly over-saturated solutions. Use of higher precipitant concentration leads to dendritic crystal formation. EcoRII is difficult to solubilize and meticulous attention must be paid to the presence of reducing agents.

  1. 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.

  2. DNA Modification Methylase Activity of Escherichia coli Restriction Endonucleases K and P

    Science.gov (United States)

    Haberman, Allan; Heywood, Janet; Meselson, Matthew

    1972-01-01

    The highly purified restriction endonucleases of E. coli K and coliphage P1 transfer methyl groups from S-adenosylmethionine to adenine residues of unmodified DNA. Incubation of unmodified DNA with endonucleases K or P and S-adenosylmethionine renders the DNA resistant to restriction. The enzymes, therefore, have both restriction endonuclease and modification methylase activities. PMID:4564204

  3. The Restriction Endonuclease Cleavage Map of Rat Liver Mitochondrial DNA

    NARCIS (Netherlands)

    Bakker, H.; Holtrop, M.; Terpstra, P.

    1977-01-01

    Mitochondrial DNA from rat liver contains six sites for cleavage by the restriction endonucleases Hind III and EcoRI. A large stretch of DNA, comprising about 40% of the mitochondrial genome is not cleaved by either of the enzymes; eight cleavage sites are located on a DNA stretch of 35% of the

  4. Type I restriction endonucleases are true catalytic enzymes.

    Science.gov (United States)

    Bianco, Piero R; Xu, Cuiling; Chi, Min

    2009-06-01

    Type I restriction endonucleases are intriguing, multifunctional complexes that restrict DNA randomly, at sites distant from the target sequence. Restriction at distant sites is facilitated by ATP hydrolysis-dependent, translocation of double-stranded DNA towards the stationary enzyme bound at the recognition sequence. Following restriction, the enzymes are thought to remain associated with the DNA at the target site, hydrolyzing copious amounts of ATP. As a result, for the past 35 years type I restriction endonucleases could only be loosely classified as enzymes since they functioned stoichiometrically relative to DNA. To further understand enzyme mechanism, a detailed analysis of DNA cleavage by the EcoR124I holoenzyme was done. We demonstrate for the first time that type I restriction endonucleases are not stoichiometric but are instead catalytic with respect to DNA. Further, the mechanism involves formation of a dimer of holoenzymes, with each monomer bound to a target sequence and, following cleavage, each dissociates in an intact form to bind and restrict subsequent DNA molecules. Therefore, type I restriction endonucleases, like their type II counterparts, are true enzymes. The conclusion that type I restriction enzymes are catalytic relative to DNA has important implications for the in vivo function of these previously enigmatic enzymes.

  5. 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.

  6. A Complete Cleavage Map of Neurospora crassa mtDNA Obtained with Endonucleases Eco RI and Bam HI

    NARCIS (Netherlands)

    Terpstra, P.; Holtrop, M.

    1977-01-01

    A physical map of Neurospora crassa mitochondrial DNA has been constructed using specific fragments obtained with restriction endonucleases. The DNA has 5 cleavage sites for endonuclease Bam HI, 12 for endonuclease Eco RI and more than 30 for endonuclease Hind III. The sequence of the Eco RI and Bam

  7. Engineering of restriction endonucleases: using methylation activity of the bifunctional endonuclease Eco57I to select the mutant with a novel sequence specificity.

    Science.gov (United States)

    Rimseliene, Renata; Maneliene, Zita; Lubys, Arvydas; Janulaitis, Arvydas

    2003-03-21

    Type II restriction endonucleases (REs) are widely used tools in molecular biology, biotechnology and diagnostics. Efforts to generate new specificities by structure-guided design and random mutagenesis have been unsuccessful so far. We have developed a new procedure called the methylation activity-based selection (MABS) for generating REs with a new specificity. MABS uses a unique property of bifunctional type II REs to methylate DNA targets they recognize. The procedure includes three steps: (1) conversion of a bifunctional RE into a monofunctional DNA-modifying enzyme by cleavage center disruption; (2) mutagenesis and selection of mutants with altered DNA modification specificity based on their ability to protect predetermined DNA targets; (3) reconstitution of the cleavage center's wild-type structure. The efficiency of the MABS technique was demonstrated by altering the sequence specificity of the bifunctional RE Eco57I from 5'-CTGAAG to 5'-CTGRAG, and thus generating the mutant restriction endonuclease (and DNA methyltransferase) of a specificity not known before. This study provides evidence that MABS is a promising technique for generation of REs with new specificities.

  8. Investigation of mutations induced by radiation and restriction endonucleases

    Science.gov (United States)

    Haworth, Kim E.

    The effects of gamma radiation and restriction endonuclease (RE) induced DNA double strand breaks (dsb) upon the mutation frequency and the surviving fraction of three Chinese hamster cell lines V79-4, CHO-K1 and an X-ray sensitive dsb repair deficient cell line xrs-5 were studied. The X-ray sensitive xrs-5 cell line was shown to be more sensitive to both the lethal and the mutagenic effects of gamma radiation having a substantially lower surviving fraction and a higher thymidine kinase (tk) mutation frequency per unit dose than the parental CHO-K1 cells. The frequency of induced hprt- mutations in the V79-4 cell line was comparable to the induced frequency of tk mutations in the CHO-K1 cells. The effect of blunt- and cohesive- ended dsb upon the surviving fraction and the induced mutation frequency was studied by porating different Chinese hamster cell lines (CHO-K1, V79-4 and xrs-5) with RE using Streptolysin O (SLO). The surviving fraction of the different cell lines was reduced with increasing concentrations of Pvu II. Increases in the concentration of Pvu II produced increases in the frequency of hypoxyanthine guanine phosphoribosyl transferase (hprt) mutations in the V79-4 cells and tk mutations in the CHO-K1 and xrs-5 cells. However, the xrs-5 cells were shown to be hypomutable to Pvu II compared with the parental CHO-K1 cells. EcoR1 was ineffective at inducing tk mutations in the CHO-Kl cells but was as effective as Pvu II at inducing hprt mutations in the V79-4 cells. None of the spontaneously induced V79-4 hprt- mutant cells were shown to have observable molecular deletions when analysed by PCR deletion screening. One third of the radiation induced hprt - mutants were shown to be deletions. However, too few mutant cells were analysed for any non-random distribution of deletions to be observed. Half of the hprt- mutants induced by SLO poration alone were shown to be due to deletions of oi\\e or more exons. The distribution of the DNA deletions in SLO hprt

  9. 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.

  10. Engineering TaqII bifunctional endonuclease DNA recognition fidelity: the effect of a single amino acid substitution within the methyltransferase catalytic site.

    Science.gov (United States)

    Zylicz-Stachula, Agnieszka; Zebrowska, Joanna; Czajkowska, Edyta; Wrese, Weronika; Sulecka, Ewa; Skowron, Piotr M

    2016-04-01

    The aim of this study was to improve a useful molecular tool-TaqII restriction endonuclease-methyltransferase-by rational protein engineering, as well as to show an application of our novel method of restriction endonuclease activity modulation through a single amino acid change in the NPPY motif of methyltransferase. An amino acid change was introduced using site-directed mutagenesis into the taqIIRM gene. The mutated gene was expressed in Escherichia coli. The protein variant was purified and characterized. Previously, we described a TspGWI variant with an amino acid change in the methyltransferase motif IV. Here, we investigate a complex, pleiotropic effect of an analogous amino acid change on its homologue-TaqII. The methyltransferase activity is reduced, but not abolished, while TaqII restriction endonuclease can be reactivated by sinefungin, with an increased DNA recognition fidelity. The general method for engineering of the IIS/IIC/IIG restriction endonuclease activity/fidelity is developed along with the generation of an improved TaqII enzyme for biotechnological applications. A successful application of our novel strategy for restriction endonuclease activity/fidelity alteration, based on bioinformatics analyses, mutagenesis and the use of cofactor-analogue activity modulation, is presented.

  11. 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

  12. Genotyping with CRISPR-Cas-derived RNA-guided endonucleases.

    Science.gov (United States)

    Kim, Jong Min; Kim, Daesik; Kim, Seokjoong; Kim, Jin-Soo

    2014-01-01

    Restriction fragment length polymorphism (RFLP) analysis is one of the oldest, most convenient and least expensive methods of genotyping, but is limited by the availability of restriction endonuclease sites. Here we present a novel method of employing CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) in RFLP analysis. We prepare RGENs by complexing recombinant Cas9 protein derived from Streptococcus pyogenes with in vitro transcribed guide RNAs that are complementary to the DNA sequences of interest. Then, we genotype recurrent mutations found in cancer and small insertions or deletions (indels) induced in cultured cells and animals by RGENs and other engineered nucleases such as transcription activator-like effector nucleases (TALENs). Unlike T7 endonuclease I or Surveyor assays that are widely used for genotyping engineered nuclease-induced mutations, RGEN-mediated RFLP analysis can detect homozygous mutant clones that contain identical biallelic indel sequences and is not limited by sequence polymorphisms near the nuclease target sites.

  13. Salient Features of Endonuclease Platforms for Therapeutic Genome Editing.

    Science.gov (United States)

    Certo, Michael T; Morgan, Richard A

    2016-03-01

    Emerging gene-editing technologies are nearing a revolutionary phase in genetic medicine: precisely modifying or repairing causal genetic defects. This may include any number of DNA sequence manipulations, such as knocking out a deleterious gene, introducing a particular mutation, or directly repairing a defective sequence by site-specific recombination. All of these edits can currently be achieved via programmable rare-cutting endonucleases to create targeted DNA breaks that can engage and exploit endogenous DNA repair pathways to impart site-specific genetic changes. Over the past decade, several distinct technologies for introducing site-specific DNA breaks have been developed, yet the different biological origins of these gene-editing technologies bring along inherent differences in parameters that impact clinical implementation. This review aims to provide an accessible overview of the various endonuclease-based gene-editing platforms, highlighting the strengths and weakness of each with respect to therapeutic applications.

  14. Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases.

    Science.gov (United States)

    Belkebir, Abdelkarim; Azeddoug, Houssine

    2013-02-22

    Most of type II restriction endonucleases show an absolute requirement for divalent metal ions as cofactors for DNA cleavage. While Mg(2+) is the natural cofactor other metal ions can substitute it and mediate the catalysis, however Ca(2+) (alone) only supports DNA binding. To investigate the role of Mg(2+) in DNA cleavage by restriction endonucleases, we have studied the Mg(2+) and Mn(2+) concentration dependence of DNA cleavage by SepMI and EhoI. Digestion reactions were carried out at different Mg(2+) and Mn(2+) concentrations at constant ionic strength. These enzymes showed different behavior regarding the ions requirement, SepMI reached near maximal level of activity between 10 and 20mM while no activity was detected in the presence of Mn(2+) and in the presence of Ca(2+) cleavage activity was significantly decreased. However, EhoI was more highly active in the presence of Mn(2+) than in the presence of Mg(2+) and can be activated by Ca(2+). Our results propose the two-metal ion mechanism for EhoI and the one-metal ion mechanism for SepMI restriction endonuclease. The analysis of the kinetic parameters under steady state conditions showed that SepMI had a K(m) value for pTrcHisB DNA of 6.15 nM and a V(max) of 1.79×10(-2)nM min(-1), while EhoI had a K(m) for pUC19 plasmid of 8.66 nM and a V(max) of 2×10(-2)nM min(-1). Copyright © 2012 Elsevier GmbH. All rights reserved.

  15. Identification of leptospiral isolates by bacterial restriction endonuclease analysis (Brenda

    Directory of Open Access Journals (Sweden)

    Venkatesha M

    2001-01-01

    Full Text Available DNA samples from 19 reference serovars belonging to 19 different serogroups of Leptospira interrogans and two serovars belonging to Leptospira biflexa were examined by bacterial restriction endonuclease analysis using EcoR I and Hae III enzymes. All the serovars gave unique restriction patterns that differed from each other. DNA from 10 local isolates digested with these enzymes produced patterns which on comparison with the standard patterns produced by reference strains could be identified to serovar level.

  16. Saccharomyces cerevisiae MutLα IS A MISMATCH REPAIR ENDONUCLEASE*

    Science.gov (United States)

    Kadyrov, Farid A.; Holmes, Shannon F.; Arana, Mercedes E.; Lukianova, Olga A.; O’Donnell, Mike; Kunkel, Thomas A.; Modrich, Paul

    2008-01-01

    MutL homologs are crucial for mismatch repair and genetic stability, but their function is not well understood. Human MutLα (MLH1-PMS2 heterodimer) harbors a latent endonuclease that is dependent on integrity of a PMS2 DQHA(X)2E(X)4E motif (Kadyrov et al. (2006) Cell 126, 297-308). This sequence element is conserved in many MutL homologs, including the PMS1 subunit of Saccharomyces cerevisiae MutLα, but is absent in MutL proteins from bacteria like Escherichia coli that rely on d(GATC) methylation for strand directionality. We show that yeast MutLα is a strand-directed endonuclease that incises DNA in a reaction that depends on a mismatch, yMutSα, yRFC, yPCNA, ATP, and a pre-existing strand break, whereas E. coli MutL is not. Amino acid substitution within the PMS1 DQHA(X)2E(X)4E motif abolishes yMutLα endonuclease activity in vitro and confers strong genetic instability in vivo, but does not affect yMutLα ATPase activity or the ability of the protein to support assembly of the yMutLα•yMutSα•heteroduplex ternary complex. The loaded form of yPCNA may play an important effector role in directing yMutLα incision to the discontinuous strand of a nicked heteroduplex. PMID:17951253

  17. The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases.

    Science.gov (United States)

    Wei, Hua; Therrien, Caitlin; Blanchard, Aine; Guan, Shengxi; Zhu, Zhenyu

    2008-05-01

    Restriction endonucleases are the basic tools of molecular biology. Many restriction endonucleases show relaxed sequence recognition, called star activity, as an inherent property under various digestion conditions including the optimal ones. To quantify this property we propose the concept of the Fidelity Index (FI), which is defined as the ratio of the maximum enzyme amount showing no star activity to the minimum amount needed for complete digestion at the cognate recognition site for any particular restriction endonuclease. Fidelity indices for a large number of restriction endonucleases are reported here. The effects of reaction vessel, reaction volume, incubation mode, substrate differences, reaction time, reaction temperature and additional glycerol, DMSO, ethanol and Mn(2+) on the FI are also investigated. The FI provides a practical guideline for the use of restriction endonucleases and defines a fundamental property by which restriction endonucleases can be characterized.

  18. Crystal structure of the crenarchaeal ExoIII AP endonuclease SisExoIII reveals a conserved disulfide bond endowing the protein with thermostability.

    Science.gov (United States)

    Yan, Zhou; Yuan, Zenglin; Ni, Jinfeng; Gu, Lichuan; Shen, Yulong

    2017-08-26

    AP endonuclease recognizes and cleaves apurinic/apyrimidinic (AP) sites and plays a critical role in base excision repair. Many ExoIII and EndoIV family AP endonucleases have been characterized both biochemically and structurally in Eukaryote and Bacteria. However, relatively fewer have been studied in Euryarchaeota and there is no such report on an AP endonuclease from Crenarchaeota. Here we report, for the first time, the crystal structure of a crenarchaeal ExoIII AP endonuclease, SisExoIII, from Sulfolobus islandicus REY15A. SisExoIII comprises a two-layer core formed by 10 β-sheets and a shell formed by 9 surrounding α-helices. A disulfide bond connecting β8 and β9 is formed by Cys142 and Cys215. This intra-molecular linkage is conserved among crenarchaeal ExoIII homologs and site-directed mutagenesis revealed that it endows the protein with thermostability, however, disruption of the disulfide bond only has a slight effect on the AP endonuclease activity. We also observed that several key residues within the catalytic center including conserved Glu35 and Asn9 show different conformation compared with known ExoIII proteins and form various intra-molecular salt bridges. The protein possesses three putative DNA binding loops with higher flexibility and hydrophobicity than those of ExoIIIs from other organisms. These features may result in low AP endonuclease activity and defect of exonuclease activity of SisExoIII. The study has deepened our understanding in the structural basis of crenarchaeal ExoIII catalysis and clarified a role of the disulfide bond in maintaining protein thermostability. Copyright © 2017. Published by Elsevier Inc.

  19. 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.

  20. Autoscreening of restriction endonucleases for PCR-restriction fragment length polymorphism identification of fungal species, with Pleurotus spp. as an example.

    Science.gov (United States)

    Yang, Zhi-Hui; Huang, Ji-Xiang; Yao, Yi-Jian

    2007-12-01

    A molecular method based on PCR-restriction fragment length polymorphism (RFLP) analysis of internal transcribed spacer (ITS) ribosomal DNA sequences was designed to rapidly identify fungal species, with members of the genus Pleurotus as an example. Based on the results of phylogenetic analysis of ITS sequences from Pleurotus, a PCR-RFLP endonuclease autoscreening (PRE Auto) program was developed to screen restriction endonucleases for discriminating multiple sequences from different species. The PRE Auto program analyzes the endonuclease recognition sites and calculates the sizes of the fragments in the sequences that are imported into the program in groups according to species recognition. Every restriction endonuclease is scored through the calculation of the average coefficient for the sequence groups and the average coefficient for the sequences within a group, and then virtual electrophoresis maps for the selected restriction enzymes, based on the results of the scoring system, are displayed for the rapid determination of the candidate endonucleases. A total of 85 haplotypes representing 151 ITS sequences were used for the analysis, and 2,992 restriction endonucleases were screened to find the candidates for the identification of species. This method was verified by an experiment with 28 samples representing 12 species of Pleurotus. The results of the digestion by the restriction enzymes showed the same patterns of DNA fragments anticipated by the PRE Auto program, apart from those for four misidentified samples. ITS sequences from 14 samples (of which nine sequences were obtained in this study), including four originally misidentified samples, confirmed the species identities revealed by the PCR-RFLP analysis. The method developed here can be used for the identification of species of other living microorganisms.

  1. Differentiation of Acanthamoeba strains from infected corneas and the environment by using restriction endonuclease digestion of whole-cell DNA.

    Science.gov (United States)

    Kilvington, S; Beeching, J R; White, D G

    1991-01-01

    Restriction endonuclease digestion of Acanthamoeba whole-cell DNA was used to study the relationship between 33 morphologically identical strains from keratitis cases (30 strains), contact lens storage containers (2 strains), and soil (1 strain). Samples digested with BglII, EcoRI, or HindIII and separated by agarose gel electrophoresis contained detectable mitochondrial DNA restriction fragment length polymorphisms (RFLPs). By comparing RFLPs, the strains could be assigned to seven multiple-strain and three single-strain groups. The largest of these contained nine strains, eight of which were isolated in keratitis cases in various locations worldwide and may indicate a group particularly associated with keratitis. Restriction endonuclease analysis of whole-cell DNA is proposed as a valuable technique for detecting mitochondrial DNA RFLPs in the differentiation of morphologically identical Acanthamoeba strains and may therefore be useful in resolving the complex taxonomy of the genus, which has hitherto been founded on subjective morphological criteria. Images PMID:1672534

  2. A domain in human EXOG converts apoptotic endonuclease to DNA-repair exonuclease

    Energy Technology Data Exchange (ETDEWEB)

    Szymanski, Michal R.; Yu, Wangsheng; Gmyrek, Aleksandra M.; White, Mark A.; Molineux, Ian J.; Lee, J. Ching; Yin, Y. Whitney

    2017-05-03

    Human EXOG (hEXOG) is a 5'-exonuclease that is crucial for mitochondrial DNA repair; the enzyme belongs to a nonspecific nuclease family that includes the apoptotic endonuclease EndoG. Here we report biochemical and structural studies of hEXOG, including structures in its apo form and in a complex with DNA at 1.81 and 1.85 Å resolution, respectively. A Wing domain, absent in other ββα-Me members, suppresses endonuclease activity, but confers on hEXOG a strong 5'-dsDNA exonuclease activity that precisely excises a dinucleotide using an intrinsic ‘tape-measure’. The symmetrical apo hEXOG homodimer becomes asymmetrical upon binding to DNA, providing a structural basis for how substrate DNA bound to one active site allosterically regulates the activity of the other. These properties of hEXOG suggest a pathway for mitochondrial BER that provides an optimal substrate for subsequent gap-filling synthesis by DNA polymerase γ.

  3. Probing the dynamics of restriction endonuclease NgoMIV-DNA interaction by single-molecule FRET.

    Science.gov (United States)

    Tutkus, Marijonas; Sasnauskas, Giedrius; Rutkauskas, Danielis

    2017-12-01

    Many type II restriction endonucleases require two copies of their recognition sequence for optimal activity. Concomitant binding of two DNA sites by such an enzyme produces a DNA loop. Here we exploit single-molecule Förster resonance energy transfer (smFRET) of surface-immobilized DNA fragments to study the dynamics of DNA looping induced by tetrameric endonuclease NgoMIV. We have employed a DNA fragment with two NgoMIV recognition sites and a FRET dye pair such that upon protein-induced DNA looping the dyes are brought to close proximity resulting in a FRET signal. The dynamics of DNA-NgoMIV interactions proved to be heterogeneous, with individual smFRET trajectories exhibiting broadly different average looped state durations. Distinct types of the dynamics were attributed to different types of DNA-protein complexes, mediated either by one NgoMIV tetramer simultaneously bound to two specific sites ("slow" trajectories) or by semi-specific interactions of two DNA-bound NgoMIV tetramers ("fast" trajectories), as well as to conformational heterogeneity of individual NgoMIV molecules. © 2017 Wiley Periodicals, Inc.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. 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

  6. Quantum Entanglement in the Genome? The Role of Quantum Effects in Catalytic Synchronization of Type II Restriction Endonucleases

    Science.gov (United States)

    Kurian, P.

    Several living systems have been examined for their exhibition of macroscopic quantum effects, showcasing biology's apparent optimization of structure and function for quantum behavior. Prevalent in lower organisms with analogues in eukaryotes, type II restriction endonucleases are the largest class of restriction enzymes. Orthodox type II endonucleases recognize four-to-eight base pair sequences of palindromic DNA, cut both strands symmetrically, and act without an external metabolite such as ATP. While it is known that these enzymes induce strand breaks by nucleophilic attack on opposing phosphodiester bonds of the DNA helix, what remains unclear is the mechanism by which cutting occurs in concert at the catalytic centers. Previous studies indicate the primacy of intimate DNA contacts made by the specifically bound enzyme in coordinating the two synchronized cuts. We propose that collective electronic behavior in the DNA helix generates coherent oscillations---quantized through boundary conditions imposed by the endonuclease---that provide the energy required to break two phosphodiester bonds. Such quanta may be preserved in the presence of thermal noise and electromagnetic interference through the specific complex's exclusion of water and ions surrounding the helix, with the enzyme serving as a decoherence shield. Clamping energy imparted by the decoherence shield is comparable with zero-point modes of the dipole-dipole oscillations in the DNA recognition sequence. The palindromic mirror symmetry of this sequence should conserve parity during the process. Experimental data corroborate that symmetric bond-breaking ceases when the symmetry of the endonuclease complex is violated, or when environmental parameters are perturbed far from biological optima. Persistent correlation between states in DNA sequence across spatial separations of any length---a characteristic signature of quantum entanglement---may be explained by such a physical mechanism.

  7. Structural basis for the substrate selectivity of PvuRts1I, a 5-hydroxymethylcytosine DNA restriction endonuclease.

    Science.gov (United States)

    Shao, Chen; Wang, Chengliang; Zang, Jianye

    2014-09-01

    5-Hydroxymethylation is a curious modification of cytosine that was discovered some decades ago, but its functional role in eukaryotes still awaits elucidation. 5-Hydroxymethylcytosine is an epigenetic marker that is crucial for multiple biological processes. The profile is altered under certain disease conditions such as cancer, Huntington's disease and Alzheimer's disease. Using the DNA-modification-dependent restriction endonuclease AbaSI coupled with sequencing (Aba-seq), the hydroxymethylome can be deciphered at the resolution of individual bases. The method is based on the enzymatic properties of AbaSI, a member of the PvuRts1I family of endonucleases. PvuRts1I is a modification-dependent endonuclease with high selectivity for 5-hydroxymethylcytosine over 5-methylcytosine and cytosine. In this study, the crystal structure of PvuRts1I was determined in order to understand and improve the substrate selectivity. A nuclease domain and an SRA-like domain are located at the N- and C-termini, respectively. Through comparison with other SRA-domain structures, the SRA-like domain was proposed to be the 5-hmC recognition module. Several mutants of PvuRts1I with enzymatic activity restricted to 5-hydroxymethylcytosine only were generated based on the structural analysis, and these enzyme variants are appropriate for separating the hydroxymethylome from the wider methylome.

  8. Quantifying DNA double-strand breaks induced by site-specific endonucleases in living cells by ligation-mediated purification.

    Science.gov (United States)

    Chailleux, Catherine; Aymard, François; Caron, Pierre; Daburon, Virginie; Courilleau, Céline; Canitrot, Yvan; Legube, Gaëlle; Trouche, Didier

    2014-03-01

    Recent advances in our understanding of the management and repair of DNA double-strand breaks (DSBs) rely on the study of targeted DSBs that have been induced in living cells by the controlled activity of site-specific endonucleases, usually recombinant restriction enzymes. Here we describe a protocol for quantifying these endonuclease-induced DSBs; this quantification is essential to an interpretation of how DSBs are managed and repaired. A biotinylated double-stranded oligonucleotide is ligated to enzyme-cleaved genomic DNA, allowing the purification of the cleaved DNA on streptavidin beads. The extent of cleavage is then quantified either by quantitative PCR (qPCR) at a given site or at multiple sites by genome-wide techniques (e.g., microarrays or high-throughput sequencing). This technique, named ligation-mediated purification, can be performed in 2 d. It is more accurate and sensitive than existing alternative methods, and it is compatible with genome-wide analysis. It allows the amount of endonuclease-mediated breaks to be precisely compared between two conditions or across the genome, thereby giving insight into the influence of a given factor or of various chromatin contexts on local repair parameters.

  9. 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...

  10. 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...

  11. Mining for Restriction Endonucleases in Nicaragua

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    Suyén S. Espinoza-Miranda

    2012-12-01

    Full Text Available The Molecular Biology Center at the University of Central America in Nicaragua (CBM-UCA was founded in 1999 to strengthen biotechnology research capacity and education in Nicaragua and the Central American region. One of the first projects launched by the CBM-UCA was bio-prospecting for key industrial enzymes. This ongoing study seeks to discover and characterize restriction enzymes (RE in bacteria, and to create a database of microorganisms isolated and identified by 16S rDNA sequencing methodology. In this paper we highlight the importance of studying the extreme environmental conditions for building knowledge of Nicaraguan biodiversity through modern molecular biology techniques such as metagenomics. The isolation of prototype enzymes such as EcoRV and ClaI is presented as an update and extension of previously undertaken work.

  12. Mechanistic insight into Type I restriction endonucleases.

    Science.gov (United States)

    Youell, James; Firman, Keith

    2012-06-01

    Restriction and modification are two opposing activities that are used to protect bacteria from cellular invasion by DNA (e.g. bacteriophage infection). Restriction activity involves cleavage of the DNA; while modification activity is the mechanism used to "mark" host DNA and involves DNA methylation. The study of Type I restriction enzymes has often been seen as an esoteric exercise and this reflects some of their more unusual properties - non-stoichiometric (non-catalytic) cleavage of the DNA substrate, random cleavage of DNA, a massive ATPase activity, and the ability to both cleave DNA and methylate DNA. Yet these enzymes have been found in many bacteria and are very efficient as a means of protecting bacteria against bacteriophage infection, indicating they are successful enzymes. In this review, we summarise recent work on the mechanisms of action, describe switching of function and review their mechanism of action. We also discuss structural rearrangements and cellular localisation, which provide powerful mechanisms for controlling the enzyme activity. Finally, we speculate as to their involvement in recombination and discuss their relationship to helicase enzymes.

  13. Restriction glycosylases: involvement of endonuclease activities in the restriction process.

    Science.gov (United States)

    Zhang, Yingbiao; Matsuzaka, Tomoyuki; Yano, Hirokazu; Furuta, Yoshikazu; Nakano, Toshiaki; Ishikawa, Ken; Fukuyo, Masaki; Takahashi, Noriko; Suzuki, Yutaka; Sugano, Sumio; Ide, Hiroshi; Kobayashi, Ichizo

    2017-02-17

    All restriction enzymes examined are phosphodiesterases generating 3΄-OH and 5΄-P ends, but one restriction enzyme (restriction glycosylase) excises unmethylated bases from its recognition sequence. Whether its restriction activity involves endonucleolytic cleavage remains unclear. One report on this enzyme, R.PabI from a hyperthermophile, ascribed the breakage to high temperature while another showed its weak AP lyase activity generates atypical ends. Here, we addressed this issue in mesophiles. We purified R.PabI homologs from Campylobacter coli (R.CcoLI) and Helicobacter pylori (R.HpyAXII) and demonstrated their DNA cleavage, DNA glycosylase and AP lyase activities in vitro at 37°C. The AP lyase activity is more coupled with glycosylase activity in R.CcoLI than in R.PabI. R.CcoLI/R.PabI expression caused restriction of incoming bacteriophage/plasmid DNA and endogenous chromosomal DNA within Escherichia coli at 37°C. The R.PabI-mediated restriction was promoted by AP endonuclease action in vivo or in vitro. These results reveal the role of endonucleolytic DNA cleavage in restriction and yet point to diversity among the endonucleases. The cleaved ends are difficult to repair in vivo, which may indicate their biological significance. These results support generalization of the concept of restriction–modification system to the concept of self-recognizing epigenetic system, which combines any epigenetic labeling and any DNA damaging.

  14. 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.

  15. Sensitive fluorescent detection of DNA methyltransferase using nicking endonuclease-mediated multiple primers-like rolling circle amplification.

    Science.gov (United States)

    Huang, Juan; Li, Xiao-Yu; Du, Yi-Chen; Zhang, Li-Na; Liu, Ke-Ke; Zhu, Li-Na; Kong, De-Ming

    2017-05-15

    Sensitive and reliable detection of DNA methyltransferase (MTase) is of great significance for both early tumor diagnosis and therapy. In this study, a simple, label-free and sensitive DNA MTase-sensing method was developed on the basis of a nicking endonuclease-mediated multiple primers-like rolling circle amplification (RCA) strategy. In this method, a dumbbell RCA template was prepared by blunt-end ligation of two molecules of hairpin DNA. In addition to the primer-binding sequence, the dumbbell template contained another three important parts: 5'-CCGG-3' sequences in double-stranded stems, nicking endonuclease recognition sites and C-rich sequences in single-stranded loops. The introduction of 5'-CCGG-3' sequences allows the dumbbell template to be destroyed by the restriction endonuclease, HpaII, but is not destroyed in the presence of the target MTase-M.SssI MTase. The introduction of nicking endonuclease recognition sites makes the M.SssI MTase-protected dumbbell template-mediated RCA proceed in a multiple primers-like exponential mode, thus providing the RCA with high amplification efficiency. The introduction of C-rich sequences may promote the folding of amplification products into a G-quadruplex structure, which is specifically recognized by the commercially available fluorescent probe thioflavin T. Improved RCA amplification efficiency and specific fluorescent recognition of RCA products provide the M.SssI MTase-sensing platform with high sensitivity. When a dumbbell template containing four nicking endonuclease sites is used, highly specific M.SssI MTase activity detection can be achieved in the range of 0.008-50U/mL with a detection limit as low as 0.0011U/mL. Simple experimental operation and mix-and-detection fluorescent sensing mode ensures that M.SssI MTase quantitation works well in a real-time RCA mode, thus further simplifying the sensing performance and making high throughput detection possible. The proposed MTase-sensing strategy was also

  16. Type III restriction endonuclease EcoP15I is a heterotrimeric complex containing one Res subunit with several DNA-binding regions and ATPase activity.

    Science.gov (United States)

    Wyszomirski, Karol H; Curth, Ute; Alves, Jürgen; Mackeldanz, Petra; Möncke-Buchner, Elisabeth; Schutkowski, Mike; Krüger, Detlev H; Reuter, Monika

    2012-04-01

    For efficient DNA cleavage, the Type III restriction endonuclease EcoP15I communicates with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of methylation (Mod) and restriction (Res) subunits forming a multifunctional enzyme complex able to methylate or to cleave DNA. In this study, we determined by different analytical methods that EcoP15I contains a single Res subunit in a Mod(2)Res stoichiometry. The Res subunit comprises a translocase (Tr) domain carrying functional motifs of superfamily 2 helicases and an endonuclease domain with a PD..D/EXK motif. We show that the isolated Tr domain retains ATP-hydrolyzing activity and binds single- and double-stranded DNA in a sequence-independent manner. To localize the regions of DNA binding, we screened peptide arrays representing the entire Res sequence for their ability to interact with DNA. We discovered four DNA-binding regions in the Tr domain and two DNA-binding regions in the endonuclease domain. Modelling of the Tr domain shows that these multiple DNA-binding regions are located on the surface, free to interact with DNA. Interestingly, the positions of the DNA-binding regions are conserved among other Type III restriction endonucleases.

  17. Creation of a type IIS restriction endonuclease with a long recognition sequence.

    Science.gov (United States)

    Lippow, Shaun M; Aha, Patti M; Parker, Matthew H; Blake, William J; Baynes, Brian M; Lipovsek, Dasa

    2009-05-01

    Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6 nt from the target site to generate homogeneous, 5', four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases.

  18. 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

  19. A web-based restriction endonuclease tool for mycobacteriophage cluster prediction.

    Science.gov (United States)

    Gissendanner, Chris R; Wiedemeier, Allison M D; Wiedemeier, Paul D; Minton, Russell L; Bhuiyan, Swapan; Harmson, Jeremy S; Findley, Ann M

    2014-10-01

    A recent explosion in the amount of genomic data has revealed a large genetic diversity in the bacteriophages that infect Mycobacterium smegmatis. In an effort to assess the novelty of newly described mycobacteriophage isolates and provide a preliminary determination of their probable cluster assignment prior to full genome sequencing, we have developed a systematic approach that relies on restriction endonuclease analysis. We demonstrate that a web-based tool, the Phage Enzyme Tool (or PET), is capable of rapidly facilitating this analysis and exhibits reliability in the putative placement of mycobacteriophages into specific clusters of previously sequenced phages. We propose that this tool represents a useful analytical step in the initial study of phage genomes and that this tool will increase the efficiency of phage genome characterization and enhance the educational activities involving mycobacteriophage discovery. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

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

    2015-11-01

    A systematic study of the cleavage of DNA sequences containing 5-fluorocytosine or 5-fluorouracil by type II restriction endonucleases (REs) was performed and the results compared with the same sequences containing natural pyrimidine bases, uracil or 5-methylcytosine. The results show that some REs recognize fluorine as a hydrogen on cytosine and cleave the corresponding sequences where the presence of m5dC leads to blocking of the cleavage. However, on uracil, the same REs recognize the F as a methyl surrogate and cleave the sequences which are not cleaved if uracil is incorporated instead of thymine. These results are interesting for understanding the recognition of DNA sequences by REs and for manipulation of the specific DNA cutting. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Cofactor requirement of HpyAV restriction endonuclease.

    Directory of Open Access Journals (Sweden)

    Siu-Hong Chan

    Full Text Available BACKGROUND: Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M systems in microorganisms. PRINCIPAL FINDINGS: We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg(++. The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity. CONCLUSIONS/SIGNIFICANCE: Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.

  2. Cofactor requirement of HpyAV restriction endonuclease.

    Science.gov (United States)

    Chan, Siu-Hong; Opitz, Lars; Higgins, Lauren; O'loane, Diana; Xu, Shuang-Yong

    2010-02-05

    Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M) systems in microorganisms. We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg(++). The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity. Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.

  3. CgII cleaves DNA using a mechanism distinct from other ATP-dependent restriction endonucleases.

    Science.gov (United States)

    Toliusis, Paulius; Zaremba, Mindaugas; Silanskas, Arunas; Szczelkun, Mark D; Siksnys, Virginijus

    2017-08-21

    The restriction endonuclease CglI from Corynebacterium glutamicum recognizes an asymmetric 5'-GCCGC-3' site and cleaves the DNA 7 and 6/7 nucleotides downstream on the top and bottom DNA strands, respectively, in an NTP-hydrolysis dependent reaction. CglI is composed of two different proteins: an endonuclease (R.CglI) and a DEAD-family helicase-like ATPase (H.CglI). These subunits form a heterotetrameric complex with R2H2 stoichiometry. However, the R2H2·CglI complex has only one nuclease active site sufficient to cut one DNA strand suggesting that two complexes are required to introduce a double strand break. Here, we report studies to evaluate the DNA cleavage mechanism of CglI. Using one- and two-site circular DNA substrates we show that CglI does not require two sites on the same DNA for optimal catalytic activity. However, one-site linear DNA is a poor substrate, supporting a mechanism where CglI complexes must communicate along the one-dimensional DNA contour before cleavage is activated. Based on experimental data, we propose that adenosine triphosphate (ATP) hydrolysis by CglI produces translocation on DNA preferentially in a downstream direction from the target, although upstream translocation is also possible. Our results are consistent with a mechanism of CglI action that is distinct from that of other ATP-dependent restriction-modification enzymes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. 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...

  5. Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases

    DEFF Research Database (Denmark)

    Crouzier, Lucile; Dubois, Camille; Wengel, Jesper

    2012-01-01

    Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving...... LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI....

  6. Exploring both sequence detection and restriction endonuclease cleavage kinetics by recognition site via single-molecule microfluidic trapping.

    Science.gov (United States)

    Xu, Weilin; Muller, Susan J

    2011-02-07

    We demonstrate the feasibility of a single-molecule microfluidic approach to both sequence detection and obtaining kinetic information for restriction endonucleases on dsDNA. In this method, a microfluidic stagnation point flow is designed to trap, hold, and linearize double-stranded (ds) genomic DNA to which a restriction endonuclease has been pre-bound sequence-specifically. By introducing the cofactor magnesium, we determine the binding location of the enzyme by the cleavage process of dsDNA as in optical restriction mapping, however here the DNA need not be immobilized on a surface. We note that no special labeling of the enzyme is required, which makes it simpler than our previous scheme using stagnation point flows for sequence detection. Our accuracy in determining the location of the recognition site is comparable to or better than other single molecule techniques due to the fidelity with which we can control the linearization of the DNA molecules. In addition, since the cleavage process can be followed in real time, information about the cleavage kinetics, and subtle differences in binding and cleavage frequencies among the recognition sites, may also be obtained. Data for the five recognition sites for the type II restriction endonuclease EcoRI on λ-DNA are presented as a model system. While the roles of the varying fluid velocity and tension along the chain backbone on the measured kinetics remain to be determined, we believe this new method holds promise for a broad range of studies of DNA-protein interactions, including the kinetics of other DNA cleavage processes, the dissociation of a restriction enzyme from the cleaved substrate, and other macromolecular cleavage processes.

  7. Genomic DNA restriction endonuclease from Pasteurella multocida isolated from Indonesia, katha strain and reference strains and analysed by PFGE

    Directory of Open Access Journals (Sweden)

    Supar

    2003-10-01

    Full Text Available Pasteurella multocida strains are the causative disease agents of wide range of domestic and wild animals in Indonesia. The most important serotypes are associated with Hemorrhagic septicaemic (HS diseases in cattle and buffaloes, cholera in ducks and chickens. The HS disease associated with P. multocia in large ruminants in Indonesia is controled by killed whole cell vaccines produced by the use of P. multocida Katha strains. There is no discriminatory data of the molecular biology technique has been applied to investigate P. multocida isolates from different geographic locations in Indonesia. The purpose of this studies were to observe the genetic diversity among P. multocida isolated from various geograpic locations and compared with Katha vaccine strain and other reference strains. A total samples of 38 isolates and strains of P. multocida were analysed by means of pulsed-field gel electrophoresis (PFGE. Each sample was grown in nutrient broth, cells were separeted by centrifugation. Whole cell pellet was mixed with agarose and then prepared agarose plugs. The genomic DNA of each sample was digested in situ (plug with either restriction endonuclease of ApaI and/or BamHI. The digested genomic DNA of each sample was analysed by PFGE, the genomic DNA restricted profile of each sample was compared with others. The use of ApaI restriction endonuclease digestion and analysed by PFGE, demonstrated that 34 out of 38 P. multocia samples could be differentiated into 16 ApaI types, whereas based on the BamHI digestion of these samples were differentiated into 20 BamHI types. Genomic DNA restriction pattern of Indonesian P. multocida isolates originated from cattle and buffaloes associated with haemorrhagic septicaemic diseases demonstrated different pattern to those of vaccine Katha strain, poultry strains as well as the reference strains currenly kept at Balitvet Culture Collection (BCC unit. Two P. multocida isolates derived from ducks with cholera

  8. Peculiarities of the interaction of the restriction endonuclease BspD6I with DNA containing its recognition site.

    Science.gov (United States)

    Abrosimova, Liudmila A; Kubareva, Elena A; Migur, Anzhela Yu; Gavshina, Aleksandra V; Ryazanova, Aleksandra Yu; Norkin, Maxim V; Perevyazova, Tatiana A; Wende, Wolfgang; Hianik, Tibor; Zheleznaya, Liudmila A; Oretskaya, Tatiana S

    2016-09-01

    Nicking endonucleases are enzymes that recognize specific sites in double-stranded DNA and cleave only one strand at a predetermined position. These enzymes are involved in DNA replication and repair; they can also function as subunits of bacterial heterodimeric restriction endonucleases. One example of such a proteins is the restriction endonuclease BspD6I (R.BspD6I) from Bacillus species strain D6, which consists of the large subunit - nicking endonuclease BspD6I (Nt.BspD6I), and the small subunit (ss.BspD6I). Nt.BspD6I can function independently. Similar enzymes are now widely used in numerous biotechnological applications. The aim of this study was to investigate the fundamental properties of two subunits of R.BspD6I and their interdependence in the course of R.BspD6I activity. The binding and hydrolysis of DNA duplexes by R.BspD6I are primary analyzed by gel electrophoresis. To elucidate the difference between Nt.BspD6I interaction with the substrate and product of hydrolysis, the thickness shear mode acoustic method is used. The thermodynamic and kinetic parameters of the Nt.BspD6I interaction with DNA are determined. For the first time we demonstrated that Nt.BspD6I bends the DNA during complex formation. Nt.BspD6I is able to form complexes with the product nicked in the top strand and ss.BspD6I cleaves the bottom strand of the DNA consecutively. Furthermore, the influence of dA methylation in the R.BspD6I recognition site on ss.BspD6I activity is analyzed. The obtained results provide evidence that Nt.BspD6I coordinates the activity of R.BspD6I by strictly coupling of the bottom strand cleavage by ss.BspD6I to the top strand cleavage. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Efficient DNA subcloning through selective restriction endonuclease digestion.

    Science.gov (United States)

    Spear, M A

    2000-04-01

    Described here is a selective restriction endonuclease digestion method that eliminates the electrophoresis step that is usually used during the subcloning of new DNA sequences into typical E. coli-based plasmids. The method increases yield while decreasing laboratory resource and time utilization. By using donor and acceptor sequences that contain unique restriction sites found only outside of the intended recombination sequences, the initial digestion products can be directly combined without electrophoresis if the ligation step is followed by a selective digestion using the unique restriction enzymes before transformation. This system is based on the several order of magnitude decrease in transformation efficiency of linearized compared to circular plasmids. As an example, this method was used to obtain recombinants between a 3.6 kb acceptor plasmid and 3.0 kb insert following one ligation reaction after the failure of nine standard reactions using similar amounts of input DNA. It is particularly applicable to situations in which low subcloning efficiencies are expected. The technique can be extended to a large percentage of planned recombinations by using nonidentical compatible cohesive or blunt-ended fragments, or site-directed mutagenesis.

  10. The wonders of flap endonucleases: structure, function, mechanism and regulation.

    Science.gov (United States)

    Finger, L David; Atack, John M; Tsutakawa, Susan; Classen, Scott; Tainer, John; Grasby, Jane; Shen, Binghui

    2012-01-01

    Processing of Okazaki fragments to complete lagging strand DNA synthesis requires coordination among several proteins. RNA primers and DNA synthesised by DNA polymerase α are displaced by DNA polymerase δ to create bifurcated nucleic acid structures known as 5'-flaps. These 5'-flaps are removed by Flap Endonuclease 1 (FEN), a structure-specific nuclease whose divalent metal ion-dependent phosphodiesterase activity cleaves 5'-flaps with exquisite specificity. FENs are paradigms for the 5' nuclease superfamily, whose members perform a wide variety of roles in nucleic acid metabolism using a similar nuclease core domain that displays common biochemical properties and structural features. A detailed review of FEN structure is undertaken to show how DNA substrate recognition occurs and how FEN achieves cleavage at a single phosphate diester. A proposed double nucleotide unpairing trap (DoNUT) is discussed with regards to FEN and has relevance to the wider 5' nuclease superfamily. The homotrimeric proliferating cell nuclear antigen protein (PCNA) coordinates the actions of DNA polymerase, FEN and DNA ligase by facilitating the hand-off intermediates between each protein during Okazaki fragment maturation to maximise through-put and minimise consequences of intermediates being released into the wider cellular environment. FEN has numerous partner proteins that modulate and control its action during DNA replication and is also controlled by several post-translational modification events, all acting in concert to maintain precise and appropriate cleavage of Okazaki fragment intermediates during DNA replication.

  11. 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.

  12. Selective microbial genomic DNA isolation using restriction endonucleases.

    Science.gov (United States)

    Barnes, Helen E; Liu, Guohong; Weston, Christopher Q; King, Paula; Pham, Long K; Waltz, Shannon; Helzer, Kimberly T; Day, Laura; Sphar, Dan; Yamamoto, Robert T; Forsyth, R Allyn

    2014-01-01

    To improve the metagenomic analysis of complex microbiomes, we have repurposed restriction endonucleases as methyl specific DNA binding proteins. As an example, we use DpnI immobilized on magnetic beads. The ten minute extraction technique allows specific binding of genomes containing the DpnI Gm6ATC motif common in the genomic DNA of many bacteria including γ-proteobacteria. Using synthetic genome mixtures, we demonstrate 80% recovery of Escherichia coli genomic DNA even when only femtogram quantities are spiked into 10 µg of human DNA background. Binding is very specific with less than 0.5% of human DNA bound. Next Generation Sequencing of input and enriched synthetic mixtures results in over 100-fold enrichment of target genomes relative to human and plant DNA. We also show comparable enrichment when sequencing complex microbiomes such as those from creek water and human saliva. The technique can be broadened to other restriction enzymes allowing for the selective enrichment of trace and unculturable organisms from complex microbiomes and the stratification of organisms according to restriction enzyme enrichment.

  13. 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.

  14. Levels of the DNA repair enzyme human apurinic/apyrimidinic endonuclease (APE1, APEX, Ref-1) are associated with the intrinsic radiosensitivity of cervical cancers.

    OpenAIRE

    Herring, C. J.; West, C. M.; Wilks, D. P.; Davidson, S. E.; Hunter, R. D.; Berry, P.; Forster, G.; MacKinnon, J.; Rafferty, J. A.; Elder, R. H.; Hendry, J. H.; Margison, G. P.

    1998-01-01

    A study was made of the relationship between the intrinsic radiosensitivity of human cervical tumours and the expression of the DNA repair enzyme human apurinic/apyrimidinic endonuclease (HAP1). The radiosensitivity of clonogenic cells in tumour biopsies was measured as surviving fraction at 2 Gy (SF2) using a soft agar assay. HAP1 expression levels were determined after staining of formalin-fixed paraffin-embedded tumour sections with a rabbit antiserum raised against recombinant HAP1. Both ...

  15. 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

  16. Molecular dynamics of the salt dependence of a cold-adapted enzyme: endonuclease I.

    Science.gov (United States)

    Benrezkallah, D; Dauchez, M; Krallafa, A M

    2015-01-01

    The effects of salt on the stability of globular proteins have been known for a long time. In the present investigations, we shall focus on the effect of the salt ions upon the structure and the activity of the endonuclease I enzyme. In the present work, we shall focus on the relationship between ion position and the structural features of the Vibrio salmonicida (VsEndA) enzyme. We will concentrate on major questions such as: how can salt ions affect the molecular structure? What is the activity of the enzyme and which specific regions are directly involved? For that purpose, we will study the behaviour of the VsEndA over different salt concentrations using molecular dynamics (MD) simulations. We report the results of MD simulations of the endonuclease I enzyme at five different salt concentrations. Analysis of trajectories in terms of the root mean square fluctuation (RMSF), radial distribution function, contact numbers and hydrogen bonding lifetimes, indicate distinct differences when changing the concentration of NaCl. Results are found to be in good agreement with experimental data, where we have noted an optimum salt concentration for activity equal to 425 mM. Under this salt concentration, the VsEndA exhibits two more flexible loop regions, compared to the other salt concentrations. When analysing the RMSF of these two specific regions, three residues were selected for their higher mobility. We find a correlation between the structural properties studied here such as the radial distribution function, the contact numbers and the hydrogen bonding lifetimes, and the structural flexibility of only two polar residues. Finally, in the light of the present work, the molecular basis of the salt adaptation of VsEndA enzyme has been explored by mean of explicit solvent and salt treatment. Our results reveal that modulation of the sodium/chloride ions interaction with some specific loop regions of the protein is the strategy followed by this type of psychrophilic enzyme

  17. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Modulating mtDNA heteroplasmy by mitochondria-targeted restriction endonucleases in a ‘differential multiple cleavage-site’ model

    Science.gov (United States)

    Bacman, SR; Williams, SL; Hernandez, D; Moraes, CT

    2009-01-01

    The ability to manipulate mitochondrial DNA (mtDNA) heteroplasmy would provide a powerful tool to treat mitochondrial diseases. Recent studies showed that mitochondria-targeted restriction endonucleases can modify mtDNA heteroplasmy in a predictable and efficient manner if it recognizes a single site in the mutant mtDNA. However, the applicability of such model is limited to mutations that create a novel cleavage site, not present in the wild-type mtDNA. We attempted to extend this approach to a ‘differential multiple cleavage site’ model, where an mtDNA mutation creates an extra restriction site to the ones normally present in the wild-type mtDNA. Taking advantage of a heteroplasmic mouse model harboring two haplotypes of mtDNA (NZB/BALB) and using adenovirus as a gene vector, we delivered a mitochondria-targeted Scal restriction endonuclease to different mouse tissues. Scal recognizes five sites in the NZB mtDNA but only three in BALB mtDNA. Our results showed that changes in mtDNA heteroplasmy were obtained by the expression of mitochondria-targeted ScaI in both liver, after intravenous injection, and in skeletal muscle, after intramuscular injection. Although mtDNA depletion was an undesirable side effect, our data suggest that under a regulated expression system, mtDNA depletion could be minimized and restriction endonucleases recognizing multiple sites could have a potential for therapeutic use. PMID:17597792

  20. 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; Sellers, Rani S.; Modrich, Paul; Scharff, Matthew D.; Edelmann, Winfried

    2010-01-01

    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. PMID:20624957

  1. Protein NCRII-18: the role of gene fusion in the molecular evolution of restriction endonucleases.

    Science.gov (United States)

    Ibryashkina, Elena M; Solonin, Alexander S; Zakharova, Marina V

    2017-06-01

    This work first constructed the fusion protein NCRII-18 by fusing the restriction endonuclease Ecl18kI gene and part of the gene coding for the N-terminal domain of the endonuclease EcoRII. The fusion of the EcoRII N-terminal domain leads to a change in the properties of the recombinant protein. Unlike Ecl18kI, which made the basis of NCRII-18, the fusion protein predominantly recognizes the CCWGG sites, having lost the capability of interacting with the CCSGG sites. Experimental data support the hypothesis of a close evolutionary relationship between type IIE and IIP restriction endonucleases via a recombination between domains with active site structure and elements for recognition with domains responsible for recognition of DNA sequences. © 2017 Federation of European Biochemical Societies.

  2. Homing endonucleases catalyze double-stranded DNA breaks and somatic transgene excision in Aedes aegypti.

    Science.gov (United States)

    Traver, B E; Anderson, M A E; Adelman, Z N

    2009-10-01

    Aedes aegypti is a major vector of arthropod-borne viruses such as yellow fever virus and dengue viruses. Efforts to discern the function of genes involved in important behaviours, such as vector competence and host seeking through reverse genetics, would greatly benefit from the ability to generate targeted gene disruptions. Homing endonucleases are selfish elements which catalyze double-stranded DNA (dsDNA) breaks in a sequence-specific manner. In this report we demonstrate that the homing endonucleases I-PpoI, I-SceI, I-CreI and I-AniI are all able to induce dsDNA breaks in adult female Ae. aegypti chromosomes as well as catalyze the somatic excision of a transgene. These experiments provide evidence that homing endonucleases can be used to manipulate the genome of this important disease vector.

  3. Human AP Endonuclease 1: A Potential Marker for the Prediction of Environmental Carcinogenesis Risk

    Directory of Open Access Journals (Sweden)

    Jae Sung Park

    2014-01-01

    Full Text Available Human apurinic/apyrimidinic endonuclease 1 (APE1 functions mainly in DNA repair as an enzyme removing AP sites and in redox signaling as a coactivator of various transcription factors. Based on these multifunctions of APE1 within cells, numerous studies have reported that the alteration of APE1 could be a crucial factor in development of human diseases such as cancer and neurodegeneration. In fact, the study on the combination of an individual’s genetic make-up with environmental factors (gene-environment interaction is of great importance to understand the development of diseases, especially lethal diseases including cancer. Recent reports have suggested that the human carcinogenic risk following exposure to environmental toxicants is affected by APE1 alterations in terms of gene-environment interactions. In this review, we initially outline the critical APE1 functions in the various intracellular mechanisms including DNA repair and redox regulation and its roles in human diseases. Several findings demonstrate that the change in expression and activity as well as genetic variability of APE1 caused by environmental chemical (e.g., heavy metals and cigarette smoke and physical carcinogens (ultraviolet and ionizing radiation is likely associated with various cancers. These enable us to ultimately suggest APE1 as a vital marker for the prediction of environmental carcinogenesis risk.

  4. Crystal Structure of the Homing Endonuclease I-CvuI Provides a New Template for Genome Modification

    DEFF Research Database (Denmark)

    Molina, Rafael; Redondo, Pilar; López-Méndez, Blanca

    2015-01-01

    of homing endonucleases have been identified, the landscape of possible target sequences is still very limited to cover the complexity of the whole eukaryotic genome. Therefore, the finding and molecular analysis of homing endonucleases identified but not yet characterized may widen the landscape...

  5. DNA-hosted Hoechst dyes: application for label-free fluorescent monitoring of endonuclease activity and inhibition.

    Science.gov (United States)

    Jiang, Xiao-Qin; Guo, Su-Miao; Zhang, Min; Zhou, Ming; Ye, Bang-Ce

    2014-11-21

    A simple and facile approach was developed for monitoring EcoRI endonuclease activity and inhibition, in which a hairpin-like DNA containing restriction cutting site for EcoRI endonuclease acts as the sensing element and Hoechst dyes as the signal indicator in a label-free format.

  6. Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases.

    Science.gov (United States)

    Crouzier, Lucile; Dubois, Camille; Wengel, Jesper; Veedu, Rakesh N

    2012-07-15

    Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Characterization of endonuclease G and mitochondria-sarcoplasmic reticulum-related proteins during cardiac hypertrophy.

    Science.gov (United States)

    Liang, Xingguang; Ma, Kuifen; Rao, Yuefeng; Hong, Dongsheng; Huo, Zhaoxia; Ye, Ziqi; Huang, Mingzhu; Zhang, Xingguo; Zhao, Qingwei

    2015-09-01

    Endonuclease G (Endo G) is a novel determinant of cardiac hypertrophy. Here, we report the characterization of Endo G and mitochondria-sarcoplasmic reticulum-related proteins during cardiac hypertrophy, and hypothesize that Endo G regulate mitochondrial function partly through Mfn2 and Jp2 during cardiac hypertrophy. Our results show that Endo G levels gradually increased at the beginning of phenylephrine-induced cardiac hypertrophy, accompanied by an abnormal mitochondrial membrane potential. The up-regulation of Mfn2, Jp2, and Endo G appeared at an early stage of cardiac hypertrophy, whereas PGC1α was not up-regulated until a later stage. Abolishing Endo G with siRNA led to the uncoupling of the mitochondrial electron transport chain from ATP production and decreased PGC1α expression, likely by affecting the juxtaposition of the mitochondria and the sarcoplasmic reticulum via Mfn2 and Jp2. Furthermore, abolishing Jp2 altered the expression of Endo G expression and induced mitochondrial dysfunction, suggesting that mitochondrial abnormalities in cardiac hypertrophy are most likely caused by Endo G. Taken together, our study established a link between Endo G and mitochondrial function during cardiac hypertrophy, partly through the effects of Endo G on Mfn2 and Jp2, and revealed a role for Endo G in the crosstalk between the processes controlled by Mfn2 and Jp2 in maladaptive cardiac hypertrophy.

  8. Polymorphism of the Flap Endonuclease 1 Gene in Keratoconus and Fuchs Endothelial Corneal Dystrophy

    Directory of Open Access Journals (Sweden)

    Katarzyna A. Wojcik

    2014-08-01

    Full Text Available Oxidative stress is implicated in the pathogenesis of many diseases, including serious ocular diseases, keratoconus (KC and Fuchs endothelial corneal dystrophy (FECD. Flap endonuclease 1 (FEN1 plays an important role in the repair of oxidative DNA damage in the base excision repair pathway. We determined the association between two single nucleotide polymorphisms (SNPs, c.–441G>A (rs174538 and g.61564299G>T (rs4246215, in the FEN1 gene and the occurrence of KC and FECD. This study involved 279 patients with KC, 225 patients with FECD and 322 control individuals. Polymerase chain reaction (PCR and length polymorphism restriction fragment analysis (RFLP were applied. The T/T genotype of the g.61564299G>T polymorphism was associated with an increased occurrence of KC and FECD. There was no association between the c.–441G>A polymorphism and either disease. However, the GG haplotype of both polymorphisms was observed more frequently and the GT haplotype less frequently in the KC group than the control. The AG haplotype was associated with increased FECD occurrence. Our findings suggest that the g.61564299G>T and c.–441G>A polymorphisms in the FEN1 gene may modulate the risk of keratoconus and Fuchs endothelial corneal dystrophy.

  9. 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.

  10. 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.

  11. Enhancement of PCR Detection Limit by Single-Tube Restriction Endonuclease-PCR (RE-PCR).

    Science.gov (United States)

    Datta, Sibnarayan; Budhauliya, Raghvendra; Chatterjee, Soumya; Vanlalhmuaka; Veer, Vijay; Chakravarty, Runu

    2016-06-01

    Polymerase chain reaction (PCR) is widely used in biological research and diagnostics because of its high sensitivity and specificity. However, the sensitivity of PCR is strongly influenced by topological characteristics of the template. Supercoiled templates are known to inhibit PCR, whereas linearized forms of the same supercoiled templates facilitate PCR. This study was conducted to compare the PCR efficiency of circular supercoiled DNA templates to their restriction endonuclease (RE)-mediated linearized forms. Additionally, we also evaluated the possibility of RE digestion of the circular supercoiled templates within the complete PCR buffer. Following a systematic approach, we demonstrated that circular supercoiled templates could be efficiently linearized by RE in the complete PCR buffer itself. This allowed linearization of circular supercoiled templates and their subsequent amplification in the PCR buffer in a single-tube format. Using this extremely simple RE-PCR approach, we documented up to tenfold increases in detection efficiency of PCR with two different circular supercoiled templates of clinical origin, including an international calibration standard. This inexpensive and easy approach to increasing PCR sensitivity can be easily adapted to any standard PCR protocol aimed at amplifying circular supercoiled genomes. Apart from its application in the development of sensitive clinical diagnostic PCR assays for a large number of organisms, this method could also prove to be very useful in simplifying the existing protocols for other applications where pre-PCR restriction digestion is required, such as mutation detection, genotyping, and selective template amplification.

  12. Insights on the Structural Details of Endonuclease EcoRI-DNA Complexes by Electron Spin Resonance

    Science.gov (United States)

    Sarver, Jessica

    2009-03-01

    Pulsed electron spin resonance (ESR) was used to probe the binding specificity of EcoRI, a restriction endonuclease. Using site-directed spin labeling, a nitroxide side chain was incorporated into the protein, enabling the use of ESR to study structural details of EcoRI. Distance measurements were performed on EcoRI mutants when bound to varying sequences of DNA using the Double Electron-Electron Resonance experiment. These distances demonstrated that the average structure in the arm regions of EcoRI, thought to play a major role in binding specificity, is the same when the protein binds to different sequences of DNA. Also, it was determined that the arms exhibit higher flexibility when bound to sequences other than the specific sequence due to the larger distance distributions acquired from these spin labeled complexes. Molecular dynamics (MD) simulations were performed on the spin-label-modified specific EcoRI-DNA crystal structure to model the average nitroxide orientation. The distance distributions from MD were found to be narrower than experiment, indicating the need for a more rigorous sampling of the nitroxide conformers in silico.

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

    Directory of Open Access Journals (Sweden)

    Masahiro Hashizume

    2014-08-01

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

  14. 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.

  15. Insights into the DNA cleavage mechanism of human LINE-1 retrotransposon endonuclease

    NARCIS (Netherlands)

    Repanas, K.; Fuentes, G.; Cohen, S.; Bonvin, A.M.J.J.; Perrakis, A.

    2008-01-01

    The human LINE-1 endonuclease (L1-EN) contributes in defining the genomic integration sites of the abundant human L1 and Alu retrotransposons. LINEs have been considered as possible vehicles for gene delivery and understanding the mechanism of L1-EN could help engineering them as genetic tools. We

  16. PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair

    Science.gov (United States)

    Pluciennik, Anna; Dzantiev, Leonid; Iyer, Ravi R.; Constantin, Nicoleta; Kadyrov, Farid A.; Modrich, Paul

    2010-01-01

    MutLα (MLH1–PMS2) is a latent endonuclease that is activated in a mismatch-, MutSα-, proliferating cell nuclear antigen (PCNA)-, replication factor C (RFC)-, and ATP-dependent manner, with nuclease action directed to the heteroduplex strand that contains a preexisting break. RFC depletion experiments and use of linear DNAs indicate that RFC function in endonuclease activation is limited to PCNA loading. Whereas nicked circular heteroduplex DNA is a good substrate for PCNA loading and for endonuclease activation on the incised strand, covalently closed, relaxed circular DNA is a poor substrate for both reactions. However, covalently closed supercoiled or bubble-containing relaxed heteroduplexes, which do support PCNA loading, also support MutLα activation, but in this case cleavage strand bias is largely abolished. Based on these findings we suggest that PCNA has two roles in MutLα function: The clamp is required for endonuclease activation, an effect that apparently involves interaction of the two proteins, and by virtue of its loading orientation, PCNA determines the strand direction of MutLα incision. These results also provide a potential mechanism for activation of mismatch repair on nonreplicating DNA, an effect that may have implications for the somatic phase of triplet repeat expansion. PMID:20713735

  17. 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

  18. 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.

  19. Problem-solving test: digestion of a plasmid with restriction endonucleases.

    Science.gov (United States)

    Szeberényi, József

    2013-01-01

    Terms to be familiar with before you start to solve the test: plasmid, restriction endonuclease, agarose gel electrophoresis, ethidium bromide staining, autoradiography, Coomassie staining, Southern blotting, linear and circular DNA, superhelical DNA, exonuclease, modification methylase, palindrome, sticky and blunt ends, nicked circular DNA. Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  20. Identification of novel restriction endonuclease-like fold families among hypothetical proteins.

    Science.gov (United States)

    Kinch, Lisa N; Ginalski, Krzysztof; Rychlewski, Leszek; Grishin, Nick V

    2005-01-01

    Restriction endonucleases and other nucleic acid cleaving enzymes form a large and extremely diverse superfamily that display little sequence similarity despite retaining a common core fold responsible for cleavage. The lack of significant sequence similarity between protein families makes homology inference a challenging task and hinders new family identification with traditional sequence-based approaches. Using the consensus fold recognition method Meta-BASIC that combines sequence profiles with predicted protein secondary structure, we identify nine new restriction endonuclease-like fold families among previously uncharacterized proteins and predict these proteins to cleave nucleic acid substrates. Application of transitive searches combined with gene neighborhood analysis allow us to confidently link these unknown families to a number of known restriction endonuclease-like structures and thus assign folds to the uncharacterized proteins. Finally, our method identifies a novel restriction endonuclease-like domain in the C-terminus of RecC that is not detected with structure-based searches of the existing PDB database.

  1. 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

  2. Arthrobacter luteus restriction endonuclease cleavage map of X174 RF DNA

    NARCIS (Netherlands)

    Vereijken, J.M.; Mansfeld, A.D.M. van; Baas, P.D.; Jansz, H.S.

    1975-01-01

    Cleavage of X174 RF DNA with the restriction endonuclease from Arthrobacter luteus (Alu I) produces 23 fragments of approximately 24–1100 base pairs in length. The order of most of these fragments has been established by digestion of Haemophilus influenzae Rd (Hind II) and Haemophilus aegyptius (Hae

  3. BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism.

    Science.gov (United States)

    Raskó, Tamás; Dér, András; Klement, Eva; Slaska-Kiss, Krystyna; Pósfai, Eszter; Medzihradszky, Katalin F; Marshak, Daniel R; Roberts, Richard J; Kiss, Antal

    2010-11-01

    The GGCC-specific restriction endonuclease BspRI is one of the few Type IIP restriction endonucleases, which were suggested to be a monomer. Amino acid sequence information obtained by Edman sequencing and mass spectrometry analysis was used to clone the gene encoding BspRI. The bspRIR gene is located adjacently to the gene of the cognate modification methyltransferase and encodes a 304 aa protein. Expression of the bspRIR gene in Escherichia coli was dependent on the replacement of the native TTG initiation codon with an ATG codon, explaining previous failures in cloning the gene using functional selection. A plasmid containing a single BspRI recognition site was used to analyze kinetically nicking and second-strand cleavage under steady-state conditions. Cleavage of the supercoiled plasmid went through a relaxed intermediate indicating sequential hydrolysis of the two strands. Results of the kinetic analysis of the first- and second-strand cleavage are consistent with cutting the double-stranded substrate site in two independent binding events. A database search identified eight putative restriction-modification systems in which the predicted endonucleases as well as the methyltransferases share high sequence similarity with the corresponding protein of the BspRI system. BspRI and the related putative restriction endonucleases belong to the PD-(D/E)XK nuclease superfamily.

  4. Direct endonuclease digestion and multi-analysis of restriction fragment length polymorphisms by microchip electrophoresis.

    Science.gov (United States)

    Akamine, Rie; Yatsushiro, Shouki; Yamamura, Shouhei; Kido, Jun-ichi; Shinohara, Yasuo; Baba, Yoshinobu; Kataoka, Masatoshi

    2009-12-05

    A high-performance multi-analysis system for genotypic mutation by means of restriction fragment length polymorphisms (RFLP) involving endonuclease treatment of PCR-amplified DNA on a microchip and subsequent analysis by microchip electrophoresis for DNA sizing was developed. A Hitachi SV1210 system, with which 12 samples can be analyzed on a plastic chip with good accuracy as to DNA sizing between 25 and 300 bp, was employed for RFLP analysis. We performed RFLP analysis of the ABO genotypes of blood donors for whom the ABO type was known. Six blood samples were analyzed by PCR to amplify two different regions of the genomic DNA, each of the amplified DNAs containing a different nucleotide polymorphism. To analyze the genes at polymorphic sites 261 and 526, restriction endonucleases Kpn I and Ban I were employed, respectively. When an amplified DNA was digested with each endonuclease on a microchip for 20 min, sequential analysis revealed the presence or absence of the respective restriction site. This analysis was performed within 7 min using a 1/10 volume of a DNA sample in comparison with the conventional method, and the estimated DNA size differed from the predicted size by less than 10 bp. The results indicate the potential of microchip electrophoresis for RFLP with on-chip direct endonuclease digestion and sequential analysis, offering high resolution in a short time.

  5. Accurate scanning of the BssHII endonuclease in search for its DNA cleavage site

    NARCIS (Netherlands)

    Berkhout, B.; van Wamel, J.

    1996-01-01

    A facilitated diffusion mechanism has been proposed to account for the kinetic efficiency with which restriction endonucleases are able to locate DNA recognition sites. Such a mechanism involves the initial formation of a nonspecific complex upon collision of the protein with the DNA, with the

  6. 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.

  7. Cleavage of adenine-modified functionalized DNA by type II restriction endonucleases

    Czech Academy of Sciences Publication Activity Database

    Macíčková-Cahová, Hana; Hocek, Michal

    2009-01-01

    Roč. 37, č. 22 (2009), s. 7612-7622 ISSN 0305-1048 R&D Projects: GA ČR GA203/09/0317; GA MŠk LC512 Institutional research plan: CEZ:AV0Z40550506 Keywords : functionalized DNA * restriction endonucleases * DNA polymerase * modified adenosines Subject RIV: CC - Organic Chemistry Impact factor: 7.479, year: 2009

  8. 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

  9. Engineering strand-specific DNA nicking enzymes from the type IIS restriction endonucleases BsaI, BsmBI, and BsmAI.

    Science.gov (United States)

    Zhu, Zhenyu; Samuelson, James C; Zhou, Jing; Dore, Andrew; Xu, Shuang-Yong

    2004-03-26

    More than 80 type IIA/IIS restriction endonucleases with different recognition specificities are now known. In contrast, only a limited number of strand-specific nicking endonucleases are currently available. To overcome this limitation, a novel genetic screening method was devised to convert type IIS restriction endonucleases into strand-specific nicking endonucleases. The genetic screen consisted of four steps: (1) random mutagenesis to create a plasmid library, each bearing an inactivated endonuclease gene; (2) restriction digestion of plasmids containing the wild-type and the mutagenized endonuclease gene; (3) back-crosses with the wild-type gene by ligation to the wild-type N-terminal or C-terminal fragment; (4) transformation of the ligated DNA into a pre-modified host and screening for nicking endonuclease activity in total cell culture or cell extracts of the transformants. Nt.BsaI and Nb.BsaI nicking endonucleases were isolated from BsaI using this genetic screen. In addition, site-directed mutagenesis was carried out to isolate BsaI nicking variants with minimal double-stranded DNA cleavage activity. The equivalent amino acid substitutions were introduced into BsmBI and BsmAI restriction endonucleases with similar recognition sequence and significant amino acid sequence identity and their nicking variants were successfully isolated. This work provides strong evidence that some type IIS restriction endonucleases carry two separate active sites. When one of the active sites is inactivated, the type IIS restriction endonuclease may nick only one strand.

  10. 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.

  11. Polymerase synthesis of DNAs bearing vinyl groups in the major groove and their cleavage by restriction endonucleases.

    Science.gov (United States)

    Mačková, Michaela; Pohl, Radek; Hocek, Michal

    2014-10-13

    DNA molecules containing 5-vinyluracil, 5-vinylcytosine, or 7-deaza-7-vinyladenine were prepared by polymerase incorporation of the corresponding vinyl-modified 2'-deoxyribonucleoside triphosphates, and the influence of the vinyl group in the major groove of DNA on the cleavage by diverse type II restriction endonucleases (REs) was studied. The presence of 5-vinyluracil was tolerated by most of the REs, whereas only some REs were able to cleave sequences containing 7-deaza-7-vinyladenine. The enzyme ScaI was found to cleave DNA containing 5-vinylcytosine efficiently but not DNA containing the related 5-ethynylcytosine. All other REs failed to cleave sequences containing any cytosine modifications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Positioning the 5'-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage

    KAUST Repository

    Song, Bo

    2018-02-09

    Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair and recombination, and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca2+ have suggested that base unpairing at the 5\\'-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps - from substrate binding to product release - during the hFEN1-catalyzed reaction in the presence of Mg2+. We found that while hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg2+-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5\\'-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5\\'-flap:template junction, indicating that local DNA distortion (e.g., disruption of base stacking observed in structures), associated with positioning the 5\\'-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34, Asp-181), steering the 5\\'-flap through the active site and binding the scissile phosphate (Lys-93, Arg-100), and stacking against the base 5\\' to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5\\'-flaps.

  13. Optimising homing endonuclease gene drive performance in a semi-refractory species: the Drosophila melanogaster experience.

    Directory of Open Access Journals (Sweden)

    Yuk-Sang Chan

    Full Text Available Homing endonuclease gene (HEG drive is a promising insect population control technique that employs meganucleases to impair the fitness of pest populations. Our previous studies showed that HEG drive was more difficult to achieve in Drosophila melanogaster than Anopheles gambiae and we therefore investigated ways of improving homing performance in Drosophila. We show that homing in Drosophila responds to increased expression of HEGs specifically during the spermatogonia stage and this could be achieved through improved construct design. We found that 3'-UTR choice was important to maximise expression levels, with HEG activity increasing as we employed Hsp70, SV40, vasa and βTub56D derived UTRs. We also searched for spermatogonium-specific promoters and found that the Rcd-1r promoter was able to drive specific expression at this stage. Since Rcd-1 is a regulator of differentiation in other species, it suggests that Rcd-1r may serve a similar role during spermatogonial differentiation in Drosophila. Contrary to expectations, a fragment containing the entire region between the TBPH gene and the bgcn translational start drove strong HEG expression only during late spermatogenesis rather than in the germline stem cells and spermatogonia as expected. We also observed that the fraction of targets undergoing homing was temperature-sensitive, falling nearly four-fold when the temperature was lowered to 18°C. Taken together, this study demonstrates how a few simple measures can lead to substantial improvements in the HEG-based gene drive strategy and reinforce the idea that the HEG approach may be widely applicable to a variety of insect control programs.

  14. 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.

  15. 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

  16. Altered endoribonuclease activity of apurinic/apyrimidinic endonuclease 1 variants identified in the human population.

    Directory of Open Access Journals (Sweden)

    Wan Cheol Kim

    Full Text Available Apurinic/apyrimidinic endonuclease 1 (APE1 is the major mammalian enzyme in the DNA base excision repair pathway and cleaves the DNA phosphodiester backbone immediately 5' to abasic sites. APE1 also has 3'-5' DNA exonuclease and 3' DNA phosphodiesterase activities, and regulates transcription factor DNA binding through its redox regulatory function. The human APE1 has recently been shown to endonucleolytically cleave single-stranded regions of RNA. Towards understanding the biological significance of the endoribonuclease activity of APE1, we examined eight different amino acid substitution variants of APE1 previously identified in the human population. Our study shows that six APE1 variants, D148E, Q51H, I64V, G241R, R237A, and G306A, exhibit a 76-85% reduction in endoribonuclease activity against a specific coding region of the c-myc RNA, yet fully retain the ability to cleave apurinic/apyrimidinic DNA. We found that two APE1 variants, L104R and E126D, exhibit a unique RNase inhibitor-resistant endoribonuclease activity, where the proteins cleave c-myc RNA 3' of specific single-stranded guanosine residues. Expression of L104R and E126D APE1 variants in bacterial Origami cells leads to a 60-80% reduction in colony formation and a 1.5-fold increase in cell doubling time, whereas the other variants, which exhibit diminished endoribonuclease activity, had no effect. These data indicate that two human APE1 variants exhibit a unique endoribonuclease activity, which correlates with their ability to induce cytotoxicity or slow down growth in bacterial cells and supports the notion of their biological functionality.

  17. 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

  18. Modulating mtDNA heteroplasmy by mitochondria-targeted restriction endonucleases in a ‘differential multiple cleavage-site’ model

    OpenAIRE

    Bacman, SR; Williams, SL; Hernandez, D; Moraes, CT

    2007-01-01

    The ability to manipulate mitochondrial DNA (mtDNA) heteroplasmy would provide a powerful tool to treat mitochondrial diseases. Recent studies showed that mitochondria-targeted restriction endonucleases can modify mtDNA heteroplasmy in a predictable and efficient manner if it recognizes a single site in the mutant mtDNA. However, the applicability of such model is limited to mutations that create a novel cleavage site, not present in the wild-type mtDNA. We attempted to extend this approach t...

  19. 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

  20. 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

  1. Homing endonucleases catalyze double-stranded DNA breaks and somatic transgene excision in Aedes aegypti

    OpenAIRE

    Traver, Brenna E.; Anderson, Michelle A. E.; Adelman, Zach N.

    2009-01-01

    Aedes aegypti is a major vector of arthropod-borne viruses such as yellow fever virus and dengue viruses. Efforts to discern the function of genes involved in important behaviors such as vector competence and host seeking through reverse genetics would greatly benefit from the ability to generate targeted gene disruptions. Homing endonucleases are selfish elements which catalyze double-stranded DNA (dsDNA) breaks in a sequence-specific manner. In this report we demonstrate that the homing end...

  2. 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

  3. Zinc finger nuclease and homing endonuclease-mediated assembly of multigene plant transformation vectors.

    Science.gov (United States)

    Zeevi, Vardit; Liang, Zhuobin; Arieli, Uri; Tzfira, Tzvi

    2012-01-01

    Binary vectors are an indispensable component of modern Agrobacterium tumefaciens-mediated plant genetic transformation systems. A remarkable variety of binary plasmids have been developed to support the cloning and transfer of foreign genes into plant cells. The majority of these systems, however, are limited to the cloning and transfer of just a single gene of interest. Thus, plant biologists and biotechnologists face a major obstacle when planning the introduction of multigene traits into transgenic plants. Here, we describe the assembly of multitransgene binary vectors by using a combination of engineered zinc finger nucleases (ZFNs) and homing endonucleases. Our system is composed of a modified binary vector that has been engineered to carry an array of unique recognition sites for ZFNs and homing endonucleases and a family of modular satellite vectors. By combining the use of designed ZFNs and commercial restriction enzymes, multiple plant expression cassettes were sequentially cloned into the acceptor binary vector. Using this system, we produced binary vectors that carried up to nine genes. Arabidopsis (Arabidopsis thaliana) protoplasts and plants were transiently and stably transformed, respectively, by several multigene constructs, and the expression of the transformed genes was monitored across several generations. Because ZFNs can potentially be engineered to digest a wide variety of target sequences, our system allows overcoming the problem of the very limited number of commercial homing endonucleases. Thus, users of our system can enjoy a rich resource of plasmids that can be easily adapted to their various needs, and since our cloning system is based on ZFN and homing endonucleases, it may be possible to reconstruct other types of binary vectors and adapt our vectors for cloning on multigene vector systems in various binary plasmids.

  4. 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...

  5. Cleavage of Functionalized DNA Containing 5-Modified Pyrimidines by Type II Restriction Endonucleases

    Czech Academy of Sciences Publication Activity Database

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

    2011-01-01

    Roč. 12, č. 3 (2011), s. 431-438 ISSN 1439-4227 R&D Projects: GA MŠk LC512; GA ČR GA203/09/0317 Institutional research plan: CEZ:AV0Z40550506 Keywords : base-modified DNA * DNA cleavage * DNA polymerases * nucleosides * restriction endonucleases Subject RIV: CC - Organic Chemistry Impact factor: 3.944, year: 2011

  6. Polymorphism in mitochondrial DNA of humans as revealed by restriction endonuclease analysis.

    OpenAIRE

    Brown, W M

    1980-01-01

    Mitochondrial DNA samples from each of 21 humans of diverse racial and geographic origin were digested with each of 18 restriction endonucleases. The sizes of the resulting DNA fragments were compared after gel electrophoresis. No differences among the samples were detected in digest with 7 of the enzymes. Analysis of digests with the remaining enzymes showed one or more differences. Each of the 21 samples could be characterized individually on the basis of these digests. All between-sample d...

  7. Structure of the endonuclease domain of MutL: unlicensed to cut

    Science.gov (United States)

    Pillon, Monica C.; Lorenowicz, Jessica J.; Uckelmann, Michael; Klocko, Andrew D.; Mitchell, Ryan R.; Chung, Yu Seon; Modrich, Paul; Walker, Graham C.; Simmons, Lyle A.; Friedhoff, Peter; Guarné, Alba

    2010-01-01

    Summary DNA mismatch repair corrects errors that have escaped polymerase proofreading, increasing replication fidelity 100- to 1000-fold in organisms ranging from bacteria to humans. The MutL protein plays a central role in mismatch repair by coordinating multiple protein-protein interactions that signal strand removal upon mismatch recognition by MutS. Here we report the crystal structure of the endonuclease domain of Bacillus subtilis MutL. The structure is organized in dimerization and regulatory subdomains connected by a helical lever spanning the conserved endonuclease motif. Additional conserved motifs cluster around the lever and define a Zn2+-binding site that is critical for MutL function in vivo. The structure unveils a powerful inhibitory mechanism to prevent undesired DNA nicking and allows us to propose a model describing how the interaction with MutS and the processivity clamp could license the endonuclease activity of MutL. The structure also provides a molecular framework to propose and test additional roles of MutL in mismatch repair. PMID:20603082

  8. 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

  9. Computational reprogramming of homing endonuclease specificity at multiple adjacent base pairs.

    Science.gov (United States)

    Ashworth, Justin; Taylor, Gregory K; Havranek, James J; Quadri, S Arshiya; Stoddard, Barry L; Baker, David

    2010-09-01

    Site-specific homing endonucleases are capable of inducing gene conversion via homologous recombination. Reprogramming their cleavage specificities allows the targeting of specific biological sites for gene correction or conversion. We used computational protein design to alter the cleavage specificity of I-MsoI for three contiguous base pair substitutions, resulting in an endonuclease whose activity and specificity for its new site rival that of wild-type I-MsoI for the original site. Concerted design for all simultaneous substitutions was more successful than a modular approach against individual substitutions, highlighting the importance of context-dependent redesign and optimization of protein-DNA interactions. We then used computational design based on the crystal structure of the designed complex, which revealed significant unanticipated shifts in DNA conformation, to create an endonuclease that specifically cleaves a site with four contiguous base pair substitutions. Our results demonstrate that specificity switches for multiple concerted base pair substitutions can be computationally designed, and that iteration between design and structure determination provides a route to large scale reprogramming of specificity.

  10. Structure and mutagenesis of the DNA modification-dependent restriction endonuclease AspBHI.

    Science.gov (United States)

    Horton, John R; Nugent, Rebecca L; Li, Andrew; Mabuchi, Megumu Yamada; Fomenkov, Alexey; Cohen-Karni, Devora; Griggs, Rose M; Zhang, Xing; Wilson, Geoffrey G; Zheng, Yu; Xu, Shuang-yong; Cheng, Xiaodong

    2014-03-07

    The modification-dependent restriction endonuclease AspBHI recognizes 5-methylcytosine (5mC) in the double-strand DNA sequence context of (C/T)(C/G)(5mC)N(C/G) (N = any nucleotide) and cleaves the two strands a fixed distance (N12/N16) 3' to the modified cytosine. We determined the crystal structure of the homo-tetrameric AspBHI. Each subunit of the protein comprises two domains: an N-terminal DNA-recognition domain and a C-terminal DNA cleavage domain. The N-terminal domain is structurally similar to the eukaryotic SET and RING-associated (SRA) domain, which is known to bind to a hemi-methylated CpG dinucleotide. The C-terminal domain is structurally similar to classic Type II restriction enzymes and contains the endonuclease catalytic-site motif of DX20EAK. To understand how specific amino acids affect AspBHI recognition preference, we generated a homology model of the AspBHI-DNA complex, and probed the importance of individual amino acids by mutagenesis. Ser41 and Arg42 are predicted to be located in the DNA minor groove 5' to the modified cytosine. Substitution of Ser41 with alanine (S41A) and cysteine (S41C) resulted in mutants with altered cleavage activity. All 19 Arg42 variants resulted in loss of endonuclease activity.

  11. On the role of steric clashes in methylation control of restriction endonuclease activity.

    Science.gov (United States)

    Mierzejewska, Karolina; Bochtler, Matthias; Czapinska, Honorata

    2016-01-08

    Restriction-modification systems digest non-methylated invading DNA, while protecting host DNA against the endonuclease activity by methylation. It is widely believed that the methylated DNA would not 'fit' into the binding site of the endonuclease in the productive orientation, and thus steric clashes should account for most of the protection. We test this concept statistically by grafting methyl groups in silico onto non-methylated DNA in co-crystal structures with restriction endonucleases. Clash scores are significantly higher for protective than non-protective methylation (P < 0.05% according to the Wilcoxon rank sum test). Structural data alone are sufficient to distinguish between protective and non-protective DNA methylation with 90% confidence and decision thresholds of 1.1 Å and 48 Å(3) for the most severe distance-based and cumulative volume-based clash with the protein, respectively (0.1 Å was deducted from each interatomic distance to allow for coordinate errors). The most severe clashes are more pronounced for protective methyl groups attached to the nitrogen atoms (N6-methyladenines and N4-methylcytosines) than for C5-methyl groups on cytosines. Cumulative clashes are comparable for all three types of protective methylation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Restriction endonuclease AgeI is a monomer which dimerizes to cleave DNA.

    Science.gov (United States)

    Tamulaitiene, Giedre; Jovaisaite, Virginija; Tamulaitis, Gintautas; Songailiene, Inga; Manakova, Elena; Zaremba, Mindaugas; Grazulis, Saulius; Xu, Shuang-Yong; Siksnys, Virginijus

    2017-04-07

    Although all Type II restriction endonucleases catalyze phosphodiester bond hydrolysis within or close to their DNA target sites, they form different oligomeric assemblies ranging from monomers, dimers, tetramers to higher order oligomers to generate a double strand break in DNA. Type IIP restriction endonuclease AgeI recognizes a palindromic sequence 5΄-A/CCGGT-3΄ and cuts it ('/' denotes the cleavage site) producing staggered DNA ends. Here, we present crystal structures of AgeI in apo and DNA-bound forms. The structure of AgeI is similar to the restriction enzymes that share in their target sites a conserved CCGG tetranucleotide and a cleavage pattern. Structure analysis and biochemical data indicate, that AgeI is a monomer in the apo-form both in the crystal and in solution, however, it binds and cleaves the palindromic target site as a dimer. DNA cleavage mechanism of AgeI is novel among Type IIP restriction endonucleases. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Translational independence between overlapping genes for a restriction endonuclease and its transcriptional regulator

    Directory of Open Access Journals (Sweden)

    Blumenthal Robert M

    2010-11-01

    Full Text Available Abstract Background Most type II restriction-modification (RM systems have two independent enzymes that act on the same DNA sequence: a modification methyltransferase that protects target sites, and a restriction endonuclease that cleaves unmethylated target sites. When RM genes enter a new cell, methylation must occur before restriction activity appears, or the host's chromosome is digested. Transcriptional mechanisms that delay endonuclease expression have been identified in some RM systems. A substantial subset of those systems is controlled by a family of small transcription activators called C proteins. In the PvuII system, C.PvuII activates transcription of its own gene, along with that of the downstream endonuclease gene. This regulation results in very low R.PvuII mRNA levels early after gene entry, followed by rapid increase due to positive feedback. However, given the lethal consequences of premature REase accumulation, transcriptional control alone might be insufficient. In C-controlled RM systems, there is a ± 20 nt overlap between the C termination codon and the R (endonuclease initiation codon, suggesting possible translational coupling, and in many cases predicted RNA hairpins could occlude the ribosome binding site for the endonuclease gene. Results Expression levels of lacZ translational fusions to pvuIIR or pvuIIC were determined, with the native pvuII promoter having been replaced by one not controlled by C.PvuII. In-frame pvuIIC insertions did not substantially decrease either pvuIIC-lacZ or pvuIIR-lacZ expression (with or without C.PvuII provided in trans. In contrast, a frameshift mutation in pvuIIC decreased expression markedly in both fusions, but mRNA measurements indicated that this decrease could be explained by transcriptional polarity. Expression of pvuIIR-lacZ was unaffected when the pvuIIC stop codon was moved 21 nt downstream from its WT location, or 25 or 40 bp upstream of the pvuIIR initiation codon. Disrupting

  14. Translational independence between overlapping genes for a restriction endonuclease and its transcriptional regulator.

    Science.gov (United States)

    Kaw, Meenakshi K; Blumenthal, Robert M

    2010-11-19

    Most type II restriction-modification (RM) systems have two independent enzymes that act on the same DNA sequence: a modification methyltransferase that protects target sites, and a restriction endonuclease that cleaves unmethylated target sites. When RM genes enter a new cell, methylation must occur before restriction activity appears, or the host's chromosome is digested. Transcriptional mechanisms that delay endonuclease expression have been identified in some RM systems. A substantial subset of those systems is controlled by a family of small transcription activators called C proteins. In the PvuII system, C.PvuII activates transcription of its own gene, along with that of the downstream endonuclease gene. This regulation results in very low R.PvuII mRNA levels early after gene entry, followed by rapid increase due to positive feedback. However, given the lethal consequences of premature REase accumulation, transcriptional control alone might be insufficient. In C-controlled RM systems, there is a ± 20 nt overlap between the C termination codon and the R (endonuclease) initiation codon, suggesting possible translational coupling, and in many cases predicted RNA hairpins could occlude the ribosome binding site for the endonuclease gene. Expression levels of lacZ translational fusions to pvuIIR or pvuIIC were determined, with the native pvuII promoter having been replaced by one not controlled by C.PvuII. In-frame pvuIIC insertions did not substantially decrease either pvuIIC-lacZ or pvuIIR-lacZ expression (with or without C.PvuII provided in trans). In contrast, a frameshift mutation in pvuIIC decreased expression markedly in both fusions, but mRNA measurements indicated that this decrease could be explained by transcriptional polarity. Expression of pvuIIR-lacZ was unaffected when the pvuIIC stop codon was moved 21 nt downstream from its WT location, or 25 or 40 bp upstream of the pvuIIR initiation codon. Disrupting the putative hairpins had no significant

  15. Expression of a LINE-1 endonuclease variant in gastric cancer: its association with clinicopathological parameters

    International Nuclear Information System (INIS)

    Wang, Gangshi; Wu, Benyan; Wang, Mengwei; Gao, Jie; Huang, Haili; Tian, Yu; Xue, Liyan; Wang, Weihua; You, Weidi; Lian, Hongwei; Duan, Xiaojian

    2013-01-01

    Long interspersed nuclear element-1 (LINE-1 or L1), the most abundant and only autonomously active family of non-LTR retrotransposons in the human genome, expressed not only in the germ lines but also in somatic tissues. It contributes to genetic instability, aging, and age-related diseases, such as cancer. Our previous study identified in human gastric adenocarcinoma an upregulated transcript GCRG213, which shared 88% homology with human L1 sequence and contained a putative conserved apurinic/apyrimidinic endonucleas1 domain. Immunohistochemistry was carried out by using a monoclonal mouse anti-human GCRG213 protein (GCRG213p) antibody produced in our laboratory, on tissue microarray constructed with specimens from 175 gastric adenocarcinoma patients. The correlation between GCRG213p expression and patient clinicopathological parameters was evaluated. GCRG213p expression in gastric cancer cell lines were studied using Western blotting analysis. L1 promoter methylation status of gastric cancer cells was tested using methylation-specific PCR. BLASTP was used at the NCBI Blast server to identify GCRG213p sequence to any alignments in the Protein Data Bank databases. Most primary gastric cancer, lymph node metastases and gastric intestinal metaplasia glands showed positive GCRG213p immunoreactivity. High GCRG213p immunostaining score in the primary gastric cancer was positively correlated with tumor differentiation (well differentiated, p = 0.001), Lauren’s classification (intestinal type, p < 0.05) and a late age onset of gastric adenocarcinoma (≥65 yrs; p < 0.05). GCRG213p expression has no association with other clinicopathological parameters, including survival. Western blotting analysis of GCRG213p expression in gastric cancer cells indicated that GCRG213p level was higher in gastric cancer cell lines than in human normal gastric epithelium immortalized cell line GES-1. Partial methylation of L1 in gastric cancer cells was confirmed by methylation

  16. Naturally-occurring, dually-functional fusions between restriction endonucleases and regulatory proteins.

    Science.gov (United States)

    Liang, Jixiao; Blumenthal, Robert M

    2013-10-02

    Restriction-modification (RM) systems appear to play key roles in modulating gene flow among bacteria and archaea. Because the restriction endonuclease (REase) is potentially lethal to unmethylated new host cells, regulation to ensure pre-expression of the protective DNA methyltransferase (MTase) is essential to the spread of RM genes. This is particularly true for Type IIP RM systems, in which the REase and MTase are separate, independently-active proteins. A substantial subset of Type IIP RM systems are controlled by an activator-repressor called C protein. In these systems, C controls the promoter for its own gene, and for the downstream REase gene that lacks its own promoter. Thus MTase is expressed immediately after the RM genes enter a new cell, while expression of REase is delayed until sufficient C protein accumulates. To study the variation in and evolution of this regulatory mechanism, we searched for RM systems closely related to the well-studied C protein-dependent PvuII RM system. Unexpectedly, among those found were several in which the C protein and REase genes were fused. The gene for CR.NsoJS138I fusion protein (nsoJS138ICR, from the bacterium Niabella soli) was cloned, and the fusion protein produced and partially purified. Western blots provided no evidence that, under the conditions tested, anything other than full-length fusion protein is produced. This protein had REase activity in vitro and, as expected from the sequence similarity, its specificity was indistinguishable from that for PvuII REase, though the optimal reaction conditions were different. Furthermore, the fusion was active as a C protein, as revealed by in vivo activation of a lacZ reporter fusion to the promoter region for the nsoJS138ICR gene. Fusions between C proteins and REases have not previously been characterized, though other fusions have (such as between REases and MTases). These results reinforce the evidence for impressive modularity among RM system proteins, and raise

  17. The NF1 gene contains hotspots for L1 endonuclease-dependent de novo insertion.

    Directory of Open Access Journals (Sweden)

    Katharina Wimmer

    2011-11-01

    Full Text Available Long interspersed (L1 and Alu elements are actively amplified in the human genome through retrotransposition of their RNA intermediates by the -100 still retrotranspositionally fully competent L1 elements. Retrotransposition can cause inherited disease if such an element is inserted near or within a functional gene. Using direct cDNA sequencing as the primary assay for comprehensive NF1 mutation analysis, we uncovered in 18 unrelated index patients splicing alterations not readily explained at the genomic level by an underlying point-mutation or deletion. Improved PCR protocols avoiding allelic drop-out of the mutant alleles uncovered insertions of fourteen Alu elements, three L1 elements, and one poly(T stretch to cause these splicing defects. Taken together, the 18 pathogenic L1 endonuclease-mediated de novo insertions represent the largest number of this type of mutations characterized in a single human gene. Our findings show that retrotransposon insertions account for as many as -0.4% of all NF1 mutations. Since altered splicing was the main effect of the inserted elements, the current finding was facilitated by the use of RNA-based mutation analysis protocols, resulting in improved detection compared to gDNA-based approaches. Six different insertions clustered in a relatively small 1.5-kb region (NF1 exons 21(16-23(18 within the 280-kb NF1 gene. Furthermore, three different specific integration sites, one of them located in this cluster region, were each used twice, i.e. NM_000267.3(NF1:c.1642-1_1642 in intron 14(10c, NM_000267.3(NF1:c.2835_2836 in exon 21(16, and NM_000267.3(NF1:c.4319_4320 in exon 33(25. Identification of three loci that each served twice as integration site for independent retrotransposition events as well as 1.5-kb cluster region harboring six independent insertions supports the notion of non-random insertion of retrotransposons in the human genome. Currently, little is known about which features make sites

  18. The N-terminal 45-kDa Domain of Dna2 Endonuclease/Helicase Targets the Enzyme to Secondary Structure DNA*

    Science.gov (United States)

    Lee, Chul-Hwan; Lee, Miju; Kang, Hyo-Jin; Kim, Do-Hyung; Kang, Young-Hoon; Bae, Sung-Ho; Seo, Yeon-Soo

    2013-01-01

    The removal of initiating primers from the 5′-ends of each Okazaki fragment, required for the generation of contiguous daughter strands, can be catalyzed by the combined action of DNA polymerase δ and Fen1. When the flaps generated by displacement of DNA synthesis activity of polymerase δ become long enough to bind replication protein A or form hairpin structures, the helicase/endonuclease enzyme, Dna2, becomes critical because of its ability to remove replication protein A-coated or secondary structure flaps. In this study, we show that the N-terminal 45-kDa domain of Dna2 binds hairpin structures, allowing the enzyme to target secondary structure flap DNA. We found that this activity was essential for the efficient removal of hairpin flaps by the endonuclease activity of Dna2 with the aid of its helicase activity. Thus, the efficient removal of hairpin structure flaps requires the coordinated action of all three functional domains of Dna2. We also found that deletion of the N-terminal 45-kDa domain of Dna2 led to a partial loss of the intra-S-phase checkpoint function and an increased rate of homologous recombination in yeast. We discuss the potential roles of the N-terminal domain of Dna2 in the maintenance of genomic stability. PMID:23344960

  19. 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)

    Higgins, Owen; Clancy, Eoin; Cormican, Martin; Boo, Teck Wee; Cunney, Robert; Smith, Terry J

    2018-02-09

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. 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.

  2. MOLECULAR STUDY OF NUCLEPROTEIN GENE OF RABIES ...

    African Journals Online (AJOL)

    Analysis of the 1400-b.p RT-PCR products of the N gene and the views by sequencing and restriction endonuclease analysis enabled division of isolates into 3 types. The conclusion from the study is that RT-PCR and restriction endonuclease analysis of the amplified products of the N gene would allow identification and ...

  3. Purification of Restriction Endonuclease EcoRII and its Co-Crystallization

    Science.gov (United States)

    Karpova, E. A.; Chen, L.; Meehan, E.; Pusey, M.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    Restriction endonuclease EcoRII (EcoRII) is a homodimeric DNA-binding protein. It belongs to the type II family of restriction-modification enzymes (subclass IIe). EcoRII recognizes the nucleotide sequence 5'-CCWGG (W=A or T) and cleaves the phosphodiester bond preceding the first cytosine. Methylation at C5 of the second cytosine inhibits cleavage. The enzyme has a unique ability to search for the presence of two substrate sites before cleavage. To the best of our knowledge no other subclass IIe restriction endonuclease has been crystallized yet, without or with a DNA-substrate. We have recently grown and characterized the crystals of this enzyme (1) Here we report on the result of co-crystallization experiments of EcoRII with an 11 b.p. oligonucleotide substrate. The dissociation constant (Kd) EcoRII: 11 b.p. was determined earlier (unpublished results). The needle-like crystals of oligonucleotide-EcoRII protein complex were obtained with this substrate by the technique of vapor diffusion hanging drops. The crystals obtained were washed and dissolved in an aliquot of 10 mM Tris-HCl buffer, pH=7.5. Running a portion of this solution on the SDS-get indicated the presence of endonuclease in the solution. A UV-spectrophotometric test of a second portion confirmed the presence of DNA. We are now working on improvement of the DNA-EcoRII protein crystals. Results obtained from these and ongoing efforts will be reported.

  4. 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.

  5. Crystallization and preliminary X-ray analysis of the type IV restriction endonuclease ScoMcrA from Streptomyces coelicolor, which cleaves both Dcm-methylated DNA and phosphorothioated DNA.

    Science.gov (United States)

    Liu, Guang; Zhang, Zhenyi; Zhao, Gong; Deng, Zixin; Wu, Geng; He, Xinyi

    2015-01-01

    ScoMcrA is a type IV modification-dependent restriction endonuclease found in the model strain Streptomyces coelicolor. Unlike type I, II and III restriction endonucleases, which cleave unmodified DNA, type IV restriction endonucleases cleave modified DNA, including methylated, hydroxymethylated, glucosyl-hydroxymethylated and phosphorothioated DNA. ScoMcrA targets both Dcm-methylated DNA and phosphorothioated DNA, and makes double-strand breaks 16-28 nt away from the modified nucleotides or the phosphorothioate links. However, the mechanism by which ScoMcrA recognizes these two entirely different types of modification remains unclear. In this study, the ScoMcrA protein was overexpressed, purified and crystallized. The crystals diffracted to 3.35 Å resolution and belonged to space group P2(1)2(1)2(1). The unit-cell parameters were determined to be a=130.19, b=139.36, c=281.01 Å, α=β=γ=90°. These results will facilitate the detailed structural analysis of ScoMcrA and further elucidation of its biochemical mechanism.

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

    Energy Technology Data Exchange (ETDEWEB)

    Meramveliotaki, Chrysi [Department of Science, Agricultural University of Athens, Athens (Greece); Department of Biology, University of Crete, PO Box 2208, GR-71110 Heraklion, Crete (Greece); Institute of Molecular Biology and Biotechnology (IMBB), PO Box 1527, GR-71110 Heraklion, Crete (Greece); Department of Agricultural Biotechnology, Agricultural University of Athens, Athens (Greece); Kotsifaki, Dina [Institute of Molecular Biology and Biotechnology (IMBB), PO Box 1527, GR-71110 Heraklion, Crete (Greece); Androulaki, Maria [Department of Science, Agricultural University of Athens, Athens (Greece); Department of Biology, University of Crete, PO Box 2208, GR-71110 Heraklion, Crete (Greece); Institute of Molecular Biology and Biotechnology (IMBB), PO Box 1527, GR-71110 Heraklion, Crete (Greece); Department of Agricultural Biotechnology, Agricultural University of Athens, Athens (Greece); Hountas, Athanasios [Department of Science, Agricultural University of Athens, Athens (Greece); Eliopoulos, Elias [Department of Agricultural Biotechnology, Agricultural University of Athens, Athens (Greece); Kokkinidis, Michael, E-mail: kokkinid@imbb.forth.gr [Department of Biology, University of Crete, PO Box 2208, GR-71110 Heraklion, Crete (Greece); Institute of Molecular Biology and Biotechnology (IMBB), PO Box 1527, GR-71110 Heraklion, Crete (Greece); Department of Science, Agricultural University of Athens, Athens (Greece)

    2007-10-01

    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{sub 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.

  7. Interdomain communication in the endonuclease/motor subunit of type I restriction-modification enzyme EcoR124I.

    Science.gov (United States)

    Sinha, Dhiraj; Shamayeva, Katsiaryna; Ramasubramani, Vyas; Řeha, David; Bialevich, Vitali; Khabiri, Morteza; Guzanová, Alena; Milbar, Niv; Weiserová, Marie; Csefalvay, Eva; Carey, Jannette; Ettrich, Rüdiger

    2014-07-01

    Restriction-modification systems protect bacteria from foreign DNA. Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA-cleavage and ATP-dependent DNA translocation activities located on endonuclease/motor subunit HsdR. The recent structure of the first intact motor subunit of the type I restriction enzyme from plasmid EcoR124I suggested a mechanism by which stalled translocation triggers DNA cleavage via a lysine residue on the endonuclease domain that contacts ATP bound between the two helicase domains. In the present work, molecular dynamics simulations are used to explore this proposal. Molecular dynamics simulations suggest that the Lys-ATP contact alternates with a contact with a nearby loop housing the conserved QxxxY motif that had been implicated in DNA cleavage. This model is tested here using in vivo and in vitro experiments. The results indicate how local interactions are transduced to domain motions within the endonuclease/motor subunit.

  8. Mutagenesis identifies the critical amino acid residues of human endonuclease G involved in catalysis, magnesium coordination, and substrate specificity

    Directory of Open Access Journals (Sweden)

    Wu Shih-Lu

    2009-01-01

    Full Text Available Abstract Background Endonuclease G (EndoG, a member of DNA/RNA nonspecific ββα-Me-finger nucleases, is involved in apoptosis and normal cellular proliferation. In this study, we analyzed the critical amino acid residues of EndoG and proposed the catalytic mechanism of EndoG. Methods To identify the critical amino acid residues of human EndoG, we replaced the conserved histidine, asparagine, and arginine residues with alanine. The catalytic efficacies of Escherichia coli-expressed EndoG variants were further analyzed by kinetic studies. Results Diethyl pyrocarbonate modification assay revealed that histidine residues were involved in EndoG activity. His-141, Asn-163, and Asn-172 in the H-N-H motif of EndoG were critical for catalysis and substrate specificity. H141A mutant required a higher magnesium concentration to achieve its activity, suggesting the unique role of His-141 in both catalysis and magnesium coordination. Furthermore, an additional catalytic residue (Asn-251 and an additional metal ion binding site (Glu-271 of human EndoG were identified. Conclusion Based on the mutational analysis and homology modeling, we proposed that human EndoG shared a similar catalytic mechanism with nuclease A from Anabaena.

  9. SegH and Hef: two novel homing endonucleases whose genes replace the mobC and mobE genes in several T4-related phages.

    Science.gov (United States)

    Sandegren, Linus; Nord, David; Sjöberg, Britt-Marie

    2005-01-01

    T4 contains two groups of genes with similarity to homing endonucleases, the seg-genes (similarity to endonucleases encoded by group I introns) containing GIY-YIG motifs and the mob-genes (similarity to mobile endonucleases) containing H-N-H motifs. The four seg-genes characterized to date encode homing endonucleases with cleavage sites close to their respective gene loci while none of the mob-genes have been shown to cleave DNA. Of 18 phages screened, only T4 was found to have mobC while mobE genes were found in five additional phages. Interestingly, three phages encoded a seg-like gene (hereby called segH) with a GIY-YIG motif in place of mobC. An additional phage has an unrelated gene called hef (homing endonuclease-like function) in place of the mobE gene. The gene products of both novel genes displayed homing endonuclease activity with cleavage site specificity close to their respective genes. In contrast to intron encoded homing endonucleases, both SegH and Hef can cleave their own DNA as well as DNA from phages without the genes. Both segH and mobE (and most likely hef) can home between phages in mixed infections. We discuss why it might be a selective advantage for phage freestanding homing endonucleases to cleave both HEG-containing and HEG-less genomes.

  10. 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

  11. Expression and Purification of BmrI Restriction Endonuclease and Its N-terminal Cleavage Domain Variants

    OpenAIRE

    Bao, Yongming; Higgins, Lauren; Zhang, Penghua; Chan, Siu-hong; Laget, Sophie; Sweeney, Suzanne; Lunnen, Keith; Xu, Shuang-yong

    2007-01-01

    BmrI (ACTGGG N5/N4) is one of the few metal-independent restriction endonucleases (REases) found in bacteria. The BmrI restriction-modification system was cloned by the methylase selection method, inverse PCR, and PCR. BmrI REase shows significant amino acid sequence identity to BfiI and a putative endonuclease MspBNCORF3798 from the sequenced Mesorhizobium sp. BNC1 genome. The EDTA-resistant BmrI REase was successfully over-expressed in a pre-modified E. coli strain from pET21a or pBAC-expIQ...

  12. Using Group II Introns for Attenuating the In Vitro and In Vivo Expression of a Homing Endonuclease.

    Directory of Open Access Journals (Sweden)

    Tuhin Kumar Guha

    Full Text Available In Chaetomium thermophilum (DSM 1495 within the mitochondrial DNA (mtDNA small ribosomal subunit (rns gene a group IIA1 intron interrupts an open reading frame (ORF encoded within a group I intron (mS1247. This arrangement offers the opportunity to examine if the nested group II intron could be utilized as a regulatory element for the expression of the homing endonuclease (HEase. Constructs were generated where the codon-optimized ORF was interrupted with either the native group IIA1 intron or a group IIB type intron. This study showed that the expression of the HEase (in vivo in Escherichia coli can be regulated by manipulating the splicing efficiency of the HEase ORF-embedded group II introns. Exogenous magnesium chloride (MgCl2 stimulated the expression of a functional HEase but the addition of cobalt chloride (CoCl2 to growth media antagonized the expression of HEase activity. Ultimately the ability to attenuate HEase activity might be useful in precision genome engineering, minimizing off target activities, or where pathways have to be altered during a specific growth phase.

  13. [Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells].

    Science.gov (United States)

    Misic, V; El-Mogy, M; Geng, S; Haj-Ahmad, Y

    2016-01-01

    Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.

  14. Comparison of whole genome sequencing to restriction endonuclease analysis and gel diffusion precipitin-based serotyping of Pasteurella multocida.

    Science.gov (United States)

    LeCount, Karen J; Schlater, Linda K; Stuber, Tod; Robbe Austerman, Suelee; Frana, Timothy S; Griffith, Ronald W; Erdman, Matthew M

    2018-01-01

    The gel diffusion precipitin test (GDPT) and restriction endonuclease analysis (REA) have commonly been used in the serotyping and genotyping of Pasteurella multocida. Whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis has become the gold standard for other organisms, offering higher resolution than previously available methods. We compared WGS to REA and GDPT on 163 isolates of P. multocida to determine if WGS produced more precise results. The isolates used represented the 16 reference serovars, isolates with REA profiles matching an attenuated fowl cholera vaccine strain, and isolates from 10 different animal species. Isolates originated from across the United States and from Chile. Identical REA profiles clustered together in the phylogenetic tree. REA profiles that differed by only a few bands had fewer SNP differences than REA profiles with more differences, as expected. The GDPT results were diverse but it was common to see a single serovar show up repeatedly within clusters. Several errors were found when examining the REA profiles. WGS was able to confirm these errors and compensate for the subjectivity in analysis of REA. Also, results of WGS and SNP analysis correlated more closely with the epidemiologic data than GDPT. In silico results were also compared to a lipopolysaccharide rapid multiplex PCR test. From the data produced in our study, WGS and SNP analysis was superior to REA and GDPT and highlighted some of the issues with the older tests.

  15. 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.

  16. Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein.

    Science.gov (United States)

    Nam, Ki Hyun; Ding, Fran; Haitjema, Charles; Huang, Qingqiu; DeLisa, Matthew P; Ke, Ailong

    2012-10-19

    The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5'-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg(2+) or Mn(2+)), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1-α1 loop.

  17. Activity on non-methylated DNA limits the use of endonuclease MspJI for epigenetic analyses

    Directory of Open Access Journals (Sweden)

    María Belén Jerez

    2018-03-01

    Full Text Available Cytosine methylation of DNA in mammals has been associated with both physiological and pathological changes in gene-expression. DNA treatment with methylation sensitive and/or dependent restriction enzymes, followed by PCR amplification is a widely used approach to test CpG methylation. Recently, restriction endonuclease MspJI has been proposed as a promising tool for epigenetic analyses. In this paper, we have tested MspJI as a tool for detecting CpG methylation on mammalian genomic DNA. For this experiment mouse genomic sequences harboring or lacking CpG sites were selected. The extent of degradation was evaluated by PCR using primers flanking the chosen genomic regions. Digestion of mouse genomic DNA, in combination with end-point and real-time PCR reactions, revealed that MspJI treatment reduced the amplification of genomic regions either containing or lacking of CpG motifs. In addition, treatment of bona fide non-methylated (in vitro amplified DNA samples definitely demonstrated that MspJI shows significant activity against non-methylated DNA. These results show that star activity can be an important concern when using MspJI, even under standard conditions. Therefore, we conclude that (in contrast to classical restriction enzymes, careful case by case evaluation of reaction conditions is mandatory for optimizing the usefulness of MspJI in epigenetic studies.

  18. Comprehensive phylogenetic analysis of bacterial group II intron-encoded ORFs lacking the DNA endonuclease domain reveals new varieties.

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    Nicolás Toro

    Full Text Available Group II introns are self-splicing RNAs that act as mobile retroelements in the organelles of plants, fungi and protists. They are also widely distributed in bacteria, and are generally assumed to be the ancestors of nuclear spliceosomal introns. Most bacterial group II introns have a multifunctional intron-encoded protein (IEP ORF within the ribozyme domain IV (DIV. This ORF encodes an N-terminal reverse transcriptase (RT domain, followed by a putative RNA-binding domain with RNA splicing or maturase activity and, in some cases, a C-terminal DNA-binding (D region followed by a DNA endonuclease (En domain. In this study, we focused on bacterial group II intron ORF phylogenetic classes containing only reverse transcriptase/maturase open reading frames, with no recognizable D/En region (classes A, C, D, E, F and unclassified introns. On the basis of phylogenetic analyses of the maturase domain and its C-terminal extension, which appears to be a signature characteristic of ORF phylogenetic class, with support from the phylogeny inferred from the RT domain, we have revised the proposed new class F, defining new intron ORF varieties. Our results increase knowledge of the lineage of group II introns encoding proteins lacking the En-domain.

  19. Continuous assays for DNA translocation using fluorescent triplex dissociation: application to type I restriction endonucleases.

    Science.gov (United States)

    McClelland, Sarah E; Dryden, David T F; Szczelkun, Mark D

    2005-05-13

    Fluorescent assays and accompanying kinetic models are described for the analysis of DNA translocation independent of duplex unwinding. A triplex binding site (TBS) was introduced into DNA substrates at precise loci downstream of recognition sequences for type IA, IB and IC restriction endonucleases (EcoKI, EcoAI and EcoR124I, respectively). Each endonuclease was incubated (without ATP) with substrates on which a hexachlorofluoroscein-labelled triplex-forming oligonucleotide (HEX-TFO) was pre-bound. Following addition of ATP, 1-D enzyme motion resulted in collision with, and displacement of, the HEX-TFO, producing a >twofold increase in fluorescent intensity. Alternatively, a decrease in anisotropy following displacement of a rhodamine-labelled TFO was monitored. Using rapid mixing in a stopped-flow fluorimeter, continuous kinetic profiles were produced in which displacement is preceded by a lag-phase, directly proportional to the distance moved. For each enzyme, we obtained not only the translocation rate but also information on slow isomerisation step(s) at initiation. Furthermore, we demonstrated that enzymes deficient in DNA cleavage but with maximal ATPase activity showed initiation and translocation rates identical to wild-type, confirming that DNA strand breaks are not a pre-requisite of motion.

  20. Assembly of Francisella novicida Cpf1 endonuclease in complex with guide RNA and target DNA.

    Science.gov (United States)

    Alcón, Pablo; Montoya, Guillermo; Stella, Stefano

    2017-07-01

    Bacteria and archaea use the CRISPR-Cas system as an adaptive response against infection by foreign nucleic acids. Owing to its remarkable flexibility, this mechanism has been harnessed and adopted as a powerful tool for genome editing. The CRISPR-Cas system includes two classes that are subdivided into six types and 19 subtypes according to conservation of the cas gene and loci organization. Recently, a new protein with endonuclease activity belonging to class 2 type V has been identified. This endonuclease, termed Cpf1, in complex with a single CRISPR RNA (crRNA) is able to recognize and cleave a target DNA preceded by a 5'-TTN-3' protospacer-adjacent motif (PAM) complementary to the RNA guide. To obtain structural insight into the inner workings of Cpf1, the crystallization of an active complex containing the full extent of the crRNA and a 31-nucleotide dsDNA target was attempted. The gene encoding Cpf1 from Francisella novicida was cloned, overexpressed and purified. The crRNA was transcribed and purified in vitro. Finally, the ternary FnCpf1-crRNA-DNA complex was assembled and purified by preparative electrophoresis before crystallization. Crystals belonging to space group C222 1 , with unit-cell parameters a = 85.2, b = 137.6, c = 320.5 Å, were obtained and subjected to preliminary diffraction experiments.

  1. In vitro Inactivation of Latent HSV by Targeted Mutagenesis Using an HSV-specific Homing Endonuclease

    Directory of Open Access Journals (Sweden)

    Martine Aubert

    2014-01-01

    Full Text Available Following acute infection, herpes simplex virus (HSV establishes latency in sensory neurons, from which it can reactivate and cause recurrent disease. Available antiviral therapies do not affect latent viral genomes; therefore, they do not prevent reactivation following therapy cessation. One possible curative approach involves the introduction of DNA double strand breaks in latent HSV genomes by rare-cutting endonucleases, leading to mutagenesis of essential viral genes. We tested this approach in an in vitro HSV latency model using the engineered homing endonuclease (HE HSV1m5, which recognizes a sequence in the HSV-1 gene UL19, encoding the virion protein VP5. Coexpression of the 3′-exonuclease Trex2 with HEs increased HE-mediated mutagenesis frequencies up to sixfold. Following HSV1m5/Trex2 delivery with adeno-associated viral (AAV vectors, the target site was mutated in latent HSV genomes with no detectable cell toxicity. Importantly, HSV production by latently infected cells after reactivation was decreased after HSV1m5/Trex2 exposure. Exposure to histone deacetylase inhibitors prior to HSV1m5/Trex2 treatment increased mutagenesis frequencies of latent HSV genomes another two- to fivefold, suggesting that chromatin modification may be a useful adjunct to gene-targeting approaches. These results support the continuing development of HEs and other nucleases (ZFNs, TALENs, CRISPRs for cure of chronic viral infections.

  2. 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.

  3. A unique family of Mrr-like modification-dependent restriction endonucleases.

    Science.gov (United States)

    Zheng, Yu; Cohen-Karni, Devora; Xu, Derrick; Chin, Hang Gyeong; Wilson, Geoffrey; Pradhan, Sriharsa; Roberts, Richard J

    2010-09-01

    Mrr superfamily of homologous genes in microbial genomes restricts modified DNA in vivo. However, their biochemical properties in vitro have remained obscure. Here, we report the experimental characterization of MspJI, a remote homolog of Escherichia coli's Mrr and show it is a DNA modification-dependent restriction endonuclease. Our results suggest MspJI recognizes (m)CNNR (R = G/A) sites and cleaves DNA at fixed distances (N(12)/N(16)) away from the modified cytosine at the 3' side (or N(9)/N(13) from R). Besides 5-methylcytosine, MspJI also recognizes 5-hydroxymethylcytosine but is blocked by 5-glucosylhydroxymethylcytosine. Several other close homologs of MspJI show similar modification-dependent endonuclease activity and display substrate preferences different from MspJI. A unique feature of these modification-dependent enzymes is that they are able to extract small DNA fragments containing modified sites on genomic DNA, for example ∼32 bp around symmetrically methylated CG sites and ∼31 bp around methylated CNG sites. The digested fragments can be directly selected for high-throughput sequencing to map the location of the modification on the genomic DNA. The MspJI enzyme family, with their different recognition specificities and cleavage properties, provides a basis on which many future methods can build to decode the epigenomes of different organisms.

  4. Natural transformation of an engineered Helicobacter pylori strain deficient in type II restriction endonucleases.

    Science.gov (United States)

    Zhang, Xue-Song; Blaser, Martin J

    2012-07-01

    Restriction-modification (RM) systems are important for bacteria to limit foreign DNA invasion. The naturally competent bacterium Helicobacter pylori has highly diverse strain-specific type II systems. To evaluate the roles of strain-specific restriction in H. pylori natural transformation, a markerless type II restriction endonuclease-deficient (REd) mutant was constructed. We deleted the genes encoding all four active type II restriction endonucleases in H. pylori strain 26695 using sacB-mediated counterselection. Transformation by donor DNA with exogenous cassettes methylated by Escherichia coli was substantially (1.7 and 2.0 log(10) for cat and aphA, respectively) increased in the REd strain. There also was significantly increased transformation of the REd strain by donor DNA from other H. pylori strains, to an extent corresponding to their shared type II R-M system strain specificity with 26695. Comparison of the REd and wild-type strains indicates that restriction did not affect the length of DNA fragment integration during natural transformation. There also were no differentials in cell growth or susceptibility to DNA damage. In total, the data indicate that the type II REd mutant has enhanced competence with no loss of growth or repair facility compared to the wild type, facilitating H. pylori mutant construction and other genetic engineering.

  5. 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.

  6. Endonuclease active site plasticity allows DNA cleavage with diverse alkaline Earth and transition metal ions.

    Science.gov (United States)

    Vasu, Kommireddy; Saravanan, Matheshwaran; Nagaraja, Valakunja

    2011-09-16

    A majority of enzymes show a high degree of specificity toward a particular metal ion in their catalytic reaction. However, Type II restriction endonuclease (REase) R.KpnI, which is the first member of the HNH superfamily of REases, exhibits extraordinary diversity in metal ion dependent DNA cleavage. Several alkaline earth and transition group metal ions induce high fidelity and promiscuous cleavage or inhibition depending upon their concentration. The metal ions having different ionic radii and co-ordination geometries readily replace each other from the enzyme's active site, revealing its plasticity. Ability of R.KpnI to cleave DNA with both alkaline earth and transition group metal ions having varied ionic radii could imply utilization of different catalytic site(s). However, mutation of the invariant His residue of the HNH motif caused abolition of the enzyme activity with all of the cofactors, indicating that the enzyme follows a single metal ion catalytic mechanism for DNA cleavage. Indispensability of His in nucleophile activation together with broad cofactor tolerance of the enzyme indicates electrostatic stabilization function of metal ions during catalysis. Nevertheless, a second metal ion is recruited at higher concentrations to either induce promiscuity or inhibit the DNA cleavage. Regulation of the endonuclease activity and fidelity by a second metal ion binding is a unique feature of R.KpnI among REases and HNH nucleases. The active site plasticity of R.KpnI opens up avenues for redesigning cofactor specificities and generation of mutants specific to a particular metal ion.

  7. Alteration of sequence specificity of the type IIS restriction endonuclease BtsI.

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    Shengxi Guan

    Full Text Available 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 the enzyme, we identified a BtsIB mutant R119A found to have a different digestion pattern from the wild type BtsI. After characterization, we found that BtsIB(R119A is a novel restriction enzyme with a previously unreported recognition sequence CAGTG(2/0, which is named as BtsI-1. Compared with wild type BtsI, BtsI-1 showed different relative activities in NEB restriction enzyme reaction buffers NEB1, NEB2, NEB3 and NEB4 and less star activity. Similar to the wild type BtsIB subunit, the BtsI-1 B subunit alone can act as a bottom nicking enzyme recognizing CAGTG(-/0. This is the first successful case of a specificity change among this restriction endonuclease type.

  8. 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

  9. 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...

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

    Science.gov (United States)

    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...

  11. 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 d...

  12. Effect of apurinic/apyrimidinic endonucleases and polyamines on DNA treated with bleomycin and neocarzinostatin: specific formation and cleavage of closely opposed lesions in complementary strands

    International Nuclear Information System (INIS)

    Povirk, L.F.; Houlgrave, C.W.

    1988-01-01

    Bleomycin and neocarzinostatin induce modified apurinic/apyrimidinic (AP) sites by oxidation of the sugar moiety in DNA. In order to quantitatively assess the susceptibility of these lesions to repair endonucleases, drug-treated 3 H-labeled colE1 DNA was mixed with 14 C-labeled heat-depurinated DNA, and endonuclease-susceptible sites in the mixture were titrated with various AP endonucleases or with polyamines. Single- and double-strand breaks were quantitated by determining the fractions of supercoiled, nicked circular, and linear molecules. Exonuclease III and endonucleases III and IV of Escherichia coli, indicating cleavage of drug-induced AP sites. Bleomycin-induced AP sites were much more sensitive to cleavage by putrescine than heat-induced sites. Treatment with putrescine or very high concentrations of endonuclease III also increased the number of double-strand breaks in bleomycin-treated DNA, suggesting a minor class of lesion consisting of an AP site accompanied by a closely opposed break in the complementary strand. These complex lesions were resistant to cleavage by endonuclease IV. These results suggest that virtually all neocarzinostatin-induced AP sites are accompanied by a closely opposed strand break. Several characteristics of the putative AP site/strand break lesions induced by neocarzinostatin suggest that they may correspond to certain AP-like lesions which were previously detected on DNA sequencing gels as endonuclease IV susceptible sites and which have been strongly implicated in neocarzinostatin-induced mutagenesis

  13. A physical map of human Alu repeats cleavage by restriction endonucleases

    Directory of Open Access Journals (Sweden)

    Chernukhin Valery A

    2008-06-01

    Full Text Available Abstract Background Alu repetitive elements are the abundant sequences in human genome. Diversity of DNA sequences of these elements makes difficult the construction of theoretical patterns of Alu repeats cleavage by restriction endonucleases. We have proposed a method of restriction analysis of Alu repeats sequences in silico. Results Simple software to analyze Alu repeats database has been suggested and Alu repeats digestion patterns for several restriction enzymes' recognition sites have been constructed. Restriction maps of Alu repeats cleavage for corresponding restriction enzymes have been calculated and plotted. Theoretical data have been compared with experimental results on DNA hydrolysis with restriction enzymes, which we obtained earlier. Conclusion Alu repeats digestions provide the main contribution to the patterns of human chromosomal DNA cleavage. This corresponds to the experimental data on total human DNA hydrolysis with restriction enzymes.

  14. Class 2 CRISPR-Cas RNA-guided endonucleases: Swiss Army knives of genome editing.

    Science.gov (United States)

    Stella, Stefano; Alcón, Pablo; Montoya, Guillermo

    2017-11-01

    CRISPR-Cas is a bacterial defense system against phage infection and nucleic acid invasion. Class 2 type II CRISPR-Cas9 has also been widely used for genome engineering. Here, we review novel insights into the CRISPR class 2 type V enzymes, specifically Cpf1 and C2c1, which display different DNA-recognition and cleavage characteristics than those of Cas9, the best-characterized member of class 2. Recent structures of these ribonucleoprotein complexes that capture several stages of the endonuclease reaction have provided molecular details of recognition, unzipping and cleavage of the target DNA, allowing their comparison with Cas9. A detailed understanding of these mechanisms is crucial for improving these genome engineering tools and expanding the genomic space that can be targeted.

  15. Insights on copper coordination and reactivity of endonuclease EcoRI by ESR spectroscopy and modeling

    Science.gov (United States)

    Ji, Ming

    2009-03-01

    The cleavage of DNA by restriction endonuclease EcoRI is catalyzed by metal ions such as Mg^2+. However, Cu^2+ does not catalyze the cleavage of DNA by EcoRI. In order to understand the functional difference between Cu^2+ and Mg^2+, coordination of Cu^2+ in the EcoRI--DNA complex was clarified by ESR and MD simulation. There are two Cu^2+ components in the specific EcoRI-DNA complex. Each component has one N atom from histidine imidazole and one oxygen atom from the phosphate backbone of DNA coordinate to Cu^2+ based on the ESR experimental results. MD simulation further confirmed that the Nδ atom of His114 imidazole and one oxygen atom from the phosphate backbone of DNA coordinate to Cu^2+. Difference in the coordination of Cu^2+ and Mg^2+ explains their different functional behaviors.

  16. Role of Magnesium Ions in DNA Recognition by the EcoRV Restriction Endonuclease

    Energy Technology Data Exchange (ETDEWEB)

    Zahran, Mai [ORNL; Berezniak, Tomasz [University of Heidelberg; Imhof, Petra [University of Heidelberg; Smith, Jeremy C [ORNL

    2011-01-01

    The restriction endonuclease EcoRV binds two magnesium ions. One of these ions, Mg2+A, binds to the phosphate group where the cleavage occurs and is required for catalysis, but the role of the other ion, Mg2+B is debated. Here, multiple independent molecular dynamics simulations suggest that Mg2+B is crucial for achieving a tightly bound protein DNA complex and stabilizing a conformation that allows cleavage. In the absence of Mg2+B in all simulations the protein DNA hydrogen bond network is significantly disrupted and the sharp kink at the central base pair step of the DNA, which is observed in the two-metal complex, is not present. Also, the active site residues rearrange in such a way that the formation of a nucleophile, required for DNA hydrolysis, is unlikely.

  17. 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.

  18. Interplay between structure-specific endonucleases for crossover control during Caenorhabditis elegans meiosis.

    Directory of Open Access Journals (Sweden)

    Takamune T Saito

    Full Text Available The number and distribution of crossover events are tightly regulated at prophase of meiosis I. The resolution of Holliday junctions by structure-specific endonucleases, including MUS-81, SLX-1, XPF-1 and GEN-1, is one of the main mechanisms proposed for crossover formation. However, how these nucleases coordinately resolve Holliday junctions is still unclear. Here we identify both the functional overlap and differences between these four nucleases regarding their roles in crossover formation and control in the Caenorhabditis elegans germline. We show that MUS-81, XPF-1 and SLX-1, but not GEN-1, can bind to HIM-18/SLX4, a key scaffold for nucleases. Analysis of synthetic mitotic defects revealed that MUS-81 and SLX-1, but not XPF-1 and GEN-1, have overlapping roles with the Bloom syndrome helicase ortholog, HIM-6, supporting their in vivo roles in processing recombination intermediates. Taking advantage of the ease of genetic analysis and high-resolution imaging afforded by C. elegans, we examined crossover designation, frequency, distribution and chromosomal morphology in single, double, triple and quadruple mutants of the structure-specific endonucleases. This revealed that XPF-1 functions redundantly with MUS-81 and SLX-1 in executing crossover formation during meiotic double-strand break repair. Analysis of crossover distribution revealed that SLX-1 is required for crossover suppression at the center region of the autosomes. Finally, analysis of chromosome morphology in oocytes at late meiosis I stages uncovered that SLX-1 and XPF-1 promote meiotic chromosomal stability by preventing formation of chromosomal abnormalities. We propose a model in which coordinate action between structure-specific nucleases at different chromosome domains, namely MUS-81, SLX-1 and XPF-1 at the arms and SLX-1 at the center region, exerts positive and negative regulatory roles, respectively, for crossover control during C. elegans meiosis.

  19. Cyanobacterial ribosomal RNA genes with multiple, endonuclease-encoding group I introns

    Directory of Open Access Journals (Sweden)

    Turner Seán

    2007-09-01

    Full Text Available Abstract Background Group I introns are one of the four major classes of introns as defined by their distinct splicing mechanisms. Because they catalyze their own removal from precursor transcripts, group I introns are referred to as autocatalytic introns. Group I introns are common in fungal and protist nuclear ribosomal RNA genes and in organellar genomes. In contrast, they are rare in all other organisms and genomes, including bacteria. Results Here we report five group I introns, each containing a LAGLIDADG homing endonuclease gene (HEG, in large subunit (LSU rRNA genes of cyanobacteria. Three of the introns are located in the LSU gene of Synechococcus sp. C9, and the other two are in the LSU gene of Synechococcus lividus strain C1. Phylogenetic analyses show that these introns and their HEGs are closely related to introns and HEGs located at homologous insertion sites in organellar and bacterial rDNA genes. We also present a compilation of group I introns with homing endonuclease genes in bacteria. Conclusion We have discovered multiple HEG-containing group I introns in a single bacterial gene. To our knowledge, these are the first cases of multiple group I introns in the same bacterial gene (multiple group I introns have been reported in at least one phage gene and one prophage gene. The HEGs each contain one copy of the LAGLIDADG motif and presumably function as homodimers. Phylogenetic analysis, in conjunction with their patchy taxonomic distribution, suggests that these intron-HEG elements have been transferred horizontally among organelles and bacteria. However, the mode of transfer and the nature of the biological connections among the intron-containing organisms are unknown.

  20. Crystallization and preliminary X-ray diffraction analysis of the small subunit of the heterodimeric restriction endonuclease R.BspD6I

    Energy Technology Data Exchange (ETDEWEB)

    Kachalova, Galina S. [Max-Planck Unit for Structural Molecular Biology, Hamburg 22607 (Germany); Yunusova, Alfiya K.; Artyukh, Rimma I.; Rogulin, Eugeny A.; Perevyazova, Tatyana A.; Zheleznaya, Ludmila A. [Institute of Theoretical and Experimental Biophysics, Pushchino 142290 (Russian Federation); Matvienko, Nickolay I. [Institute of Protein Research, Pushchino 14229 (Russian Federation); Bartunik, Hans D., E-mail: bartunik@mpghdb.desy.de [Max-Planck Unit for Structural Molecular Biology, Hamburg 22607 (Germany)

    2007-09-01

    The crystallization of the small subunit of the heterodimeric restriction endonuclease R.BspD6I and diffraction data collection to 1.5 Å resolution are reported. The heterodimeric restriction endonuclease R.BspD6I is composed of a small subunit with a cleavage site and a large subunit, containing a recognition domain and a cleavage domain, that may function separately as a monomeric nicking endonuclease. Here, the crystallization of the small subunit and diffraction data collection to 1.5 Å resolution are reported.

  1. The anti-inflammatory role of extranuclear apurinic/apyrimidinic endonuclease 1/redox effector factor-1 in reactive astrocytes.

    Science.gov (United States)

    Baek, Hyunjung; Lim, Chae Seong; Byun, Hee Sun; Cho, Hyun Sil; Lee, Yu Ran; Shin, Yong Sup; Kim, Hyun-Woo; Jeon, Byeong Hwa; Kim, Dong Woon; Hong, Jinpyo; Hur, Gang Min; Park, Jin Bong

    2016-12-16

    Apurinic/apyrimidinic endonuclease 1 (APE1), a ubiquitous multipurpose protein, is also known as redox effector factor-1 (Ref-1). It is involved in DNA repair and redox signaling and, in turn, oxidative stress-induced neurodegeneration. Although previous studies have demonstrated that APE1/Ref-1 functions as a negative regulator of inflammatory response via several mechanisms in neuronal cells, little is known about the roles of APE1/Ref-1 in glial cells. In this study, we found that cytoplasmic APE1/Ref-1 expression was upregulated in reactive astrocytes of the kainic acid- or lipopolysaccharide (LPS)-injected hippocampus. Analysis of the inflammatory response induced by extranuclear APE1/Ref-1 (ΔNLS-Ref-1) in cultured primary astrocytes revealed that it markedly suppressed inducible nitric oxide synthase (iNOS) expression and tumor necrosis factor-α (TNF-α) secretion induced by LPS to a similar extent as did wild type APE1/Ref-1 (WT-Ref-1), supporting the concept an anti-inflammatory role of extranuclear APE1/Ref-1 in astrocytes. Additionally, overexpression of WT- and ΔNLS-Ref-1 suppressed the transcriptional activity of nuclear factor-κB (NF-κB), although it effectively enhanced activator protein 1 (AP-1) activity. The blunting effect of APE1/Ref-1 on LPS-induced NF-κB activation was not mediated by IκB kinase (IKK) activity. Instead, APE1/Ref-1 inhibited p300-mediated acetylation of p65 by suppressing intracellular reactive oxygen species (ROS) levels following LPS treatment. Taken together, our results showed that altered expression and/or subcellular distribution of APE1/Ref-1 in activated astrocytes regulated the neuroinflammatory response to excitotoxin and endotoxin insults used in model of neurodegenerative brain diseases.

  2. Nuclear depletion of apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) is an indicator of energy disruption in neurons.

    Science.gov (United States)

    Singh, Shilpee; Englander, Ella W

    2012-11-01

    Apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) is a multifunctional protein critical for cellular survival. Its involvement in adaptive survival responses includes key roles in redox sensing, transcriptional regulation, and repair of DNA damage via the base excision repair (BER) pathway. Ape1 is abundant in most cell types and central in integrating the first BER step catalyzed by different DNA glycosylases. BER is the main process for removal of oxidative DNA lesions in postmitotic brain cells, and after ischemic brain injury preservation of Ape1 coincides with neuronal survival, while its loss has been associated with neuronal death. Here, we report that in cultured primary neurons, diminution of cellular ATP by either oligomycin or H(2)O(2) is accompanied by depletion of nuclear Ape1, while other BER proteins are unaffected and retain their nuclear localization under these conditions. Importantly, while H(2)O(2) induces γH2AX phosphorylation, indicative of chromatin rearrangements in response to DNA damage, oligomycin does not. Furthermore, despite comparable diminution of ATP content, H(2)O(2) and oligomycin differentially affect critical parameters of mitochondrial respiration that ultimately determine cellular ATP content. Taken together, our findings demonstrate that in neurons, nuclear compartmentalization of Ape1 depends on ATP and loss of nuclear Ape1 reflects disruption of neuronal energy homeostasis. Energy crisis is a hallmark of stroke and other ischemic/hypoxic brain injuries. In vivo studies have shown that Ape1 deficit precedes neuronal loss in injured brain regions. Thus, our findings bring to light the possibility that energy failure-induced Ape1 depletion triggers neuronal death in ischemic brain injuries. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Natural C-independent expression of restriction endonuclease in a C protein-associated restriction-modification system.

    Science.gov (United States)

    Rezulak, Monika; Borsuk, Izabela; Mruk, Iwona

    2016-04-07

    Restriction-modification (R-M) systems are highly prevalent among bacteria and archaea, and appear to play crucial roles in modulating horizontal gene transfer and protection against phage. There is much to learn about these diverse enzymes systems, especially their regulation. Type II R-M systems specify two independent enzymes: a restriction endonuclease (REase) and protective DNA methyltransferase (MTase). Their activities need to be finely balanced in vivo Some R-M systems rely on specialized transcription factors called C (controller) proteins. These proteins play a vital role in the temporal regulation of R-M gene expression, and function to indirectly modulate the horizontal transfer of their genes across the species. We report novel regulation of a C-responsive R-M system that involves a C protein of a poorly-studied structural class - C.Csp231I. Here, the C and REase genes share a bicistronic transcript, and some of the transcriptional auto-control features seen in other C-regulated R-M systems are conserved. However, separate tandem promoters drive most transcription of the REase gene, a distinctive property not seen in other tested C-linked R-M systems. Further, C protein only partially controls REase expression, yet plays a role in system stability and propagation. Consequently, high REase activity was observed after deletion of the entire C gene, and cells bearing the ΔC R-M system were outcompeted in mixed culture assays by those with the WT R-M system. Overall, our data reveal unexpected regulatory variation among R-M systems. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Bifunctional TaqII restriction endonuclease: redefining the prototype DNA recognition site and establishing the Fidelity Index for partial cleaving.

    Science.gov (United States)

    Zylicz-Stachula, Agnieszka; Zołnierkiewicz, Olga; Sliwińska, Katarzyna; Jeżewska-Frąckowiak, Joanna; Skowron, Piotr M

    2011-12-05

    The TaqII enzyme is a member of the Thermus sp. enzyme family that we propounded previously within Type IIS restriction endonucleases, containing related thermophilic bifunctional endonucleases-methyltransferases from various Thermus sp.: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI and TsoI. These enzymes show significant nucleotide and amino acid sequence similarities, a rare phenomenon among restriction endonucleases, along with similarities in biochemical properties, molecular size, DNA recognition sequences and cleavage sites. They also feature some characteristics of Types I and III. Barker et al. reported the Type IIS/IIC restriction endonuclease TaqII as recognizing two distinct cognate site variants (5'-GACCGA-3' and 5'-CACCCA-3') while cleaving 11/9 nucleotides downstream. We used four independent methods, namely, shotgun cloning and sequencing, restriction pattern analysis, digestion of particular custom substrates and GeneScan analysis, to demonstrate that the recombinant enzyme recognizes only 5'-GACCGA-3' sites and cleaves 11/9 nucleotides downstream. We did not observe any 5'-CACCCA-3' cleavage under a variety of conditions and site arrangements tested. We also characterized the enzyme biochemically and established new digestion conditions optimal for practical enzyme applications. Finally, we developed and propose a new version of the Fidelity Index - the Fidelity Index for Partial Cleavage (FI-PC). The DNA recognition sequence of the bifunctional prototype TaqII endonuclease-methyltransferase from Thermus aquaticus has been redefined as recognizing only 5'-GACCGA-3' cognate sites. The reaction conditions (pH and salt concentrations) were designed either to minimize (pH = 8.0 and 10 mM ammonium sulphate) or to enhance star activity (pH = 6.0 and no salt). Redefinition of the recognition site and reaction conditions makes this prototype endonuclease a useful tool for DNA manipulation; as yet, this enzyme has no practical applications. The extension of

  5. Characterization of the restriction enzyme-like endonuclease encoded by the Entamoeba histolytica non-long terminal repeat retrotransposon EhLINE1.

    Science.gov (United States)

    Yadav, Vijay Pal; Mandal, Prabhat Kumar; Rao, Desirazu N; Bhattacharya, Sudha

    2009-12-01

    The genome of the human pathogen Entamoeba histolytica, a primitive protist, contains non-long terminal repeat retrotransposable elements called EhLINEs. These encode reverse transcriptase and endonuclease required for retrotransposition. The endonuclease shows sequence similarity with bacterial restriction endonucleases. Here we report the salient enzymatic features of one such endonuclease. The kinetics of an EhLINE1-encoded endonuclease catalyzed reaction, determined under steady-state and single-turnover conditions, revealed a significant burst phase followed by a slower steady-state phase, indicating that release of product could be the slower step in this reaction. For circular supercoiled DNA the K(m) was 2.6 x 10(-8) M and the k(cat) was 1.6 x 10(-2) sec(-1). For linear E. histolytica DNA substrate the K(m) and k(cat) values were 1.3 x 10(-8) M and 2.2 x 10(-4) sec(-1) respectively. Single-turnover reaction kinetics suggested a noncooperative mode of hydrolysis. The enzyme behaved as a monomer. While Mg(2+) was required for activity, 60% activity was seen with Mn(2+) and none with other divalent metal ions. Substitution of PDX(12-14)D (a metal-binding motif) with PAX(12-14)D caused local conformational change in the protein tertiary structure, which could contribute to reduced enzyme activity in the mutated protein. The protein underwent conformational change upon the addition of DNA, which is consistent with the known behavior of restriction endonucleases. The similarities with bacterial restriction endonucleases suggest that the EhLINE1-encoded endonuclease was possibly acquired from bacteria through horizontal gene transfer. The loss of strict sequence specificity for nicking may have been subsequently selected to facilitate spread of the retrotransposon to intergenic regions of the E. histolytica genome.

  6. Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase

    Directory of Open Access Journals (Sweden)

    Klingenspor Martin

    2007-11-01

    Full Text Available Abstract Background The subcloning of a DNA fragment from an entry vector into a destination vector is a routinely performed task in molecular biology labs. Results We here present a novel benchtop procedure to achieve rapid recombination into any destination vector of choice with the sole requirement of an endonuclease recognition site. The method relies on a specifically designed entry vector and the combined action of type II and type IIs endonucleases with ligase. The formulation leads to accumulation of a single stable cloning product representing the desired insert carrying destination vector. Conclusion The described method provides a fast single step procedure for routine subcloning from an entry vector into a series of destination vectors with the same restriction enzyme recognition site.

  7. Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase.

    Science.gov (United States)

    Fromme, Tobias; Klingenspor, Martin

    2007-11-26

    The subcloning of a DNA fragment from an entry vector into a destination vector is a routinely performed task in molecular biology labs. We here present a novel benchtop procedure to achieve rapid recombination into any destination vector of choice with the sole requirement of an endonuclease recognition site. The method relies on a specifically designed entry vector and the combined action of type II and type IIs endonucleases with ligase. The formulation leads to accumulation of a single stable cloning product representing the desired insert carrying destination vector. The described method provides a fast single step procedure for routine subcloning from an entry vector into a series of destination vectors with the same restriction enzyme recognition site.

  8. Catalytic and non-catalytic roles of the CtIP endonuclease in double-strand break end resection

    Science.gov (United States)

    Makharashvili, Nodar; Tubbs, Anthony T.; Yang, Soo-Hyun; Wang, Hailong; Barton, Olivia; Zhou, Yi; Deshpande, Rajashree A.; Lee, Ji-Hoon; Lobrich, Markus; Sleckman, Barry P.; Wu, Xiaohua; Paull, Tanya T.

    2014-01-01

    Summary The CtIP protein is known to function in 5′ strand resection during homologous recombination similar to the budding yeast Sae2 protein, although its role in this process is unclear. Here we characterize recombinant human CtIP and find that it exhibits 5′ flap endonuclease activity on branched DNA structures, independent of the MRN complex. Phosphorylation of CtIP at known ATM-dependent sites and other sites is essential for its catalytic activity, although the S327 and T847 phosphorylation sites are dispensable. A catalytic mutant of CtIP that is deficient in endonuclease activity exhibits wild-type levels of homologous recombination at restriction enzyme-generated breaks but is deficient in processing topoisomerase adducts and radiation-induced breaks in human cells, suggesting that the nuclease activity of CtIP is specifically required for the removal of DNA adducts at sites of DNA breaks. PMID:24837676

  9. Epigenetic Segregation of Microbial Genomes from Complex Samples Using Restriction Endonucleases HpaII and McrB.

    Science.gov (United States)

    Liu, Guohong; Weston, Christopher Q; Pham, Long K; Waltz, Shannon; Barnes, Helen; King, Paula; Sphar, Dan; Yamamoto, Robert T; Forsyth, R Allyn

    2016-01-01

    We describe continuing work to develop restriction endonucleases as tools to enrich targeted genomes of interest from diverse populations. Two approaches were developed in parallel to segregate genomic DNA based on cytosine methylation. First, the methyl-sensitive endonuclease HpaII was used to bind non-CG methylated DNA. Second, a truncated fragment of McrB was used to bind CpG methylated DNA. Enrichment levels of microbial genomes can exceed 100-fold with HpaII allowing improved genomic detection and coverage of otherwise trace microbial genomes from sputum. Additionally, we observe interesting enrichment results that correlate with the methylation states not only of bacteria, but of fungi, viruses, a protist and plants. The methods presented here offer promise for testing biological samples for pathogens and global analysis of population methylomes.

  10. Epigenetic Segregation of Microbial Genomes from Complex Samples Using Restriction Endonucleases HpaII and McrB.

    Directory of Open Access Journals (Sweden)

    Guohong Liu

    Full Text Available We describe continuing work to develop restriction endonucleases as tools to enrich targeted genomes of interest from diverse populations. Two approaches were developed in parallel to segregate genomic DNA based on cytosine methylation. First, the methyl-sensitive endonuclease HpaII was used to bind non-CG methylated DNA. Second, a truncated fragment of McrB was used to bind CpG methylated DNA. Enrichment levels of microbial genomes can exceed 100-fold with HpaII allowing improved genomic detection and coverage of otherwise trace microbial genomes from sputum. Additionally, we observe interesting enrichment results that correlate with the methylation states not only of bacteria, but of fungi, viruses, a protist and plants. The methods presented here offer promise for testing biological samples for pathogens and global analysis of population methylomes.

  11. 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)

  12. Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease.

    Science.gov (United States)

    Yiu, Glenn; Tieu, Eric; Nguyen, Anthony T; Wong, Brittany; Smit-McBride, Zeljka

    2016-10-01

    To employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells. CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcuspyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect ARPE-19 cells, a human RPE cell line. Frequency of insertion or deletion (indel) mutations was assessed by T7 endonuclease 1 mismatch detection assay; mRNA levels were assessed with quantitative real-time PCR; and VEGF-A protein levels were determined by ELISA. In vitro angiogenesis was measured using an endothelial cell tube formation assay. Five gRNAs targeting VEGF-A were selected based on the highest predicted on-target probabilities, lowest off-target probabilities, or combined average of both scores. Lentiviral delivery of the top-scoring gRNAs with SpCas9 resulted in indel formation in the VEGF-A gene at frequencies up to 37.0% ± 4.0% with corresponding decreases in secreted VEGF-A protein up to 41.2% ± 7.4% (P CRISPR-Cas9 endonuclease system may reduce VEGF-A secretion from human RPE cells and suppress angiogenesis, supporting the possibility of employing gene editing for antiangiogenesis therapy in ocular diseases.

  13. Ligation-mediated PCR amplification of specific fragments from a class-II restriction endonuclease total digest.

    OpenAIRE

    Guilfoyle, R A; Leeck, C L; Kroening, K D; Smith, L M; Guo, Z

    1997-01-01

    A method is described which permits the ligation- mediated PCR amplification of specific fragments from a Class-II restriction endonuclease total digest. Feasibility was tested using Bcl I and phage lambda DNA as a model enzyme and amplicon system, respectively. Bcl I is one of many widely used restriction enzymes which cleave at palindromic recognition sequences and leave 5'-protruding ends of defined sequence. Using a single pair of universal primers, a given fragment can be specifically am...

  14. Structural Features and Functional Dependency on β-Clamp Define Distinct Subfamilies of Bacterial Mismatch Repair Endonuclease MutL*

    Science.gov (United States)

    Fukui, Kenji; Baba, Seiki; Kumasaka, Takashi; Yano, Takato

    2016-01-01

    In early reactions of DNA mismatch repair, MutS recognizes mismatched bases and activates MutL endonuclease to incise the error-containing strand of the duplex. DNA sliding clamp is responsible for directing the MutL-dependent nicking to the newly synthesized/error-containing strand. In Bacillus subtilis MutL, the β-clamp-interacting motif (β motif) of the C-terminal domain (CTD) is essential for both in vitro direct interaction with β-clamp and in vivo repair activity. A large cluster of negatively charged residues on the B. subtilis MutL CTD prevents nonspecific DNA binding until β clamp interaction neutralizes the negative charge. We found that there are some bacterial phyla whose MutL endonucleases lack the β motif. For example, the region corresponding to the β motif is completely missing in Aquifex aeolicus MutL, and critical amino acid residues in the β motif are not conserved in Thermus thermophilus MutL. We then revealed the 1.35 Å-resolution crystal structure of A. aeolicus MutL CTD, which lacks the β motif but retains the metal-binding site for the endonuclease activity. Importantly, there was no negatively charged cluster on its surface. It was confirmed that CTDs of β motif-lacking MutLs, A. aeolicus MutL and T. thermophilus MutL, efficiently incise DNA even in the absence of β-clamp and that β-clamp shows no detectable enhancing effect on their activity. In contrast, CTD of Streptococcus mutans, a β motif-containing MutL, required β-clamp for the digestion of DNA. We propose that MutL endonucleases are divided into three subfamilies on the basis of their structural features and dependence on β-clamp. PMID:27369079

  15. In vitro enzymatic studies on the nature and repair of x-ray induced lesions in DNA

    International Nuclear Information System (INIS)

    Wallace, S.S.

    1979-01-01

    Areas studied include: purification and properties of enzyme probes for x-ray induced DNA lesions using E. Coli x-ray endonuclease and S. cerevisiae endonuclease E; use of enzymes probes; and use of physical, chemical and enzymatic probes to quantify x-ray-induced lesions in viruses and cells

  16. Structural and functional analysis of the symmetrical Type I restriction endonuclease R.EcoR124I(NT.

    Directory of Open Access Journals (Sweden)

    James E Taylor

    Full Text Available Type I restriction-modification (RM systems are comprised of two multi-subunit enzymes, the methyltransferase (∼160 kDa, responsible for methylation of DNA, and the restriction endonuclease (∼400 kDa, responsible for DNA cleavage. Both enzymes share a number of subunits. An engineered RM system, EcoR124I(NT, based on the N-terminal domain of the specificity subunit of EcoR124I was constructed that recognises the symmetrical sequence GAAN(7TTC and is active as a methyltransferase. Here, we investigate the restriction endonuclease activity of R. EcoR124I(NTin vitro and the subunit assembly of the multi-subunit enzyme. Finally, using small-angle neutron scattering and selective deuteration, we present a low-resolution structural model of the endonuclease and locate the motor subunits within the multi-subunit enzyme. We show that the covalent linkage between the two target recognition domains of the specificity subunit is not required for subunit assembly or enzyme activity, and discuss the implications for the evolution of Type I enzymes.

  17. Characterization of LlaKI, a New Metal Ion-Independent Restriction Endonuclease from Lactococcus lactis KLDS4.

    Science.gov (United States)

    Belkebir, Abdelkarim; Azeddoug, Houssine

    2012-01-01

    Requirement of divalent cations for DNA cleavage is a general feature of type II restriction enzymes with the exception of few members of this group. A new type II restriction endonuclease has been partially purified from Lactococcus lactis KLDS4. The enzyme was denoted as LlaKI and showed to recognize and cleave the same site as FokI. The enzyme displayed a denatured molecular weight of 50 kDa and behaved as a dimer in solution as evidenced by the size exclusion chromatography. To investigate the role of divalent cations in DNA cleavage by LlaKI, digestion reactions were carried out at different Mg(2+), Mn(2+), and Ca(2+) concentrations. Unlike most of type II restriction endonucleases, LlaKI did not require divalent metal ions to cleave DNA and is one of the few metal-independent restriction endonucleases found in bacteria. The enzyme showed near-maximal levels of activity in 10 mM Tris-HCl pH 7.9, 50 mM NaCl, 10 mM MgCl2, and 1 mM dithiothreitol at 30°C. The presence of DNA modification was also determined and was correlated with the correspondent restriction enzyme.

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

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    Yan Li

    2015-01-01

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

  19. Characterization of Naegleria species by restriction endonuclease digestion of whole-cell DNA.

    Science.gov (United States)

    De Jonckheere, J F

    1987-05-01

    Whole-cell DNA in Naegleria spp. and two related genera was examined by restriction endonuclease digestion and fractionation of the fragments by agarose gel electrophoresis. Visual inspection of ethidium bromide-stained gels shows differences in banding pattern between N. fowleri, N. lovaniensis, N. gruberi, N. jadini, N. australiensis, Didasculus thorntoni and Willaertia magna, and between the two subspecies of N. australiensis. Even between strains belonging to the same species differences could be observed. Significant differences were seen between strains of N. fowleri according to the continent of origin, and a hypothesis on the ancestry and the dispersal of N. fowleri was deduced from it. A N. fowleri strain isolated from one of the very few cured human infections showed the most distinct pattern within the species. The considerable variation detected with serological and biochemical techniques between strains of N. australiensis as well as between strains of N. gruberi, was confirmed in the analysis of their whole-cell DNA. With this technique the existence of N. jadini, D. thorntoni, W. magna and two Naegleria strains as separate systematic entities is substantiated.

  20. Purification of restriction endonuclease from Acetobacter aceti IFO 3281 (AatII) and its properties.

    Science.gov (United States)

    Sato, H; Suzuki, T; Yamada, Y

    1990-12-01

    The restriction endonuclease AatII was purified from cell-free extracts of Acetobacter aceti IFO 3281 by streptomycin treatment, ammonium sulfate fractionation, combined column chromatographies on DEAE-Toyopearl 650S, heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and FPLC on Mono Q and on Superose 12 (gel filtration). The purified enzyme was homogeneous on SDS-polyacrylamide gel disk electrophoresis. The relative molecular mass of the purified enzyme was 190,000 daltons by gel filtration. The SDS-polyacrylamide gel disk electrophoresis gave the relative molecular mass of 47,500 daltons. These data indicated that the purified, native enzyme is a tetramer (190,000 daltons) composed of four 47,500-dalton subunits. The isoelectric point of the enzyme was 6.0. The purified enzyme was intensely activated by manganese ion (50-fold increase or more when compared with magnesium ion). The enzyme worked best at 37 degrees C and pH 8.5 in a reaction mixture (50 microliters) containing 1.0 micrograms lambda DNA, 10 mM Tris-HCl, 7 mM 2-mercaptoethanol, 7 mM MnCl2 and 50 mM NaCl. The enzyme recognizes the same palindromic hexanucleotide sequence 5'-GACGTC-3', cuts between T and C and produces a 3'-tetranucleotide extension in the presence of MnCl2, as it does in the presence of MgCl2.

  1. Sequencing by ligation variation with endonuclease V digestion and deoxyinosine-containing query oligonucleotides

    Directory of Open Access Journals (Sweden)

    Ho Antoine

    2011-12-01

    Full Text Available Abstract Background Sequencing-by-ligation (SBL is one of several next-generation sequencing methods that has been developed for massive sequencing of DNA immobilized on arrayed beads (or other clonal amplicons. SBL has the advantage of being easy to implement and accessible to all because it can be performed with off-the-shelf reagents. However, SBL has the limitation of very short read lengths. Results To overcome the read length limitation, research groups have developed complex library preparation processes, which can be time-consuming, difficult, and result in low complexity libraries. Herein we describe a variation on traditional SBL protocols that extends the number of sequential bases that can be sequenced by using Endonuclease V to nick a query primer, thus leaving a ligatable end extended into the unknown sequence for further SBL cycles. To demonstrate the protocol, we constructed a known DNA sequence and utilized our SBL variation, cyclic SBL (cSBL, to resequence this region. Using our method, we were able to read thirteen contiguous bases in the 3' - 5' direction. Conclusions Combining this read length with sequencing in the 5' - 3' direction would allow a read length of over twenty bases on a single tage. Implementing mate-paired tags and this SBL variation could enable > 95% coverage of the genome.

  2. Structure-guided sequence specificity engineering of the modification-dependent restriction endonuclease LpnPI.

    Science.gov (United States)

    Sasnauskas, Giedrius; Zagorskaitė, Evelina; Kauneckaitė, Kotryna; Tamulaitiene, Giedre; Siksnys, Virginijus

    2015-07-13

    The eukaryotic Set and Ring Associated (SRA) domains and structurally similar DNA recognition domains of prokaryotic cytosine modification-dependent restriction endonucleases recognize methylated, hydroxymethylated or glucosylated cytosine in various sequence contexts. Here, we report the apo-structure of the N-terminal SRA-like domain of the cytosine modification-dependent restriction enzyme LpnPI that recognizes modified cytosine in the 5'-C(mC)DG-3' target sequence (where mC is 5-methylcytosine or 5-hydroxymethylcytosine and D = A/T/G). Structure-guided mutational analysis revealed LpnPI residues involved in base-specific interactions and demonstrated binding site plasticity that allowed limited target sequence degeneracy. Furthermore, modular exchange of the LpnPI specificity loops by structural equivalents of related enzymes AspBHI and SgrTI altered sequence specificity of LpnPI. Taken together, our results pave the way for specificity engineering of the cytosine modification-dependent restriction enzymes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. High pressure activation of the Mrr restriction endonuclease in Escherichia coli involves tetramer dissociation.

    Science.gov (United States)

    Bourges, Anaïs C; Torres Montaguth, Oscar E; Ghosh, Anirban; Tadesse, Wubishet M; Declerck, Nathalie; Aertsen, Abram; Royer, Catherine A

    2017-05-19

    A sub-lethal hydrostatic pressure (HP) shock of ∼100 MPa elicits a RecA-dependent DNA damage (SOS) response in Escherichia coli K-12, despite the fact that pressure cannot compromise the covalent integrity of DNA. Prior screens for HP resistance identified Mrr (Methylated adenine Recognition and Restriction), a Type IV restriction endonuclease (REase), as instigator for this enigmatic HP-induced SOS response. Type IV REases tend to target modified DNA sites, and E. coli Mrr activity was previously shown to be elicited by expression of the foreign M.HhaII Type II methytransferase (MTase), as well. Here we measured the concentration and stoichiometry of a functional GFP-Mrr fusion protein using in vivo fluorescence fluctuation microscopy. Our results demonstrate that Mrr is a tetramer in unstressed cells, but shifts to a dimer after HP shock or co-expression with M.HhaII. Based on the differences in reversibility of tetramer dissociation observed for wild-type GFP-Mrr and a catalytic mutant upon HP shock compared to M.HhaII expression, we propose a model by which (i) HP triggers Mrr activity by directly pushing inactive Mrr tetramers to dissociate into active Mrr dimers, while (ii) M.HhaII triggers Mrr activity by creating high affinity target sites on the chromosome, which pull the equilibrium from inactive tetrameric Mrr toward active dimer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. The dynamics of the monomeric restriction endonuclease BcnI during its interaction with DNA.

    Science.gov (United States)

    Kostiuk, Georgij; Dikic, Jasmina; Schwarz, Friedrich W; Sasnauskas, Giedrius; Seidel, Ralf; Siksnys, Virginijus

    2017-06-02

    Endonucleases that generate DNA double strand breaks often employ two independent subunits such that the active site from each subunit cuts either DNA strand. Restriction enzyme BcnI is a remarkable exception. It binds to the 5΄-CC/SGG-3΄ (where S = C or G, '/' designates the cleavage position) target as a monomer forming an asymmetric complex, where a single catalytic center approaches the scissile phosphodiester bond in one of DNA strands. Bulk kinetic measurements have previously shown that the same BcnI molecule cuts both DNA strands at the target site without dissociation from the DNA. Here, we analyse the BcnI DNA binding and target recognition steps at the single molecule level. We find, using FRET, that BcnI adopts either 'open' or 'closed' conformation in solution. Next, we directly demonstrate that BcnI slides over long distances on DNA using 1D diffusion and show that sliding is accompanied by occasional jumping events, where the enzyme leaves the DNA and rebinds immediately at a distant site. Furthermore, we quantify the dynamics of the BcnI interactions with cognate and non-cognate DNA, and determine the preferred binding orientation of BcnI to the target site. These results provide new insights into the intricate dynamics of BcnI-DNA interactions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Crystallization and preliminary X-ray diffraction analysis of restriction endonuclease EcoRII

    Science.gov (United States)

    Karpova, E. A.; Meehan, E.; Pusey, M. L.; Chen, L.

    1999-01-01

    Crystals of the restriction endonuclease EcoRII have been obtained by the vapor-diffusion technique in the presence of ammonium sulfate or polyethylene glycol. The best crystals were grown with ammonium sulfate as a precipitant. Crystals with dimensions of up to 0.6 x 0. 6 x 0.6 mm have been observed. The crystals diffract to about 4.0 A resolution at a cryo-temperature of 100 K using a rotating-anode X-ray source and a Rigaku R-AXIS IV imaging-plate detector. The space group has been determined to be either I23 or I2(1)3, with unit-cell parameters a = b = c = 160.3 A, alpha = beta = gamma = 90 degrees. The crystal asymmetric unit contains two protein molecules, and self-rotation function analysis shows a pseudo-twofold symmetry relating the two monomers. Attempts to improve the resolution of crystal diffraction and to search for heavy-atom derivatives are under way.

  6. 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.

  7. 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.

  8. 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.

    2014-01-01

    The 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. 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 employs PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate dsDNA scission. PMID:24476820

  9. Restriction endonuclease triggered bacterial apoptosis as a mechanism for long time survival.

    Science.gov (United States)

    Nagamalleswari, Easa; Rao, Sandhya; Vasu, Kommireddy; Nagaraja, Valakunja

    2017-08-21

    Programmed cell death (PCD) under certain conditions is one of the features of bacterial altruism. Given the bacterial diversity and varied life style, different PCD mechanisms must be operational that remain largely unexplored. We describe restriction endonuclease (REase) mediated cell death by an apoptotic pathway, beneficial for isogenic bacterial communities. Cell death is pronounced in stationary phase and when the enzyme exhibits promiscuous DNA cleavage activity. We have elucidated the molecular mechanism of REase mediated cell killing and demonstrate that released nutrients from dying cells support the growth of the remaining cells in the population. These findings illustrate a new intracellular moonlighting role for REases which are otherwise established host defence arsenals. REase induced PCD appears to be a cellular design to replenish nutrients for cells undergoing starvation stress and the phenomenon could be wide spread in bacteria, given the abundance of restriction-modification (R-M) systems in the microbial population. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. 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.

  11. One-tube loop-mediated isothermal amplification combined with restriction endonuclease digestion and ELISA for colorimetric detection of resistance to isoniazid, ethambutol and streptomycin in Mycobacterium tuberculosis isolates.

    Science.gov (United States)

    Lee, Mei-Feng; Chen, Yen-Hsu; Hsu, Hui-Jine; Peng, Chien-Fang

    2010-10-01

    In this study, we designed a simple and rapid colorimetric detection method, a one-tube loop-mediated isothermal amplification (LAMP)-PCR-hybridization-restriction endonuclease-ELISA [one-tube LAMP-PCR-HY-RE-ELISA] system, to detect resistance to isoniazid, ethambutol and streptomycin in strains of Mycobacterium tuberculosis isolated from clinical specimens. The clinical performance of this method for detecting isoniazid-resistant, ethambutol-resistant and streptomycin-resistant isolates of M. tuberculosis showed 98.9%, 94.3% and 93.8%, respectively. This assay is rapid and convenient that can be performed within one working day. One-tube LAMP-PCR-HY-RE-ELISA system was designed based on hot spot point mutations in target drug-resistant genes, using LAMP-PCR, hybridization, digestion with restriction endonuclease and colorimetric method of ELISA. In this study, LAMP assay was used to amplify DNA from drug-resistant M. tuberculosis, and ELISA was used for colorimetrical determination. This assay will be a useful tool for rapid diagnosis of mutant codons in strains of M. tuberculosis for isoniazid at katG 315 and katG 463, ethambutol at embB 306 and embB 497, and streptomycin at rpsL 43. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Stella, Stefano; Alcón, Pablo; Montoya, Guillermo

    2017-06-22

    Cpf1 is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we provide insight into its DNA-targeting mechanism by determining the structure of Francisella novicida Cpf1 with the triple-stranded R-loop generated after DNA cleavage. The structure reveals the machinery 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 into the double-stranded DNA. Unzipping of the double-stranded DNA occurs in a cleft arranged by acidic and hydrophobic residues facilitating the crRNA-DNA hybrid formation. The PAM single-stranded DNA is funnelled towards the nuclease site through a mixed hydrophobic and basic cavity. In this catalytic conformation, the PAM-interacting domain and the helix-loop-helix motif in the REC1 domain adopt a 'rail' shape and 'flap-on' conformations, respectively, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms the 'septum', separating the displaced PAM strand 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.

  13. Three Metal Ions Participate in the Reaction Catalyzed by T5 Flap Endonuclease*S⃞

    Science.gov (United States)

    Syson, Karl; Tomlinson, Christopher; Chapados, Brian R.; Sayers, Jon R.; Tainer, John A.; Williams, Nicholas H.; Grasby, Jane A.

    2008-01-01

    Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the rapid hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are widely proposed to catalyze phosphate diester hydrolysis using a “two-metal-ion mechanism.” Yet, analyses of flap endonuclease (FEN) family members, which occur in all domains of life and act in DNA replication and repair, exemplify controversies regarding the classical two-metal-ion mechanism for phosphate diester hydrolysis. Whereas substrate-free structures of FENs identify two active site metal ions, their typical separation of >4 Å appears incompatible with this mechanism. To clarify the roles played by FEN metal ions, we report here a detailed evaluation of the magnesium ion response of T5FEN. Kinetic investigations reveal that overall the T5FEN-catalyzed reaction requires at least three magnesium ions, implying that an additional metal ion is bound. The presence of at least two ions bound with differing affinity is required to catalyze phosphate diester hydrolysis. Analysis of the inhibition of reactions by calcium ions is consistent with a requirement for two viable cofactors (Mg2+ or Mn2+). The apparent substrate association constant is maximized by binding two magnesium ions. This may reflect a metal-dependent unpairing of duplex substrate required to position the scissile phosphate in contact with metal ion(s). The combined results suggest that T5FEN primarily uses a two-metal-ion mechanism for chemical catalysis, but that its overall metallobiochemistry is more complex and requires three ions. PMID:18697748

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Extrahelical (CAG)/(CTG) triplet repeat elements support proliferating cell nuclear antigen loading and MutLα endonuclease activation

    Science.gov (United States)

    Pluciennik, Anna; Burdett, Vickers; Baitinger, Celia; Iyer, Ravi R.; Shi, Kevin; Modrich, Paul

    2013-01-01

    MutLα endonuclease can be activated on covalently continuous DNA that contains a MutSα- or MutSβ-recognizable lesion and a helix perturbation that supports proliferating cell nuclear antigen (PCNA) loading by replication factor C, providing a potential mechanism for triggering mismatch repair on nonreplicating DNA. Because mouse models for somatic expansion of disease-associated (CAG)n/(CTG)n triplet repeat sequences have implicated both MutSβ and MutLα and have suggested that expansions can occur in the absence of replication, we have asked whether an extrahelical (CAG)n or (CTG)n element is sufficient to trigger MutLα activation. (CAG)n and (CTG)n extrusions in relaxed closed circular DNA do in fact support MutSβ-, replication factor C-, and PCNA-dependent activation of MutLα endonuclease, which can incise either DNA strand. Extrahelical elements of two or three repeat units are the preferred substrates for MutLα activation, and extrusions of this size also serve as moderately effective sites for loading the PCNA clamp. Relaxed heteroduplex DNA containing a two or three-repeat unit extrusion also triggers MutSβ- and MutLα-endonuclease-dependent mismatch repair in nuclear extracts of human cells. This reaction occurs without obvious strand bias at about 10% the rate of that observed with otherwise identical nicked heteroduplex DNA. These findings provide a mechanism for initiation of triplet repeat processing in nonreplicating DNA that is consistent with several features of the model of Gomes-Pereira et al. [Gomes-Pereira M, Fortune MT, Ingram L, McAbney JP, Monckton DG (2004) Hum Mol Genet 13(16):1815–1825]. They may also have implications for triplet repeat processing at a replication fork. PMID:23840062

  16. The PA Endonuclease Inhibitor RO-7 Protects Mice from Lethal Challenge with Influenza A or B Viruses.

    Science.gov (United States)

    Jones, Jeremy C; Marathe, Bindumadhav M; Vogel, Peter; Gasser, Rodolfo; Najera, Isabel; Govorkova, Elena A

    2017-05-01

    Current influenza treatment relies on a single class of antiviral drugs, the neuraminidase inhibitors (NAIs), raising concern over the potential emergence of resistant variants and necessitating the development of novel drugs. In recent years, investigational inhibitors targeting the endonuclease activity of the influenza acidic polymerase (PA) protein have yielded encouraging results, although there are only limited data on their in vivo efficacy. Here, we examined the antiviral potential of the PA endonuclease inhibitor RO-7 in prophylactic and therapeutic regimens in BALB/c mice inoculated with influenza A/California/04/2009 (H1N1)pdm09 or B/Brisbane/60/2008 viruses, which represent currently circulating antigenic variants. RO-7 was administered to mice intraperitoneally twice daily at dosages of 6, 15, or 30 mg/kg/day for 5 days, starting 4 h before or 24 or 48 h after virus inoculation, and showed no adverse effects. Prophylactic administration completely protected mice from lethal infection by influenza A or B virus. The level of therapeutic protection conferred depended upon the time of treatment initiation and RO-7 dosage, resulting in 60 to 100% and 80 to 100% survival with influenza A and B viruses, respectively. RO-7 treatment significantly decreased virus titers in the lung and lessened the extent and severity of lung damage. No PA endonuclease-inhibitor resistance was observed in viruses isolated from lungs of RO-7-treated mice, and the viruses remained susceptible to the drug at nanomolar concentrations in phenotypic assays. These in vivo efficacy results further highlight the potential of RO-7 for development as antiviral therapy for influenza A and B virus infections. Copyright © 2017 American Society for Microbiology.

  17. Type II restriction endonuclease R.Eco29kI is a member of the GIY-YIG nuclease superfamily

    Directory of Open Access Journals (Sweden)

    Feder Marcin

    2007-07-01

    Full Text Available Abstract Background The majority of experimentally determined crystal structures of Type II restriction endonucleases (REases exhibit a common PD-(D/EXK fold. Crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI and half-pipe (R.PabI, and bioinformatics analyses supported by mutagenesis suggested that some REases belong to the HNH fold. Our previous bioinformatic analysis suggested that REase R.Eco29kI shares sequence similarities with one more unrelated nuclease superfamily, GIY-YIG, however so far no experimental data were available to support this prediction. The determination of a crystal structure of the GIY-YIG domain of homing endonuclease I-TevI provided a template for modeling of R.Eco29kI and prompted us to validate the model experimentally. Results Using protein fold-recognition methods we generated a new alignment between R.Eco29kI and I-TevI, which suggested a reassignment of one of the putative catalytic residues. A theoretical model of R.Eco29kI was constructed to illustrate its predicted three-dimensional fold and organization of the active site, comprising amino acid residues Y49, Y76, R104, H108, E142, and N154. A series of mutants was constructed to generate amino acid substitutions of selected residues (Y49A, R104A, H108F, E142A and N154L and the mutant proteins were examined for their ability to bind the DNA containing the Eco29kI site 5'-CCGCGG-3' and to catalyze the cleavage reaction. Experimental data reveal that residues Y49, R104, E142, H108, and N154 are important for the nuclease activity of R.Eco29kI, while H108 and N154 are also important for specific DNA binding by this enzyme. Conclusion Substitutions of residues Y49, R104, H108, E142 and N154 predicted by the model to be a part of the active site lead to mutant proteins with strong defects in the REase activity. These results are in very good agreement with the structural model presented in this work and with our

  18. The human Rad9–Rad1–Hus1 checkpoint complex stimulates flap endonuclease 1

    OpenAIRE

    Wang, Wensheng; Brandt, Patrick; Rossi, Marie L.; Lindsey-Boltz, Laura; Podust, Vladimir; Fanning, Ellen; Sancar, Aziz; Bambara, Robert A.

    2004-01-01

    The toroidal damage checkpoint complex Rad9–Rad1–Hus1 (9-1-1) has been characterized as a sensor of DNA damage. Flap endonuclease 1 (FEN1) is a structure-specific nuclease involved both in removing initiator RNA from Okazaki fragments and in DNA repair pathways. FEN1 activity is stimulated by proliferating cell nuclear antigen (PCNA), a toroidal sliding clamp that acts as a platform for DNA replication and repair complexes. We show that 9-1-1 also binds and stimulates FEN1. Stimulation is obs...

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

    Science.gov (United States)

    Andersen, Jeppe D; Pereira, Vania; Pietroni, Carlotta; Mikkelsen, Martin; Johansen, Peter; Børsting, Claus; Morling, Niels

    2014-08-01

    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.

  20. High-resolution crystal structure reveals a HEPN domain at the C-terminal region of S. cerevisiae RNA endonuclease Swt1

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Shuxia, E-mail: pengsx@ihep.ac.cn; Zhou, Ke; Wang, Wenjia; Gao, Zengqiang; Dong, Yuhui; Liu, Quansheng

    2014-10-31

    Highlights: • Crystal structure of the C-terminal (CT) domain of Swt1 was determined at 2.3 Å. • Structure of the CT domain was identified as HEPN domain superfamily member. • Low-resolution envelope of Swt1 full-length in solution was analyzed by SAXS. • The middle and CT domains gave good fit to SAXS structural model. - Abstract: Swt1 is an RNA endonuclease that plays an important role in quality control of nuclear messenger ribonucleoprotein particles (mRNPs) in eukaryotes; however, its structural details remain to be elucidated. Here, we report the crystal structure of the C-terminal (CT) domain of Swt1 from Saccharomyces cerevisiae, which shares common characteristics of higher eukaryotes and prokaryotes nucleotide binding (HEPN) domain superfamily. To study in detail the full-length protein structure, we analyzed the low-resolution architecture of Swt1 in solution using small angle X-ray scattering (SAXS) method. Both the CT domain and middle domain exhibited a good fit upon superimposing onto the molecular envelope of Swt1. Our study provides the necessary structural information for detailed analysis of the functional role of Swt1, and its importance in the process of nuclear mRNP surveillance.

  1. Epidemiologic study of Taylorella equigenitalis strains by field inversion gel electrophoresis of genomic restriction endonuclease fragments.

    Science.gov (United States)

    Bleumink-Pluym, N; ter Laak, E A; van der Zeijst, B A

    1990-09-01

    Contagious equine metritis (CEM), a sexually transmitted bacterial disease, was first described in thoroughbred horses. It also occurs in nonthoroughbred horses, in which it produces isolated, apparently unrelated outbreaks. Thirty-two strains of Taylorella equigenitalis, the causative agent of CEM, from all over the world were characterized by field inversion gel electrophoresis of fragments of genomic DNA obtained by digestion with low-cleavage-frequency restriction enzymes. This resulted in a division into five clearly distinct groups. Strains from thoroughbred horses from all continents belonged to one group. Strains from nonthoroughbred horses from various countries were different from strains from thoroughbred horses; four groups could be determined. Two groups contained both streptomycin-resistant and streptomycin-susceptible strains. The data indicate that CEM in nonthoroughbreds did not originate from the thoroughbred population; also, the reverse was not demonstrated. Thus, extensive international transportation directives regarding the testing of nonthoroughbred horses for CEM may need reconsideration.

  2. Association of thymine glycol lesioned DNA with repair enzyme endonuclease III-molecular dynamics study

    International Nuclear Information System (INIS)

    Pinak, Miroslav

    2001-07-01

    The 2 nanoseconds molecular dynamics (MD) simulation has been performed for the system consisting of repair enzyme and DNA 30-mer with native thymine at position 16 replaced by thymine glycol (TG) solvated in water environment. After 950 picoseconds of MD the enzyme and DNA associated together to form complex that lasted stable up to 2 ns when simulation was terminated. At the contact area of enzyme and DNA there is glutamic acid located as close as 1.6 A to the C3' atom of phosphodiester bond of TG. Initial B-DNA molecule was bent and kinked at the TG during MD. This distortion caused that phosphodiester bond was easier accessible by amino acids of enzyme. The negative value of electrostatic energy (-26 kcal/mol) discriminates TG from nearly neutral native thymine and contributes to the specific recognition of this lesion. Higher number of close water molecules at TG site before formation of complex (compared with other nucleotides) indicates that glycosyl bond of the lesion is easily approached by repair enzyme during scanning of DNA surface and suggests the importance of specific hydration at the lesion during recognition process. (author)

  3. Creation of a novel telomere-cutting endonuclease based on the EN domain of telomere-specific non-long terminal repeat retrotransposon, TRAS1

    Directory of Open Access Journals (Sweden)

    Yoshitake Kazutoshi

    2010-04-01

    Full Text Available Abstract Background The ends of chromosomes, termed telomeres consist of repetitive DNA. The telomeric sequences shorten with cell division and, when telomeres are critically abbreviated, cells stop proliferating. However, in cancer cells, by the expression of telomerase which elongates telomeres, the cells can continue proliferating. Many approaches for telomere shortening have been pursued in the past, but to our knowledge, cutting telomeres in vivo has not so far been demonstrated. In addition, there is lack of information on the cellular effects of telomere shortening in human cells. Results Here, we created novel chimeric endonucleases to cut telomeres by fusing the endonuclease domain (TRAS1EN of the silkworm's telomere specific non-long terminal repeat retrotransposon TRAS1 to the human telomere-binding protein, TRF1. An in vitro assay demonstrated that the TRAS1EN-TRF1 chimeric endonucleases (T-EN and EN-T cut the human (TTAGGGn repeats specifically. The concentration of TRAS1EN-TRF1 chimeric endonucleases necessary for the cleavage of (TTAGGGn repeats was about 40-fold lower than that of TRAS1EN alone. When TRAS1EN-TRF1 endonucleases were introduced into human U2OS cancer cells using adenovirus vectors, the enzymes localized at telomeres of nuclei, cleaved and shortened the telomeric DNA by double-strand breaks. When human U2OS and HFL-1 fibroblast cells were infected with EN-T recombinant adenovirus, their cellular proliferation was suppressed for about 2 weeks after infection. In contrast, the TRAS1EN mutant (H258A chimeric endonuclease fused with TRF1 (ENmut-T did not show the suppression effect. The EN-T recombinant adenovirus induced telomere shortening in U2OS cells, activated the p53-dependent pathway and caused the senescence associated cellular responses, while the ENmut-T construct did not show such effects. Conclusions A novel TRAS1EN-TRF1 chimeric endonuclease (EN-T cuts the human telomeric repeats (TTAGGGn specifically in

  4. DNA Polymerase α (swi7) and the Flap Endonuclease Fen1 (rad2) Act Together in the S-Phase Alkylation Damage Response in S. pombe

    Science.gov (United States)

    Koulintchenko, Milana; Vengrova, Sonya; Eydmann, Trevor; Arumugam, Prakash; Dalgaard, Jacob Z.

    2012-01-01

    Polymerase α is an essential enzyme mainly mediating Okazaki fragment synthesis during lagging strand replication. A specific point mutation in Schizosaccharomyces pombe polymerase α named swi7-1, abolishes imprinting required for mating-type switching. Here we investigate whether this mutation confers any genome-wide defects. We show that the swi7-1 mutation renders cells hypersensitive to the DNA damaging agents methyl methansulfonate (MMS), hydroxyurea (HU) and UV and incapacitates activation of the intra-S checkpoint in response to DNA damage. In addition we show that, in the swi7-1 background, cells are characterized by an elevated level of repair foci and recombination, indicative of increased genetic instability. Furthermore, we detect novel Swi1-, -Swi3- and Pol α- dependent alkylation damage repair intermediates with mobility on 2D-gel that suggests presence of single-stranded regions. Genetic interaction studies showed that the flap endonuclease Fen1 works in the same pathway as Pol α in terms of alkylation damage response. Fen1 was also required for formation of alkylation- damage specific repair intermediates. We propose a model to explain how Pol α, Swi1, Swi3 and Fen1 might act together to detect and repair alkylation damage during S-phase. PMID:23071723

  5. Use of DNA restriction endonuclease digest and ribosomal RNA gene probe patterns to fingerprint Helicobacter pylori and Helicobacter mustelae isolated from human and animal hosts.

    Science.gov (United States)

    Morgan, D D; Owen, R J

    1990-08-01

    Variation amongst strains of Helicobacter pylori and Helicobacter mustelae was examined by DNA restriction endonuclease digestion and rRNA gene patterns generated using a non-radioactive probe. Chromosomal DNA was extracted from 30 cultures of H. pylori from human, Rhesus monkey and pig gastric mucosa, and from three H. mustelae isolates from ferret gastric mucosa. DNA fingerprinting with Hae III and Hind III showed H. mustelae was relatively homogeneous but revealed genomic heterogeneity within H. pylori with at least 18 different DNA patterns identifiable amongst the 30 isolates. Five sets of strains other than duplicates with matching DNA fingerprints were identified within H. pylori. The Peruvian isolates were the largest identical set and comprised eight isolates from four different patients with five consecutive isolates from one patient. The Rhesus monkey strains were a relatively homogeneous set as were several Australian human isolates. The study demonstrates that rRNA gene restriction patterns provide a simple but highly discriminatory electrophoretic fingerprint for H. pylori with potential for use as a novel epidemiological marker in addition to total DNA digest analysis.

  6. AKT2 Blocks Nucleus Translocation of Apoptosis-Inducing Factor (AIF and Endonuclease G (EndoG While Promoting Caspase Activation during Cardiac Ischemia

    Directory of Open Access Journals (Sweden)

    Shuai Yang

    2017-03-01

    Full Text Available The AKT (protein kinase B, PKB family has been shown to participate in diverse cellular processes, including apoptosis. Previous studies demonstrated that protein kinase B2 (AKT2−/− mice heart was sensitized to apoptosis in response to ischemic injury. However, little is known about the mechanism and apoptotic signaling pathway. Here, we show that AKT2 inhibition does not affect the development of cardiomyocytes but increases cell death during cardiomyocyte ischemia. Caspase-dependent apoptosis of both the extrinsic and intrinsic pathway was inactivated in cardiomyocytes with AKT2 inhibition during ischemia, while significant mitochondrial disruption was observed as well as intracytosolic translocation of cytochrome C (Cyto C together with apoptosis-inducing factor (AIF and endonuclease G (EndoG, both of which are proven to conduct DNA degradation in a range of cell death stimuli. Therefore, mitochondria-dependent cell death was investigated and the results suggested that AIF and EndoG nucleus translocation causes cardiomyocyte DNA degradation during ischemia when AKT2 is blocked. These data are the first to show a previous unrecognized function and mechanism of AKT2 in regulating cardiomyocyte survival during ischemia by inducing a unique mitochondrial-dependent DNA degradation pathway when it is inhibited.

  7. Resolution of the EcoRII restriction endonuclease-DNA complex structure in solution using fluorescence spectroscopy.

    Science.gov (United States)

    Subach, Fedor; Kirsanova, Olga; Liquier, Jean; Gromova, Elizaveta S

    2008-12-01

    The X-ray structure for the type IIE EcoRII restriction endonuclease has been resolved [X.E. Zhou, Y. Wang, M. Reuter, M. Mucke, D.H. Kruger, E.J. Meehan and L. Chen. Crystal structure of type IIE restriction endonuclease EcoRII reveals an autoinhibition mechanism by a novel effector-binding fold. J. Mol. Biol. 335 (2004) 307-319.], but the structure of the R.EcoRII-DNA complex is still unknown. The aim of this article was to examine the structure of the pre-reactive R.EcoRII-DNA complex in solution by fluorescence spectroscopy. The structure for the R.EcoRII-DNA complex was resolved by determining the fluorescence resonance energy transfer (FRET) between two fluorescent dyes, covalently attached near the EcoRII recognition sites, that were located at opposite ends of a lengthy two-site DNA molecule. Analysis of the FRET data from the two-site DNA revealed a likely model for the arrangement of the two EcoRII recognition sites relative to each other in the R.EcoRII-DNA complex in the presence of Ca(2+) ions. According to this model, the R.EcoRII binds the two-site DNA and forms a DNA loop in which the EcoRII recognition sites are 20+/-10 A distant to each other and situated at an angle of 70+/-10 degrees.

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

    Directory of Open Access Journals (Sweden)

    Josep M. Comeron

    2016-04-01

    Full Text Available 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.

  9. 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.

  10. Functional significance of protein assemblies predicted by the crystal structure of the restriction endonuclease BsaWI.

    Science.gov (United States)

    Tamulaitis, Gintautas; Rutkauskas, Marius; Zaremba, Mindaugas; Grazulis, Saulius; Tamulaitiene, Giedre; Siksnys, Virginijus

    2015-09-18

    Type II restriction endonuclease BsaWI recognizes a degenerated sequence 5'-W/CCGGW-3' (W stands for A or T, '/' denotes the cleavage site). It belongs to a large family of restriction enzymes that contain a conserved CCGG tetranucleotide in their target sites. These enzymes are arranged as dimers or tetramers, and require binding of one, two or three DNA targets for their optimal catalytic activity. Here, we present a crystal structure and biochemical characterization of the restriction endonuclease BsaWI. BsaWI is arranged as an 'open' configuration dimer and binds a single DNA copy through a minor groove contacts. In the crystal primary BsaWI dimers form an indefinite linear chain via the C-terminal domain contacts implying possible higher order aggregates. We show that in solution BsaWI protein exists in a dimer-tetramer-oligomer equilibrium, but in the presence of specific DNA forms a tetramer bound to two target sites. Site-directed mutagenesis and kinetic experiments show that BsaWI is active as a tetramer and requires two target sites for optimal activity. We propose BsaWI mechanism that shares common features both with dimeric Ecl18kI/SgrAI and bona fide tetrameric NgoMIV/SfiI enzymes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Catalytic and noncatalytic roles of the CtIP endonuclease in double-strand break end resection.

    Science.gov (United States)

    Makharashvili, Nodar; Tubbs, Anthony T; Yang, Soo-Hyun; Wang, Hailong; Barton, Olivia; Zhou, Yi; Deshpande, Rajashree A; Lee, Ji-Hoon; Lobrich, Markus; Sleckman, Barry P; Wu, Xiaohua; Paull, Tanya T

    2014-06-19

    The carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) is known to function in 5' strand resection during homologous recombination, similar to the budding yeast Sae2 protein, but its role in this process is unclear. Here, we characterize recombinant human CtIP and find that it exhibits 5' flap endonuclease activity on branched DNA structures, independent of the MRN complex. Phosphorylation of CtIP at known damage-dependent sites and other sites is essential for its catalytic activity, although the S327 and T847 phosphorylation sites are dispensable. A catalytic mutant of CtIP that is deficient in endonuclease activity exhibits wild-type levels of homologous recombination at restriction enzyme-generated breaks but is deficient in processing topoisomerase adducts and radiation-induced breaks in human cells, suggesting that the nuclease activity of CtIP is specifically required for the removal of DNA adducts at sites of DNA breaks. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. 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.

  13. Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA.

    Directory of Open Access Journals (Sweden)

    Guang Liu

    2010-12-01

    Full Text Available Many taxonomically diverse prokaryotes enzymatically modify their DNA by replacing a non-bridging oxygen with a sulfur atom at specific sequences. The biological implications of this DNA S-modification (phosphorothioation were unknown. We observed that simultaneous expression of the dndA-E gene cluster from Streptomyces lividans 66, which is responsible for the DNA S-modification, and the putative Streptomyces coelicolor A(32 Type IV methyl-dependent restriction endonuclease ScoA3McrA (Sco4631 leads to cell death in the same host. A His-tagged derivative of ScoA3McrA cleaved S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Double-strand cleavage occurred 16-28 nucleotides away from the phosphorothioate links. DNase I footprinting demonstrated binding of ScoA3McrA to the Dcm methylation site, but no clear binding could be detected at the S-modified site under cleavage conditions. This is the first report of in vitro endonuclease activity of a McrA homologue and also the first demonstration of an enzyme that specifically cleaves S-modified DNA.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Continuous monitoring of restriction endonuclease cleavage activity by universal molecular beacon light quenching coupled with real-time polymerase chain reaction.

    Science.gov (United States)

    Li, Xiaomin; Song, Chen; Zhao, Meiping; Li, Yuanzong

    2008-10-01

    We describe a method for sensitive monitoring of restriction endonuclease kinetics and activity by use of a universal molecular beacon (U-MB) coupled with real-time polymerase chain reaction (PCR). The method is used to monitor the progress of DNA cleavage in a sealed reaction tube and offers more accurate and high-throughput detection. The template has a universal tail hybridized with the U-MB and the remaining sequence is complementary to one of the restriction endonuclease digestion products. The U-MB is replaced by the extension of digested product and the fluorescence quenches. With this concept, one universal fluorescence probe can be used in different enzyme analytical systems. In the work described here, homogenous assays were performed with the restriction endonucleases AluI, EcoRI, XhoI, and SacI at smoothly controlled temperature. Cleavage efficiencies were determined, and the potential applications of this method were discussed. Furthermore, the AluI and EcoRI cleavage reactions were monitored online at varying substrate concentrations at the molecular level, and K(m), V(max), and K(cat) values were calculated. The results suggest that U-MB monitoring of restriction endonuclease assays based on real-time PCR will be very useful for high-throughput, sensitive, and precise assays for enzyme activity screening and evolutionary biotechnology analysis.

  16. 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

  17. Defective mitochondrial respiration, altered dNTP pools and reduced AP endonuclease 1 activity in peripheral blood mononuclear cells of Alzheimer's disease patients

    DEFF Research Database (Denmark)

    Maynard, Scott; Hejl, Anne-Mette; Dinh, Thuan-Son T

    2015-01-01

    , and APE1 incision activity (in cell lysates) on a DNA substrate containing an AP site (to estimate DNA repair efficiency). RESULTS: In the PBMCs of AD patients, we found reduced basal mitochondrial oxygen consumption, reduced proton leak, higher dATP level, and lower AP endonuclease 1 activity, depending...

  18. Nonenzymatic release of N7-methylguanine channels repair of abasic sites into an AP endonuclease-independent pathway inArabidopsis.

    Science.gov (United States)

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

    2018-01-30

    Abasic (apurinic/apyrimidinic, AP) sites in DNA arise from spontaneous base loss or by enzymatic removal during base excision repair. It is commonly accepted that both classes of AP site have analogous biochemical properties and are equivalent substrates for AP endonucleases and AP lyases, although the relative roles of these two types of enzymes are not well understood. We provide here genetic and biochemical evidence that, in Arabidopsis , AP sites generated by spontaneous loss of N7-methylguanine (N7-meG) are exclusively repaired through an AP endonuclease-independent pathway initiated by FPG, a bifunctional DNA glycosylase with AP lyase activity. Abasic site incision catalyzed by FPG generates a single-nucleotide gap with a 3'-phosphate terminus that is processed by the DNA 3'-phosphatase ZDP before repair is completed. We further show that the major AP endonuclease in Arabidopsis (ARP) incises AP sites generated by enzymatic N7-meG excision but, unexpectedly, not those resulting from spontaneous N7-meG loss. These findings, which reveal previously undetected differences between products of enzymatic and nonenzymatic base release, may shed light on the evolution and biological roles of AP endonucleases and AP lyases. Copyright © 2018 the Author(s). Published by PNAS.

  19. Structures of the rare-cutting restriction endonuclease NotI reveal a unique metal binding fold involved in DNA binding.

    Science.gov (United States)

    Lambert, Abigail R; Sussman, Django; Shen, Betty; Maunus, Robert; Nix, Jay; Samuelson, James; Xu, Shuang-Yong; Stoddard, Barry L

    2008-04-01

    The structure of the rare-cutting restriction endonuclease NotI, which recognizes the 8 bp target 5'-GCGGCCGC-3', has been solved with and without bound DNA. Because of its specificity (recognizing a site that occurs once per 65 kb), NotI is used to generate large genomic fragments and to map DNA methylation status. NotI contains a unique metal binding fold, found in a variety of putative endonucleases, occupied by an iron atom coordinated within a tetrahedral Cys4 motif. This domain positions nearby protein elements for DNA recognition, and serves a structural role. While recognition of the central six base pairs of the target is accomplished via a saturated hydrogen bond network typical of restriction enzymes, the most peripheral base pairs are engaged in a single direct contact in the major groove, reflecting reduced pressure to recognize those positions. NotI may represent an evolutionary intermediate between mobile endonucleases (which recognize longer target sites) and canonical restriction endonucleases.

  20. DNA recognition by the SwaI restriction endonuclease involves unusual distortion of an 8 base pair A:T-rich target.

    Science.gov (United States)

    Shen, Betty W; Heiter, Daniel F; Lunnen, Keith D; Wilson, Geoffrey G; Stoddard, Barry L

    2017-02-17

    R.SwaI, a Type IIP restriction endonuclease, recognizes a palindromic eight base pair (bp) symmetric sequence, 5΄-ATTTAAAT-3΄, and cleaves that target at its center to generate blunt-ended DNA fragments. Here, we report three crystal structures of SwaI: unbound enzyme, a DNA-bound complex with calcium ions; and a DNA-bound, fully cleaved complex with magnesium ions. We compare these structures to two structurally similar ‘PD-D/ExK’ restriction endonucleases (EcoRV and HincII) that also generate blunt-ended products, and to a structurally distinct enzyme (the HNH endonuclease PacI) that also recognizes an 8-bp target site consisting solely of A:T base pairs. Binding by SwaI induces an extreme bend in the target sequence accompanied by un-pairing and re-ordering of its central A:T base pairs. This result is reminiscent of a more dramatic target deformation previously described for PacI, implying that long A:T-rich target sites might display structural or dynamic behaviors that play a significant role in endonuclease recognition and cleavage.

  1. Role of AP-endonuclease (Ape1) active site residues in stabilization of the reactant enzyme-DNA complex.

    Science.gov (United States)

    Batebi, Hossein; Dragelj, Jovan; Imhof, Petra

    2018-04-01

    Apurinic/apyrimidinic endonuclease 1 (Ape1) is an important metal-dependent enzyme in the base excision repair mechanism, responsible for the backbone cleavage of abasic DNA through a phosphate hydrolysis reaction. Molecular dynamics simulations of Ape1 complexed to its substrate DNA performed for models containing 1 or 2 Mg 2+ -ions as cofactor located at different positions show a complex with 1 metal ion bound on the leaving group site of the scissile phosphate to be the most likely reaction-competent conformation. Active-site residue His309 is found to be protonated based on pKa calculations and the higher conformational stability of the Ape1-DNA substrate complex compared to scenarios with neutral His309. Simulations of the D210N mutant further support the prevalence of protonated His309 and strongly suggest Asp210 as the general base for proton acceptance by a nucleophilic water molecule. © 2018 Wiley Periodicals, Inc.

  2. MmoSTI restriction endonuclease, isolated from Morganella morganii infecting a tropical moth, Actias selene, cleaving 5'-|CCNGG-3' sequences.

    Science.gov (United States)

    Skowron, Marta A; Zebrowska, Joanna; Wegrzyn, Grzegorz; Skowron, Piotr M

    2016-02-01

    A type II restriction endonuclease, MmoSTI, from the pathogenic bacterium Morganella morganii infecting a tropical moth, Actias selene, has been detected and biochemically characterized, as a potential etiological differentiation factor. The described REase recognizes interrupted palindromes, i.e., 5'-CCNGG-3' sequences and cleaves DNA leaving 5-nucleotide (nt) long, single-stranded (ss), 5'-cohesive ends, which was determined by three complementary methods: (i) cleavage of custom and standard DNA substrates, (ii) run-off sequencing of cleavage products, and (iii) shotgun cloning and sequencing of bacteriophage lambda (λ) DNA digested with MmoSTI. MmoSTI, the first 5'-CCNGG-3' REase characterized from M. morganii, is a neoschizomer of ScrFI, which cleaves DNA leaving 1-nt long, ss, 5'-cohesive ends. It is a high-frequency cutter and can be isolated from easily cultured bacteria, thus it can potentially serve as a tool for DNA manipulations.

  3. 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.

  4. 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.

  5. Organ-specific shifts in mtDNA heteroplasmy following systemic delivery of a mitochondria-targeted restriction endonuclease.

    Science.gov (United States)

    Bacman, S R; Williams, S L; Garcia, S; Moraes, C T

    2010-06-01

    Most pathogenic mtDNA mutations are heteroplasmic and there is a clear correlation between high levels of mutated mtDNA in a tissue and pathology. We have found that in vivo double-strand breaks (DSBs) in mtDNA lead to digestion of cleaved mtDNA and replication of residual mtDNA. Therefore, if DSB could be targeted to mutations in mtDNA, mutant genomes could be eliminated and the wild-type mtDNA would repopulate the cells. This can be achieved by using mitochondria-targeted restriction endonucleases as a means to degrade specific mtDNA haplotypes in heteroplasmic cells or tissues. In this work, we investigated the potential of systemic delivery of mitochondria-targeted restriction endonucleases to reduce the proportion of mutant mtDNA in specific tissues. Using the asymptomatic NZB/BALB mtDNA heteroplasmic mouse as a model, we found that a mitochondria-targeted ApaLI (that cleaves BALB mtDNA at a single site and does not cleave NZB mtDNA) increased the proportion of NZB mtDNA in target tissues. This was observed in heart, using a cardiotropic adeno-associated virus type-6 (AAV6) and in liver, using the hepatotropic adenovirus type-5 (Ad5). No mtDNA depletion or loss of cytochrome c oxidase activity was observed in any of these tissues. These results show the potential of systemic delivery of viral vectors to specific organs for the therapeutic application of mitochondria-targeted restriction enzymes in mtDNA disorders.

  6. Crystal structure of the R-protein of the multisubunit ATP-dependent restriction endonuclease NgoAVII.

    Science.gov (United States)

    Tamulaitiene, Giedre; Silanskas, Arunas; Grazulis, Saulius; Zaremba, Mindaugas; Siksnys, Virginijus

    2014-12-16

    The restriction endonuclease (REase) NgoAVII is composed of two proteins, R.NgoAVII and N.NgoAVII, and shares features of both Type II restriction enzymes and Type I/III ATP-dependent restriction enzymes (see accompanying paper Zaremba et al., 2014). Here we present crystal structures of the R.NgoAVII apo-protein and the R.NgoAVII C-terminal domain bound to a specific DNA. R.NgoAVII is composed of two domains: an N-terminal nucleolytic PLD domain; and a C-terminal B3-like DNA-binding domain identified previously in BfiI and EcoRII REases, and in plant transcription factors. Structural comparison of the B3-like domains of R.NgoAVII, EcoRII, BfiI and the plant transcription factors revealed a conserved DNA-binding surface comprised of N- and C-arms that together grip the DNA. The C-arms of R.NgoAVII, EcoRII, BfiI and plant B3 domains are similar in size, but the R.NgoAVII N-arm which makes the majority of the contacts to the target site is much longer. The overall structures of R.NgoAVII and BfiI are similar; however, whilst BfiI has stand-alone catalytic activity, R.NgoAVII requires an auxiliary cognate N.NgoAVII protein and ATP hydrolysis in order to cleave DNA at the target site. The structures we present will help formulate future experiments to explore the molecular mechanisms of intersubunit crosstalk that control DNA cleavage by R.NgoAVII and related endonucleases. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Drosophila DNA polymerase zeta interacts with recombination repair protein 1, the Drosophila homologue of human abasic endonuclease 1.

    Science.gov (United States)

    Takeuchi, Ryo; Ruike, Tatsushi; Nakamura, Ryo-ichi; Shimanouchi, Kaori; Kanai, Yoshihiro; Abe, Yoko; Ihara, Ayumi; Sakaguchi, Kengo

    2006-04-28

    Abasic (AP) sites are a threat to cellular viability and genomic integrity, since they impede transcription and DNA replication. In mammalian cells, DNA polymerase (pol) beta plays an important role in the repair of AP sites. However, it is known that many organisms, including Drosophila melanogaster, do not have a pol beta homologue, and it is unclear how they repair AP sites. Here, we screened for DNA polymerases that interact with the Drosophila AP endonuclease 1 homologue, Rrp1 (recombination repair protein 1), and found that Drosophila pol zeta (Dmpol zeta), DmREV3 and DmREV7 bound to Rrp1 in a protein affinity column. Rrp1 directly interacted with DmREV7 in vitro and in vivo but not with DmREV3. These findings suggest that the DNA polymerase partner for Rrp1 is Dmpol zeta and that this interaction occurs through DmREV7. Interestingly, DmREV7 bound to the N-terminal region of Rrp1, which has no known protein homologue, suggesting that this binding is a species-specific event. Moreover, DmREV7 could stimulate the AP endonuclease activity of Rrp1, but not the 3'-exonuclease activity, and form a homomultimer. DmREV3 could not incorporate nucleotides at the 5'-incised tetrahydrofran sites but did show strand displacement activity for one-nucleotide-gapped DNA, which was not influenced by either DmREV7 or Rrp1. Methyl methanesulfonate and hydrogen peroxide treatments increased mRNA levels of DmREV3 and DmREV7. On the basis of the direct interaction between DmREV7 and Rrp1, we suggest that Dmpol zeta may be involved in the repair pathway of AP sites in DNA.

  8. Neutralizing mutations of carboxylates that bind metal 2 in T5 flap endonuclease result in an enzyme that still requires two metal ions.

    Science.gov (United States)

    Tomlinson, Christopher G; Syson, Karl; Sengerová, Blanka; Atack, John M; Sayers, Jon R; Swanson, Linda; Tainer, John A; Williams, Nicholas H; Grasby, Jane A

    2011-09-02

    Flap endonucleases (FENs) are divalent metal ion-dependent phosphodiesterases. Metallonucleases are often assigned a "two-metal ion mechanism" where both metals contact the scissile phosphate diester. The spacing of the two metal ions observed in T5FEN structures appears to preclude this mechanism. However, the overall reaction catalyzed by wild type (WT) T5FEN requires three Mg(2+) ions, implying that a third ion is needed during catalysis, and so a two-metal ion mechanism remains possible. To investigate the positions of the ions required for chemistry, a mutant T5FEN was studied where metal 2 (M2) ligands are altered to eliminate this binding site. In contrast to WT T5FEN, the overall reaction catalyzed by D201I/D204S required two ions, but over the concentration range of Mg(2+) tested, maximal rate data were fitted to a single binding isotherm. Calcium ions do not support FEN catalysis and inhibit the reactions supported by viable metal cofactors. To establish participation of ions in stabilization of enzyme-substrate complexes, dissociation constants of WT and D201I/D204S-substrate complexes were studied as a function of [Ca(2+)]. At pH 9.3 (maximal rate conditions), Ca(2+) substantially stabilized both complexes. Inhibition of viable cofactor supported reactions of WT, and D201I/D204S T5FENs was biphasic with respect to Ca(2+) and ultimately dependent on 1/[Ca(2+)](2). By varying the concentration of viable metal cofactor, Ca(2+) ions were shown to inhibit competitively displacing two catalytic ions. Combined analyses imply that M2 is not involved in chemical catalysis but plays a role in substrate binding, and thus a two-metal ion mechanism is plausible.

  9. Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions*

    Science.gov (United States)

    Tomlinson, Christopher G.; Syson, Karl; Sengerová, Blanka; Atack, John M.; Sayers, Jon R.; Swanson, Linda; Tainer, John A.; Williams, Nicholas H.; Grasby, Jane A.

    2011-01-01

    Flap endonucleases (FENs) are divalent metal ion-dependent phosphodiesterases. Metallonucleases are often assigned a “two-metal ion mechanism” where both metals contact the scissile phosphate diester. The spacing of the two metal ions observed in T5FEN structures appears to preclude this mechanism. However, the overall reaction catalyzed by wild type (WT) T5FEN requires three Mg2+ ions, implying that a third ion is needed during catalysis, and so a two-metal ion mechanism remains possible. To investigate the positions of the ions required for chemistry, a mutant T5FEN was studied where metal 2 (M2) ligands are altered to eliminate this binding site. In contrast to WT T5FEN, the overall reaction catalyzed by D201I/D204S required two ions, but over the concentration range of Mg2+ tested, maximal rate data were fitted to a single binding isotherm. Calcium ions do not support FEN catalysis and inhibit the reactions supported by viable metal cofactors. To establish participation of ions in stabilization of enzyme-substrate complexes, dissociation constants of WT and D201I/D204S-substrate complexes were studied as a function of [Ca2+]. At pH 9.3 (maximal rate conditions), Ca2+ substantially stabilized both complexes. Inhibition of viable cofactor supported reactions of WT, and D201I/D204S T5FENs was biphasic with respect to Ca2+ and ultimately dependent on 1/[Ca2+]2. By varying the concentration of viable metal cofactor, Ca2+ ions were shown to inhibit competitively displacing two catalytic ions. Combined analyses imply that M2 is not involved in chemical catalysis but plays a role in substrate binding, and thus a two-metal ion mechanism is plausible. PMID:21734257

  10. Rapid identification of clinically significant species and taxa of aerobic actinomycetes, including Actinomadura, Gordona, Nocardia, Rhodococcus, Streptomyces, and Tsukamurella isolates, by DNA amplification and restriction endonuclease analysis.

    Science.gov (United States)

    Steingrube, V A; Wilson, R W; Brown, B A; Jost, K C; Blacklock, Z; Gibson, J L; Wallace, R J

    1997-04-01

    A previously described PCR-restriction fragment length polymorphism (RFLP) identification schema for Nocardia that used an amplified 439-bp segment (amplicon) of the 65-kDa heat shock protein gene was evaluated for potential use with isolates of all clinically significant aerobic actinomycetes. The study included 28 reference (American Type Culture Collection) strains and 198 clinical isolates belonging to 20 taxonomic groups. Of these 198 isolates, 188 could be differentiated by this PCR-RFLP method. Amplicons from all aerobic actinomycete isolates lacked BstEII recognition sites, thereby distinguishing them from those of mycobacteria that contain one or more such sites. Of 29 restriction endonucleases, MspI plus HinfI produced RFLP patterns that differentiated 16 of the 20 taxa. A single RFLP pattern was observed for 15 of 20 taxa that included 65% of phenotypically clustered isolates. Multiple patterns were seen with Gordona bronchialis, Nocardia asteroides complex type VI, Nocardia otitidiscaviarum, Nocardia transvalensis, and Streptomyces spp. Streptomyces RFLP patterns were the most heterogeneous (five patterns among 19 isolates), but exhibited a unique HinfI fragment of > 320 bp. RFLP patterns that matched those from type strains of Streptomyces albus, Streptomyces griseus, or Streptomyces somaliensis were obtained from 14 of 19 Streptomyces isolates. Only 10 of 28 isolates of N. otitidiscaviarum failed to yield satisfactory amplicons, while only 6 of 188 (3.2%) clinical isolates exhibited patterns that failed to match one of the 21 defined RFLP patterns. These studies extended the feasibility of using PCR-RFLP analysis as a rapid method for the identification of all clinically significant species and taxa of aerobic actinomycetes.

  11. Chemical Display of Pyrimidine Bases Flipped Out by Modification-Dependent Restriction Endonucleases of MspJI and PvuRts1I Families

    Science.gov (United States)

    Zagorskaitė, Evelina; Sasnauskas, Giedrius

    2014-01-01

    The epigenetic DNA modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in eukaryotes are recognized either in the context of double-stranded DNA (e.g., by the methyl-CpG binding domain of MeCP2), or in the flipped-out state (e.g., by the SRA domain of UHRF1). The SRA-like domains and the base-flipping mechanism for 5(h)mC recognition are also shared by the recently discovered prokaryotic modification-dependent endonucleases of the MspJI and PvuRts1I families. Since the mechanism of modified cytosine recognition by many potential eukaryotic and prokaryotic 5(h)mC “readers” is still unknown, a fast solution based method for the detection of extrahelical 5(h)mC would be very useful. In the present study we tested base-flipping by MspJI- and PvuRts1I-like restriction enzymes using several solution-based methods, including fluorescence measurements of the cytosine analog pyrrolocytosine and chemical modification of extrahelical pyrimidines with chloroacetaldehyde and KMnO4. We find that only KMnO4 proved an efficient probe for the positive display of flipped out pyrimidines, albeit the method required either non-physiological pH (4.3) or a substitution of the target cytosine with thymine. Our results imply that DNA recognition mechanism of 5(h)mC binding proteins should be tested using a combination of all available methods, as the lack of a positive signal in some assays does not exclude the base flipping mechanism. PMID:25486533

  12. Chemical display of pyrimidine bases flipped out by modification-dependent restriction endonucleases of MspJI and PvuRts1I families.

    Science.gov (United States)

    Zagorskaitė, Evelina; Sasnauskas, Giedrius

    2014-01-01

    The epigenetic DNA modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in eukaryotes are recognized either in the context of double-stranded DNA (e.g., by the methyl-CpG binding domain of MeCP2), or in the flipped-out state (e.g., by the SRA domain of UHRF1). The SRA-like domains and the base-flipping mechanism for 5(h)mC recognition are also shared by the recently discovered prokaryotic modification-dependent endonucleases of the MspJI and PvuRts1I families. Since the mechanism of modified cytosine recognition by many potential eukaryotic and prokaryotic 5(h)mC "readers" is still unknown, a fast solution based method for the detection of extrahelical 5(h)mC would be very useful. In the present study we tested base-flipping by MspJI- and PvuRts1I-like restriction enzymes using several solution-based methods, including fluorescence measurements of the cytosine analog pyrrolocytosine and chemical modification of extrahelical pyrimidines with chloroacetaldehyde and KMnO4. We find that only KMnO4 proved an efficient probe for the positive display of flipped out pyrimidines, albeit the method required either non-physiological pH (4.3) or a substitution of the target cytosine with thymine. Our results imply that DNA recognition mechanism of 5(h)mC binding proteins should be tested using a combination of all available methods, as the lack of a positive signal in some assays does not exclude the base flipping mechanism.

  13. Alkylation base damage is converted into repairable double-strand breaks and complex intermediates in G2 cells lacking AP endonuclease.

    Directory of Open Access Journals (Sweden)

    Wenjian Ma

    2011-04-01

    Full Text Available DNA double-strand breaks (DSBs are potent sources of genome instability. While there is considerable genetic and molecular information about the disposition of direct DSBs and breaks that arise during replication, relatively little is known about DSBs derived during processing of single-strand lesions, especially for the case of single-strand breaks (SSBs with 3'-blocked termini generated in vivo. Using our recently developed assay for detecting end-processing at random DSBs in budding yeast, we show that single-strand lesions produced by the alkylating agent methyl methanesulfonate (MMS can generate DSBs in G2-arrested cells, i.e., S-phase independent. These derived DSBs were observed in apn1/2 endonuclease mutants and resulted from aborted base excision repair leading to 3' blocked single-strand breaks following the creation of abasic (AP sites. DSB formation was reduced by additional mutations that affect processing of AP sites including ntg1, ntg2, and, unexpectedly, ogg1, or by a lack of AP sites due to deletion of the MAG1 glycosylase gene. Similar to direct DSBs, the derived DSBs were subject to MRX (Mre11, Rad50, Xrs2-determined resection and relied upon the recombinational repair genes RAD51, RAD52, as well as on the MCD1 cohesin gene, for repair. In addition, we identified a novel DNA intermediate, detected as slow-moving chromosomal DNA (SMD in pulsed field electrophoresis gels shortly after MMS exposure in apn1/2 cells. The SMD requires nicked AP sites, but is independent of resection/recombination processes, suggesting that it is a novel structure generated during processing of 3'-blocked SSBs. Collectively, this study provides new insights into the potential consequences of alkylation base damage in vivo, including creation of novel structures as well as generation and repair of DSBs in nonreplicating cells.

  14. Curcumin and EGCG Suppress Apurinic/Apyrimidinic Endonuclease 1 and Induce Complete Remission in B-cell Non-Hodgkin's lymphoma Patients

    Directory of Open Access Journals (Sweden)

    Hashem M. Neenaa

    2011-12-01

    Full Text Available ABSTRACT:Background: Follicular lymphoma (FL is the most common subtype of indolent lymphoma. FL is still considered to be an incurable disease and palliation of symptoms is an acceptable approach to the expected pattern of repeated relapses due to developing resistance to chemotherapy agents. Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1 is a multifunctional protein involved in DNA base excision repair (BER of oxidative DNA damage and in redox regulation of a number of transcription factors. It was observed that cytoplasmic APE1 induced COX-2 expression through NF-êB activation. It has been shown that chemopreventive agents potentiate the efficacy of chemotherapy through the regulation of multiple signaling pathways, including NF-êB, c-Myc, cyclooxygenase-2, apoptosis, and others, suggesting a multitargeted nature of chemopreventive agents. We hypothesized that curcumin, a polyphenolic antioxidant derived from the spice turmeric, and epigallocatechin gallate (EGCG from green tea would potentiate the effect of chemotherapy in B-cell lymphoma.Objective: We examined the role of human apurinic/apyrimidinic endonuclease 1 (APE1 in resistance and prognosis in patients with FL. Our major objective was to update the safety and efficacy results of the antitumor effect of combination of curcumin and EGCG therapy in relapsed or resistant indolent or transformed non-Hodgkin follicular lymphoma patients and their peripheral blood mononuclear cells (PBMCs compared with healthy donors’ controls.Methods: Thirty patients with FL with over-expression of constitutive active NF-êB in their PBMCs received regular CHOP and consumed capsules compatible with curcumin doses between 0.9 and 5.4 g daily for up to 9 months and 9.0 g/day green tea whole extract "1000 mg tablets of green tea whole extract containing 200 mg EGCG. We designed a dose-escalation Functional Foods in Health and Disease 2011, 1(12:525-544 study to explore the efficacy of CHOP

  15. 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...

  16. 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

    The green alga Scenedesmus pupukensis and the red alga Porphyra spiralis contain large group-IC1 introns in their nuclear small subunit ribosomal RNA genes due to the presence of open reading frames at the 5' end of the introns. The putative 555 amino-acid Scenedesmus-encoded protein harbors...... a sequence motif resembling the bacterial S9 ribosomal proteins. The Porphyra intron self-splices in vitro, and generates both ligated exons and a full-length intron RNA circle. The Porphyra intron has an unusual structural organization by encoding a potential 149 amino-acid homing-endonuclease-like protein...... 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...

  17. Modified 16S-23S rRNA intergenic region restriction endonuclease analysis for species identification of Enterococcus strains isolated from pigs, compared with identification using classical methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Nowakiewicz, Aneta; Ziółkowska, Grażyna; Zięba, Przemysław; Trościańczyk, Aleksandra; Banach, Tomasz; Kowalski, Cezary

    2015-03-01

    Fast and reliable identification of bacteria to at least the species level is currently the basis for correct diagnosis and appropriate treatment of infections. This is particularly important in the case of bacteria of the genus Enterococcus, whose resistance profile is often correlated with their species (e.g. resistance to vancomycin). In this study, we evaluated restriction endonuclease analysis of the 16S-23S rRNA gene intergenic transcribed spacer (ITS) region for species identification of Enterococcus. The utility of the method was compared with that of phenotypic methods [biochemical profile evaluation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)]. Identification was based on 21 Enterococcus reference strains, of the species E. faecalis, E. faecium, E. hirae, E. durans, E. casseliflavus, E. gallinarum, E. avium, E. cecorum and E. columbae, and 47 Enterococcus field strains isolated from pigs. Restriction endonuclease analysis of the ITS-PCR product using HinfI, RsaI and MboI, in the order specified, enabled species differentiation of the Enterococcus reference and field strains, and in the case of the latter, the results of species identification were identical (47/47) to those obtained by MALDI-TOF MS. Moreover, as a result of digestion with MboI, a unique restriction profile was also obtained for the strains (3/3) identified by MALDI-TOF MS as E. thailandicus. In our opinion, restriction endonuclease analysis of the 16S-23S rRNA gene ITS region of Enterococcus may be a simple and relatively fast (less than 4 h) alternative method for identifying the species occurring most frequently in humans and animals. © 2015 The Authors.

  18. 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

  19. Human apurinic/apyrimidinic endonuclease (APE1) is a prognostic factor in ovarian, gastro-oesophageal and pancreatico-biliary cancers

    OpenAIRE

    Al-Attar, A; Gossage, L; Fareed, K R; Shehata, M; Mohammed, M; Zaitoun, A M; Soomro, I; Lobo, D N; Abbotts, R; Chan, S; Madhusudan, S

    2010-01-01

    Background: Altered DNA repair may be associated with aggressive tumour biology and impact upon response to chemotherapy and radiotherapy. We investigated whether expression of human AP endonuclease (APE1), a key multifunctional protein involved in DNA BER, would impact on clinicopathological outcomes in ovarian, gastro-oesophageal, and pancreatico-biliary cancer. Methods: Formalin-fixed human ovarian, gastro-oesophageal, and pancreatico-biliary cancers were constructed into TMAs. Expression ...

  20. Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification: A Novel Analytically Rapid, Sensitive, Multiplex Loop-Mediated Isothermal Amplification Detection Technique.

    Science.gov (United States)

    Wang, Yi; Wang, Yan; Lan, Ruiting; Xu, Huaqing; Ma, Aijing; Li, Dongxun; Dai, Hang; Yuan, Xuejiao; Xu, Jianguo; Ye, Changyun

    2015-07-01

    Loop-mediated isothermal amplification (LAMP) is restricted to detecting a single target, limiting the usefulness of this method. To achieve multiplex LAMP-based detection, we developed a novel approach we called the multiple endonuclease restriction real-time-LAMP assay. In this system, the LAMP forward or backward inner primers contain 5' end short sequences that are recognized by the restriction endonuclease Nb.BsrDI, and the new forward or backward inner primers were modified at the 5' end with a fluorophore and in the middle with a dark quencher. Nb.BsrDI digests the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which releases the quenching, resulting in a gain of signal. The assay permitted real-time detection of single or multiple target sequences in a single tube, and the positive results can be obtained in as short as 12 minutes. The novel methodology is highly efficient and specific, detecting down to 250 fg of DNA per reaction of Listeria DNA tested, and was successful in evaluating raw meat samples. The multiple endonuclease restriction real-time-LAMP technology, which is an extension of LAMP to accommodate robust, target-specific, and multiplex detection, provides a molecular diagnostic tool with less detection time and high sensitivity and specificity compared with those of LAMP and quantitative real-time PCR. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  1. A general fluorescent sensor design strategy for "turn-on" activity detection of exonucleases and restriction endonucleases based on graphene oxide.

    Science.gov (United States)

    Zhang, Qi; Kong, De-Ming

    2013-11-07

    Using graphene oxide (GO) as a nanoquencher, a universal sensor design strategy was developed on the basis of significantly different binding affinities of GO to single-stranded DNAs (ss-DNAs) with different lengths. The proposed sensors could be used for the activity detection of both exonucleases and restriction endonucleases. To achieve this, a single-labeled fluorescent oligonucleotide probe, which had a single-stranded structure or a hairpin structure with a long single-stranded loop, was used. Such a probe could be efficiently absorbed on the surface of GO, resulting in the quenching of the fluorescent signal. Excision of the single-stranded probe by exonucleases or site-specific cleavage at the double-stranded stem of the hairpin probe by restriction endonuclease released fluorophore-labeled nucleotide, which could not be efficiently absorbed by GO, thus leading to increase in fluorescence of the corresponding sensing system. As examples, three sensors, which were used for activity detection of the exonuclease Exo 1 and the restriction endonucleases EcoR I and Hind III, were developed. These three sensors could specifically and sensitively detect the activities of Exo 1, EcoR I and Hind III with detection limits of 0.03 U mL(-1), 0.06 U mL(-1) and 0.04 U mL(-1), respectively. Visual detection was also possible.

  2. The human Rad9–Rad1–Hus1 checkpoint complex stimulates flap endonuclease 1

    Science.gov (United States)

    Wang, Wensheng; Brandt, Patrick; Rossi, Marie L.; Lindsey-Boltz, Laura; Podust, Vladimir; Fanning, Ellen; Sancar, Aziz; Bambara, Robert A.

    2004-01-01

    The toroidal damage checkpoint complex Rad9–Rad1–Hus1 (9-1-1) has been characterized as a sensor of DNA damage. Flap endonuclease 1 (FEN1) is a structure-specific nuclease involved both in removing initiator RNA from Okazaki fragments and in DNA repair pathways. FEN1 activity is stimulated by proliferating cell nuclear antigen (PCNA), a toroidal sliding clamp that acts as a platform for DNA replication and repair complexes. We show that 9-1-1 also binds and stimulates FEN1. Stimulation is observed on a variety of flap, nick, and gapped substrates simulating repair intermediates. Blocking 9-1-1 entry to the double strands prevents a portion of the stimulation. Like PCNA stimulation, 9-1-1 stimulation cannot circumvent the tracking mechanism by which FEN1 enters the substrate; however, 9-1-1 does not substitute for PCNA in the stimulation of DNA polymerase β. This suggests that 9-1-1 is a damage-specific activator of FEN1. PMID:15556996

  3. Loss of mitochondrial exo/endonuclease EXOG affects mitochondrial respiration and induces ROS-mediated cardiomyocyte hypertrophy.

    Science.gov (United States)

    Tigchelaar, Wardit; Yu, Hongjuan; de Jong, Anne Margreet; van Gilst, Wiek H; van der Harst, Pim; Westenbrink, B Daan; de Boer, Rudolf A; Silljé, Herman H W

    2015-01-15

    Recently, a locus at the mitochondrial exo/endonuclease EXOG gene, which has been implicated in mitochondrial DNA repair, was associated with cardiac function. The function of EXOG in cardiomyocytes is still elusive. Here we investigated the role of EXOG in mitochondrial function and hypertrophy in cardiomyocytes. Depletion of EXOG in primary neonatal rat ventricular cardiomyocytes (NRVCs) induced a marked increase in cardiomyocyte hypertrophy. Depletion of EXOG, however, did not result in loss of mitochondrial DNA integrity. Although EXOG depletion did not induce fetal gene expression and common hypertrophy pathways were not activated, a clear increase in ribosomal S6 phosphorylation was observed, which readily explains increased protein synthesis. With the use of a Seahorse flux analyzer, it was shown that the mitochondrial oxidative consumption rate (OCR) was increased 2.4-fold in EXOG-depleted NRVCs. Moreover, ATP-linked OCR was 5.2-fold higher. This increase was not explained by mitochondrial biogenesis or alterations in mitochondrial membrane potential. Western blotting confirmed normal levels of the oxidative phosphorylation (OXPHOS) complexes. The increased OCR was accompanied by a 5.4-fold increase in mitochondrial ROS levels. These increased ROS levels could be normalized with specific mitochondrial ROS scavengers (MitoTEMPO, mnSOD). Remarkably, scavenging of excess ROS strongly attenuated the hypertrophic response. In conclusion, loss of EXOG affects normal mitochondrial function resulting in increased mitochondrial respiration, excess ROS production, and cardiomyocyte hypertrophy. Copyright © 2015 the American Physiological Society.

  4. Recombinant expression, purification, and characterization of XorKII: a restriction endonuclease from Xanthomonas oryzae pv. oryzae.

    Science.gov (United States)

    Moon, Won Jae; Cho, Jae-Yong; Chae, Young Kee

    2008-12-01

    An endonuclease from Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, XorKII, was recombinantly produced in Escherichia coli by applying the stationary state induction method, which was necessary to prevent the unwanted lysis of E. coli cells. XorKII was purified by immobilized metal affinity chromatography on an FPLC system. The yield was 3.5mg of XorKII per liter of LB medium. The purified recombinant XorKII showed that it recognized and cleaved to the same site as PstI. It behaved as a dimer as evidenced by the size exclusion chromatography. The specific activity of the purified XorKII was determined to be 31,300 U/mg. The enzyme activity was monitored by cleaving lambda DNA or YEp24 plasmid as substrates. The enzyme was the most active at 10mM Tris-HCl pH 7.0, 10 mM MgCl(2), 1mM dithiothreitol at 37 degrees C. XorKII was easily inactivated by heating at 65 degrees C for 5 min, but retained most of the original activity after incubation at 37 degrees C for 24h.

  5. 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

  6. A molecular switch sensor for detection of PRSS1 genotype based on site-specific DNA cleavage of restriction endonuclease.

    Science.gov (United States)

    Liu, Qicai; Gao, Feng; Weng, Shaohuang; Peng, Huaping; Lin, Liqing; Zhao, Chengfei; Lin, Xinhua

    2015-01-01

    PRSS1 mutations or polymorphism in the peripheral blood of patients can be used as susceptible molecular markers to pancreatic cancer. A sensor for selective electrochemical detection of PRSS1 genotypes was developed based on site-specific DNA cleavage of restriction endonuclease EcoRI. A mercapto-modified hairpin probe was immobilized on a gold electrode. The probe's neck can be cleaved by EcoRI in the absence of rs10273639 C/C of PRSS1 genotype, but it cannot be cleaved in the presence of T/T. The difference in quantity of electric charge was monitored by biosensors before and after enzymatic cleavage. Electrochemical signals are generated by differential pulse voltammetry interrogation of methylene blue (MB) that quantitatively binds to surface-confined hairpin probe via electrostatic interactions. The results suggested this method had a good specificity in distinguishing PRSS1 genotypes. There was a good linear relationship between the charge and the logarithmic function of PRSS1 rs10273639 T/T type DNA concentration (current=120.6303+8.8512log C, R=0.9942). The detection limit was estimated at 0.5 fM. The molecular switch sensor has several advantages, and it is possible to qualitatively, quantitatively, and noninvasively detect PRSS1 genotypes in the blood of patients with pancreatic cancer. © 2015 by the Association of Clinical Scientists, Inc.

  7. A fluorescence method for detection of DNA and DNA methylation based on graphene oxide and restriction endonuclease HpaII.

    Science.gov (United States)

    Wei, Wei; Gao, Chunyan; Xiong, Yanxiang; Zhang, Yuanjian; Liu, Songqin; Pu, Yuepu

    2015-01-01

    DNA methylation plays an important role in many biological events and is associated with various diseases. Most traditional methods for detection of DNA methylation are based on the complex and expensive bisulfite method. In this paper, we report a novel fluorescence method to detect DNA and DNA methylation based on graphene oxide (GO) and restriction endonuclease HpaII. The skillfully designed probe DNA labeled with 5-carboxyfluorescein (FAM) and optimized GO concentration keep the probe/target DNA still adsorbed on the GO. After the cleavage action of HpaII the labeled FAM is released from the GO surface and its fluorescence recovers, which could be used to detect DNA in the linear range of 50 pM-50 nM with a detection limit of 43 pM. DNA methylation induced by transmethylase (Mtase) or other chemical reagents prevents HpaII from recognizing and cleaving the specific site; as a result, fluorescence cannot recover. The fluorescence recovery efficiency is closely related to the DNA methylation level, which can be used to detect DNA methylation by comparing it with the fluorescence in the presence of intact target DNA. The method for detection of DNA and DNA methylation is simple, reliable and accurate. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Phosphate steering by Flap Endonuclease 1 promotes 5′-flap specificity and incision to prevent genome instability

    KAUST Repository

    Tsutakawa, Susan E.

    2017-06-27

    DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5\\'-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5\\'-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 5\\'polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via phosphate steering\\', basic residues energetically steer an inverted ss 5\\'-flap through a gateway over FEN1\\'s active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA) repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 5\\'-flap specificity and catalysis, preventing genomic instability.

  9. PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Hui; Gao, Pu; Rajashankar, Kanagalaghatta R.; Patel, Dinshaw J. (MSKCC); (Cornell); (Chinese Aca. Sci.)

    2016-12-01

    C2c1 is a newly identified guide RNA-mediated type V-B CRISPR-Cas endonuclease that site-specifically targets and cleaves both strands of target DNA. We have determined crystal structures of Alicyclobacillus acidoterrestris C2c1 (AacC2c1) bound to sgRNA as a binary complex and to target DNAs as ternary complexes, thereby capturing catalytically competent conformations of AacC2c1 with both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. Moreover, C2c1-mediated cleavage results in a staggered seven-nucleotide break of target DNA. crRNA adopts a pre-ordered five-nucleotide A-form seed sequence in the binary complex, with release of an inserted tryptophan, facilitating zippering up of 20-bp guide RNA:target DNA heteroduplex on ternary complex formation. Notably, the PAM-interacting cleft adopts a “locked” conformation on ternary complex formation. Structural comparison of C2c1 ternary complexes with their Cas9 and Cpf1 counterparts highlights the diverse mechanisms adopted by these distinct CRISPR-Cas systems, thereby broadening and enhancing their applicability as genome editing tools.

  10. Lundep, a sand fly salivary endonuclease increases Leishmania parasite survival in neutrophils and inhibits XIIa contact activation in human plasma.

    Directory of Open Access Journals (Sweden)

    Andrezza C Chagas

    2014-02-01

    Full Text Available Neutrophils are the host's first line of defense against infections, and their extracellular traps (NET were recently shown to kill Leishmania parasites. Here we report a NET-destroying molecule (Lundep from the salivary glands of Lutzomyia longipalpis. Previous analysis of the sialotranscriptome of Lu. longipalpis showed the potential presence of an endonuclease. Indeed, not only was the cloned cDNA (Lundep shown to encode a highly active ss- and dsDNAse, but also the same activity was demonstrated to be secreted by salivary glands of female Lu. longipalpis. Lundep hydrolyzes both ss- and dsDNA with little sequence specificity with a calculated DNase activity of 300000 Kunitz units per mg of protein. Disruption of PMA (phorbol 12 myristate 13 acetate- or parasite-induced NETs by treatment with recombinant Lundep or salivary gland homogenates increases parasite survival in neutrophils. Furthermore, co-injection of recombinant Lundep with metacyclic promastigotes significantly exacerbates Leishmania infection in mice when compared with PBS alone or inactive (mutagenized Lundep. We hypothesize that Lundep helps the parasite to establish an infection by allowing it to escape from the leishmanicidal activity of NETs early after inoculation. Lundep may also assist blood meal intake by lowering the local viscosity caused by the release of host DNA and as an anticoagulant by inhibiting the intrinsic pathway of coagulation.

  11. Cardiomyocyte hypertrophy induced by Endonuclease G deficiency requires reactive oxygen radicals accumulation and is inhibitable by the micropeptide humanin.

    Science.gov (United States)

    Blasco, Natividad; Cámara, Yolanda; Núñez, Estefanía; Beà, Aida; Barés, Gisel; Forné, Carles; Ruíz-Meana, Marisol; Girón, Cristina; Barba, Ignasi; García-Arumí, Elena; García-Dorado, David; Vázquez, Jesús; Martí, Ramon; Llovera, Marta; Sanchis, Daniel

    2018-03-01

    The endonuclease G gene (Endog), which codes for a mitochondrial nuclease, was identified as a determinant of cardiac hypertrophy. How ENDOG controls cardiomyocyte growth is still unknown. Thus, we aimed at finding the link between ENDOG activity and cardiomyocyte growth. Endog deficiency induced reactive oxygen species (ROS) accumulation and abnormal growth in neonatal rodent cardiomyocytes, altering the AKT-GSK3β and Class-II histone deacethylases (HDAC) signal transduction pathways. These effects were blocked by ROS scavengers. Lack of ENDOG reduced mitochondrial DNA (mtDNA) replication independently of ROS accumulation. Because mtDNA encodes several subunits of the mitochondrial electron transport chain, whose activity is an important source of cellular ROS, we investigated whether Endog deficiency compromised the expression and activity of the respiratory chain complexes but found no changes in these parameters nor in ATP content. MtDNA also codes for humanin, a micropeptide with possible metabolic functions. Nanomolar concentrations of synthetic humanin restored normal ROS levels and cell size in Endog-deficient cardiomyocytes. These results support the involvement of redox signaling in the control of cardiomyocyte growth by ENDOG and suggest a pathway relating mtDNA content to the regulation of cell growth probably involving humanin, which prevents reactive oxygen radicals accumulation and hypertrophy induced by Endog deficiency. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage

    Science.gov (United States)

    Josephs, Eric A.; Kocak, D. Dewran; Fitzgibbon, Christopher J.; McMenemy, Joshua; Gersbach, Charles A.; Marszalek, Piotr E.

    2015-01-01

    CRISPR-associated endonuclease Cas9 cuts DNA at variable target sites designated by a Cas9-bound RNA molecule. Cas9's ability to be directed by single ‘guide RNA’ molecules to target nearly any sequence has been recently exploited for a number of emerging biological and medical applications. Therefore, understanding the nature of Cas9's off-target activity is of paramount importance for its practical use. Using atomic force microscopy (AFM), we directly resolve individual Cas9 and nuclease-inactive dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind with different guide RNA variants to targeted or off-target sequences. Mapping the structural properties of Cas9 and dCas9 to their respective binding sites reveals a progressive conformational transformation at DNA sites with increasing sequence similarity to its target. With kinetic Monte Carlo (KMC) simulations, these results provide evidence of a ‘conformational gating’ mechanism driven by the interactions between the guide RNA and the 14th–17th nucleotide region of the targeted DNA, the stabilities of which we find correlate significantly with reported off-target cleavage rates. KMC simulations also reveal potential methodologies to engineer guide RNA sequences with improved specificity by considering the invasion of guide RNAs into targeted DNA duplex. PMID:26384421

  13. Group I introns and associated homing endonuclease genes reveals a clinal structure for Porphyra spiralis var. amplifolia (Bangiales, Rhodophyta) along the Eastern coast of South America.

    Science.gov (United States)

    Milstein, Daniela; Oliveira, Mariana C; Martins, Felipe M; Matioli, Sergio R

    2008-11-07

    Group I introns are found in the nuclear small subunit ribosomal RNA gene (SSU rDNA) of some species of the genus Porphyra (Bangiales, Rhodophyta). Size polymorphisms in group I introns has been interpreted as the result of the degeneration of homing endonuclease genes (HEG) inserted in peripheral loops of intron paired elements. In this study, intron size polymorphisms were characterized for different Porphyra spiralis var. amplifolia (PSA) populations on the Southern Brazilian coast, and were used to infer genetic relationships and genetic structure of these PSA populations, in addition to cox2-3 and rbcL-S regions. Introns of different sizes were tested qualitatively for in vitro self-splicing. Five intron size polymorphisms within 17 haplotypes were obtained from 80 individuals representing eight localities along the distribution of PSA in the Eastern coast of South America. In order to infer genetic structure and genetic relationships of PSA, these polymorphisms and haplotypes were used as markers for pairwise Fst analyses, Mantel's test and median joining network. The five cox2-3 haplotypes and the unique rbcL-S haplotype were used as markers for summary statistics, neutrality tests Tajima's D and Fu's Fs and for median joining network analyses. An event of demographic expansion from a population with low effective number, followed by a pattern of isolation by distance was obtained for PSA populations with the three analyses. In vitro experiments have shown that introns of different lengths were able to self-splice from pre-RNA transcripts. The findings indicated that degenerated HEGs are reminiscent of the presence of a full-length and functional HEG, once fixed for PSA populations. The cline of HEG degeneration determined the pattern of isolation by distance. Analyses with the other markers indicated an event of demographic expansion from a population with low effective number. The different degrees of degeneration of the HEG do not refrain intron self

  14. Lys98 substitution in human AP endonuclease 1 affects the kinetic mechanism of enzyme action in base excision and nucleotide incision repair pathways.

    Directory of Open Access Journals (Sweden)

    Nadezhda A Timofeyeva

    Full Text Available Human apurinic/apyrimidinic endonuclease 1 (APE1 is a key enzyme in the base excision repair (BER and nucleotide incision repair (NIR pathways. We recently analyzed the conformational dynamics and kinetic mechanism of wild-type (wt protein, in a stopped-flow fluorescence study. In this study, we investigated the mutant enzyme APE1K98A using the same approach. Lys98 was known to hydrogen bond to the carboxyl group of Asp70, a residue implicated in binding the divalent metal ion. Our data suggested that the conformational selection and induced fit occur during the enzyme action. We expanded upon the evidence that APE1 can pre-exist in two conformations. The isomerization of an enzyme-product complex in the BER process and the additional isomerization stage of enzyme-substrate complex in the NIR process were established for APE1K98A. These stages had not been registered for the wtAPE1. We found that the K98A substitution resulted in a 12-fold reduction of catalytic constant of 5'-phosphodiester bond hydrolysis in (3-hydroxytetrahydrofuran-2-ylmethyl phosphate (F, tetrahydrofuran containing substrate, and in 200-fold reduction in 5,6-dihydrouridine (DHU containing substrate. Thus, the K98A substitution influenced NIR more than BER. We demonstrated that the K98A mutation influenced the formation of primary unspecific enzyme-substrate complex in a complicated manner, depending on the Mg(2+ concentration and pH. This mutation obstructed the induced fit of enzyme in the complex with undamaged DNA and F-containing DNA and appreciably decreased the stability of primary complex upon interaction of enzyme with DNA, containing the natural apurinic/apyrimidinic (AP site. Furthermore, it significantly delayed the activation of the less active form of enzyme during NIR and slowed down the conformational conversion of the complex of enzyme with the cleavage product of DHU-substrate. Our data revealed that APE1 uses the same active site to catalyze the cleavage

  15. Group I introns and associated homing endonuclease genes reveals a clinal structure for Porphyra spiralis var. amplifolia (Bangiales, Rhodophyta along the Eastern coast of South America

    Directory of Open Access Journals (Sweden)

    Matioli Sergio R

    2008-11-01

    Full Text Available Abstract Background Group I introns are found in the nuclear small subunit ribosomal RNA gene (SSU rDNA of some species of the genus Porphyra (Bangiales, Rhodophyta. Size polymorphisms in group I introns has been interpreted as the result of the degeneration of homing endonuclease genes (HEG inserted in peripheral loops of intron paired elements. In this study, intron size polymorphisms were characterized for different Porphyra spiralis var. amplifolia (PSA populations on the Southern Brazilian coast, and were used to infer genetic relationships and genetic structure of these PSA populations, in addition to cox2-3 and rbcL-S regions. Introns of different sizes were tested qualitatively for in vitro self-splicing. Results Five intron size polymorphisms within 17 haplotypes were obtained from 80 individuals representing eight localities along the distribution of PSA in the Eastern coast of South America. In order to infer genetic structure and genetic relationships of PSA, these polymorphisms and haplotypes were used as markers for pairwise Fst analyses, Mantel's test and median joining network. The five cox2-3 haplotypes and the unique rbcL-S haplotype were used as markers for summary statistics, neutrality tests Tajima's D and Fu's Fs and for median joining network analyses. An event of demographic expansion from a population with low effective number, followed by a pattern of isolation by distance was obtained for PSA populations with the three analyses. In vitro experiments have shown that introns of different lengths were able to self-splice from pre-RNA transcripts. Conclusion The findings indicated that degenerated HEGs are reminiscent of the presence of a full-length and functional HEG, once fixed for PSA populations. The cline of HEG degeneration determined the pattern of isolation by distance. Analyses with the other markers indicated an event of demographic expansion from a population with low effective number. The different degrees of

  16. Group I introns and associated homing endonuclease genes reveals a clinal structure for Porphyra spiralis var. amplifolia (Bangiales, Rhodophyta) along the Eastern coast of South America

    Science.gov (United States)

    2008-01-01

    Background Group I introns are found in the nuclear small subunit ribosomal RNA gene (SSU rDNA) of some species of the genus Porphyra (Bangiales, Rhodophyta). Size polymorphisms in group I introns has been interpreted as the result of the degeneration of homing endonuclease genes (HEG) inserted in peripheral loops of intron paired elements. In this study, intron size polymorphisms were characterized for different Porphyra spiralis var. amplifolia (PSA) populations on the Southern Brazilian coast, and were used to infer genetic relationships and genetic structure of these PSA populations, in addition to cox2-3 and rbcL-S regions. Introns of different sizes were tested qualitatively for in vitro self-splicing. Results Five intron size polymorphisms within 17 haplotypes were obtained from 80 individuals representing eight localities along the distribution of PSA in the Eastern coast of South America. In order to infer genetic structure and genetic relationships of PSA, these polymorphisms and haplotypes were used as markers for pairwise Fst analyses, Mantel's test and median joining network. The five cox2-3 haplotypes and the unique rbcL-S haplotype were used as markers for summary statistics, neutrality tests Tajima's D and Fu's Fs and for median joining network analyses. An event of demographic expansion from a population with low effective number, followed by a pattern of isolation by distance was obtained for PSA populations with the three analyses. In vitro experiments have shown that introns of different lengths were able to self-splice from pre-RNA transcripts. Conclusion The findings indicated that degenerated HEGs are reminiscent of the presence of a full-length and functional HEG, once fixed for PSA populations. The cline of HEG degeneration determined the pattern of isolation by distance. Analyses with the other markers indicated an event of demographic expansion from a population with low effective number. The different degrees of degeneration of the HEG

  17. Identification and analysis of candidate fungal tRNA 3'-end processing endonucleases tRNase Zs, homologs of the putative prostate cancer susceptibility protein ELAC2

    Directory of Open Access Journals (Sweden)

    Zhao Wei

    2010-09-01

    Full Text Available Abstract Background tRNase Z is the endonuclease that is responsible for the 3'-end processing of tRNA precursors, a process essential for tRNA 3'-CCA addition and subsequent tRNA aminoacylation. Based on their sizes, tRNase Zs can be divided into the long (tRNase ZL and short (tRNase ZS forms. tRNase ZL is thought to have arisen from a tandem gene duplication of tRNase ZS with further sequence divergence. The species distribution of tRNase Z is complex. Fungi represent an evolutionarily diverse group of eukaryotes. The recent proliferation of fungal genome sequences provides an opportunity to explore the structural and functional diversity of eukaryotic tRNase Zs. Results We report a survey and analysis of candidate tRNase Zs in 84 completed fungal genomes, spanning a broad diversity of fungi. We find that tRNase ZL is present in all fungi we have examined, whereas tRNase ZS exists only in the fungal phyla Basidiomycota, Chytridiomycota and Zygomycota. Furthermore, we find that unlike the Pezizomycotina and Saccharomycotina, which contain a single tRNase ZL, Schizosaccharomyces fission yeasts (Taphrinomycotina contain two tRNase ZLs encoded by two different tRNase ZL genes. These two tRNase ZLs are most likely localized to the nucleus and mitochondria, respectively, suggesting partitioning of tRNase Z function between two different tRNase ZLs in fission yeasts. The fungal tRNase Z phylogeny suggests that tRNase ZSs are ancestral to tRNase ZLs. Additionally, the evolutionary relationship of fungal tRNase ZLs is generally consistent with known phylogenetic relationships among the fungal species and supports tRNase ZL gene duplication in certain fungal taxa, including Schizosaccharomyces fission yeasts. Analysis of tRNase Z protein sequences reveals putative atypical substrate binding domains in most fungal tRNase ZSs and in a subset of fungal tRNase ZLs. Finally, we demonstrate the presence of pseudo-substrate recognition and catalytic motifs at

  18. Differential interaction kinetics of a bipolar structure-specific endonuclease with DNA flaps revealed by single-molecule imaging.

    Directory of Open Access Journals (Sweden)

    Rachid Rezgui

    Full Text Available As DNA repair enzymes are essential for preserving genome integrity, understanding their substrate interaction dynamics and the regulation of their catalytic mechanisms is crucial. Using single-molecule imaging, we investigated the association and dissociation kinetics of the bipolar endonuclease NucS from Pyrococcus abyssi (Pab on 5' and 3'-flap structures under various experimental conditions. We show that association of the PabNucS with ssDNA flaps is largely controlled by diffusion in the NucS-DNA energy landscape and does not require a free 5' or 3' extremity. On the other hand, NucS dissociation is independent of the flap length and thus independent of sliding on the single-stranded portion of the flapped DNA substrates. Our kinetic measurements have revealed previously unnoticed asymmetry in dissociation kinetics from these substrates that is markedly modulated by the replication clamp PCNA. We propose that the replication clamp PCNA enhances the cleavage specificity of NucS proteins by accelerating NucS loading at the ssDNA/dsDNA junctions and by minimizing the nuclease interaction time with its DNA substrate. Our data are also consistent with marked reorganization of ssDNA and nuclease domains occurring during NucS catalysis, and indicate that NucS binds its substrate directly at the ssDNA-dsDNA junction and then threads the ssDNA extremity into the catalytic site. The powerful techniques used here for probing the dynamics of DNA-enzyme binding at the single-molecule have provided new insight regarding substrate specificity of NucS nucleases.

  19. Mm19, a Mycoplasma meleagridis Major Surface Nuclease that Is Related to the RE_AlwI Superfamily of Endonucleases.

    Directory of Open Access Journals (Sweden)

    Elhem Yacoub

    Full Text Available Mycoplasma meleagridis infection is widespread in turkeys, causing poor growth and feathering, airsacculitis, osteodystrophy, and reduction in hatchability. Like most mycoplasma species, M. meleagridis is characterized by its inability to synthesize purine and pyrimidine nucleotides de novo. Consistent with this intrinsic deficiency, we here report the cloning, expression, and characterization of a M. meleagridis gene sequence encoding a major surface nuclease, referred to as Mm19. Mm19 consists of a 1941-bp ORF encoding a 646-amino-acid polypeptide with a predicted molecular mass of 74,825 kDa. BLASTP analysis revealed a significant match with the catalytic/dimerization domain of type II restriction enzymes of the RE_AlwI superfamily. This finding is consistent with the genomic location of Mm19 sequence, which dispalys characteristics of a typical type II restriction-modification locus. Like intact M. meleagridis cells, the E. coli-expressed Mm19 fusion product was found to exhibit a nuclease activity against plasmid DNA, double-stranded DNA, single-stranded DNA, and RNA. The Mm19-associated nuclease activity was consistently enhanced with Mg2+ divalent cations, a hallmark of type II restriction enzymes. A rabbit hyperimmune antiserum raised against the bacterially expressed Mm19 strongly reacted with M. meleagridis intact cells and fully neutralized the surface-bound nuclease activity. Collectively, the results show that M. meleagridis expresses a strong surface-bound nuclease activity, which is the product of a single gene sequence that is related to the RE_AlwI superfamily of endonucleases.

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

    Science.gov (United States)

    Ivanov, Yury V; Shariat, Nikki; Register, Karen B; Linz, Bodo; Rivera, Israel; Hu, Kai; Dudley, Edward G; Harvill, Eric T

    2015-10-26

    Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) are widely distributed among bacteria. These systems provide adaptive immunity against mobile genetic elements specified by the spacer sequences stored within the CRISPR. The CRISPR-Cas system has been identified using Basic Local Alignment Search Tool (BLAST) against other sequenced and annotated genomes and confirmed via CRISPRfinder program. Using Polymerase Chain Reactions (PCR) and Sanger DNA sequencing, we discovered CRISPRs in additional bacterial isolates of the same species of Bordetella. Transcriptional activity and processing of the CRISPR have been assessed via RT-PCR. Here we describe a novel Type II-C CRISPR and its associated genes-cas1, cas2, and cas9-in several isolates of a newly discovered Bordetella species. The CRISPR-cas locus, which is absent in all other Bordetella species, has a significantly lower GC-content than the genome-wide average, suggesting acquisition of this locus via horizontal gene transfer from a currently unknown source. The CRISPR array is transcribed and processed into mature CRISPR RNAs (crRNA), some of which have homology to prophages found in closely related species B. hinzii. Expression of the CRISPR-Cas system and processing of crRNAs with perfect homology to prophages present in closely related species, but absent in that containing this CRISPR-Cas system, suggest it provides protection against phage predation. The 3,117-bp cas9 endonuclease gene from this novel CRISPR-Cas system is 990 bp smaller than that of Streptococcus pyogenes, the 4,017-bp allele currently used for genome editing, and which may make it a useful tool in various CRISPR-Cas technologies.

  1. CHIP has a protective role against oxidative stress-induced cell death through specific regulation of Endonuclease G

    Science.gov (United States)

    Lee, J S; Seo, T W; Yi, J H; Shin, K S; Yoo, S J

    2013-01-01

    Oxidative stress is implicated in carcinogenesis, aging, and neurodegenerative diseases. The E3 ligase C terminus of Hsc-70 interacting protein (CHIP) has a protective role against various stresses by targeting damaged proteins for proteasomal degradation, and thus maintains protein quality control. However, the detailed mechanism by which CHIP protects cells from oxidative stress has not been demonstrated. Here, we show that depletion of CHIP led to elevated Endonuclease G (EndoG) levels and enhanced cell death upon oxidative stress. In contrast, CHIP overexpression reduced EndoG levels, and resulted in reduced or no oxidative stress-induced cell death in cancer cells and primary rat cortical neurons. Under normal conditions Hsp70 mediated the interaction between EndoG and CHIP, downregulating EndoG levels in a Hsp70/proteasome-dependent manner. However, under oxidative stress Hsp70 no longer interacted with EndoG, and the stabilized EndoG translocated to the nucleus and degraded chromosomal DNA. Our data suggest that regulation of the level of EndoG by CHIP in normal conditions may determine the sensitivity to cell death upon oxidative stress. Indeed, injection of H2O2 into the rat brain markedly increased cell death in aged mice compared with young mice, which correlated with elevated levels of EndoG and concurrent downregulation of CHIP in aged mice. Taken together, our findings demonstrate a novel protective mechanism of CHIP against oxidative stress through regulation of EndoG, and provide an opportunity to modulate oxidative stress-induced cell death in cancer and aging. PMID:23764847

  2. 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

  3. 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.

  4. Unusual evolutionary history of the tRNA splicing endonuclease EndA: relationship to the LAGLIDADG and PD-(D/E)XK deoxyribonucleases.

    Science.gov (United States)

    Bujnicki, J M; Rychlewski, L

    2001-03-01

    The tRNA splicing endoribonuclease EndA from Methanococcus jannaschii is a homotetramer formed via heterologous interaction between the two pairs of homodimers. Each monomer consists of two alpha/beta domains, the N-terminal domain (NTD) and the C-terminal domain (CTD) containing the RNase A-like active site. Comparison of the EndA coordinates with the publicly available protein structure database revealed the similarity of both domains to site-specific deoxyribonucleases: the NTD to the LAGLIDADG family and the CTD to the PD-(D/E)XK family. Superposition of the NTD on the catalytic domain of LAGLIDADG homing endonucleases allowed a suggestion to be made about which amino acid residues of the tRNA splicing nuclease might participate in formation of a presumptive cryptic deoxyribonuclease active site. On the other hand, the CTD and PD-(D/E)XK endonucleases, represented by restriction enzymes and a phage lambda exonuclease, were shown to share extensive similarities of the structural framework, to which entirely different active sites might be attached in two alternative locations. These findings suggest that EndA evolved from a fusion protein with at least two distinct endonuclease activities: the ribonuclease, which made it an essential "antitoxin" for the cells whose RNA genes were interrupted by introns, and the deoxyribonuclease, which provided the means for homing-like mobility. The residues of the noncatalytic CTDs from the positions corresponding to the catalytic side chains in PD-(D/E)XK deoxyribonucleases map to the surface at the opposite side to the tRNA binding site, for which no function has been implicated. Many restriction enzymes from the PD-(D/E)XK superfamily might have the potential to maintain an additional active or binding site at the face opposite the deoxyribonuclease active site, a property that can be utilized in protein engineering.

  5. 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

    The green alga Scenedesmus pupukensis and the red alga Porphyra spiralis contain large group-IC1 introns in their nuclear small subunit ribosomal RNA genes due to the presence of open reading frames at the 5' end of the introns. The putative 555 amino-acid Scenedesmus-encoded protein harbors...... a sequence motif resembling the bacterial S9 ribosomal proteins. The Porphyra intron self-splices in vitro, and generates both ligated exons and a full-length intron RNA circle. The Porphyra intron has an unusual structural organization by encoding a potential 149 amino-acid homing-endonuclease-like protein...

  6. Genetic discrimination for three gynogenetic clones of silver carp Hypophthalmichthys molitrix, based on restriction endonuclease analysis of Nd5-Nd6 region of mitochondrial DNA

    Science.gov (United States)

    Zhou, Jianfeng; Ye, Yuzhen; Wu, Qingjiang

    2005-03-01

    Three artificial gynogenetic clones of silver carp were produced for the analysis of restriction enzyme digestion patterns of ND5-ND6 region from mtDNA of the clones. It is revealed that all intraclonal individuals shared completely the same digestion patterns but among interclonal individuals did not. The three clones were mixed and cultured in a pond together for two years, and restriction endonuclease digestion patterns of ND5 ND6 were used as genetic markers to assess the growth performance of each clone.

  7. Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines

    DEFF Research Database (Denmark)

    Mohammed, M Z; Vyjayanti, V N; Laughton, C A

    2011-01-01

    Modulation of DNA base excision repair (BER) has the potential to enhance response to chemotherapy and improve outcomes in tumours such as melanoma and glioma. APE1, a critical protein in BER that processes potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer....... In the current study, we aimed to develop small molecule inhibitors of APE1 for cancer therapy....

  8. 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

  9. 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

  10. Single-Base Pair Genome Editing in Human Cells by Using Site-Specific Endonucleases

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    Hiroshi Ochiai

    2015-09-01

    Full Text Available Genome-wide association studies have identified numerous single-nucleotide polymorphisms (SNPs associated with human diseases or phenotypes. However, causal relationships between most SNPs and the associated disease have not been established, owing to technical challenges such as unavailability of suitable cell lines. Recently, efficient editing of a single base pair in the genome was achieved using programmable site-specific nucleases. This technique enables experimental confirmation of the causality between SNPs and disease, and is potentially valuable in clinical applications. In this review, I introduce the molecular basis and describe examples of single-base pair editing in human cells. I also discuss the challenges associated with the technique, as well as possible solutions.

  11. A homology model of restriction endonuclease SfiI in complex with DNA

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    Skowronek Krzysztof J

    2005-01-01

    Full Text Available Abstract Background Restriction enzymes (REases are commercial reagents commonly used in recombinant DNA technologies. They are attractive models for studying protein-DNA interactions and valuable targets for protein engineering. They are, however, extremely divergent: the amino acid sequence of a typical REase usually shows no detectable similarities to any other proteins, with rare exceptions of other REases that recognize identical or very similar sequences. From structural analyses and bioinformatics studies it has been learned that some REases belong to at least four unrelated and structurally distinct superfamilies of nucleases, PD-DxK, PLD, HNH, and GIY-YIG. Hence, they are extremely hard targets for structure prediction and homology-based inference of sequence-function relationships and the great majority of REases remain structurally and evolutionarily unclassified. Results SfiI is a REase which recognizes the interrupted palindromic sequence 5'GGCCNNNN^NGGCC3' and generates 3 nt long 3' overhangs upon cleavage. SfiI is an archetypal Type IIF enzyme, which functions as a tetramer and cleaves two copies of the recognition site in a concerted manner. Its sequence shows no similarity to other proteins and nothing is known about the localization of its active site or residues important for oligomerization. Using the threading approach for protein fold-recognition, we identified a remote relationship between SfiI and BglI, a dimeric Type IIP restriction enzyme from the PD-DxK superfamily of nucleases, which recognizes the 5'GCCNNNN^NGGC3' sequence and whose structure in complex with the substrate DNA is available. We constructed a homology model of SfiI in complex with its target sequence and used it to predict residues important for dimerization, tetramerization, DNA binding and catalysis. Conclusions The bioinformatics analysis suggest that SfiI, a Type IIF enzyme, is more closely related to BglI, an "orthodox" Type IIP restriction enzyme

  12. Restriction endonuclease analysis of mitochondrial DNA of Acanthamoeba sp. YM-4 (Korean isolate).

    Science.gov (United States)

    Shin, H J; Im, K; Jeon, K W

    1997-06-01

    Acanthamoeba sp. YM-4 is similar to A. culbertsoni based upon morphological characteristics of trophozoites and cysts. However, based on other characteristics, pathogenicity to mice, in vitro cytotoxicity and isoenzyme patterns. Acanthamoeba sp. YM-4 was quite different from A. culbertsoni. Restriction fragment length polymorphism (RFLP) analysis of mtDNA is useful in the classification of members belonging to the genus Acanthamoeba. Therefore, in this study, RFLP analysis of Acanthamoeba mtDNAs was accomplished using five restriction enzymes: HaeIII, HindIII, ClaI, PvuII and SalI. Each restriction enzyme produced approximately 3-15 fragments (range: from 0.6 kbp to 34.4 kbp). The mtDNA genome size, calculated by the summation of restriction fragments, averaged 46.4 kbp in Acanthamoeba sp. YM-4, 48.3 kbp in A. culbertsoni and 48.8 kbp in A. polyphaga, respectively. Digested mtDNA fragments of Acanthamoeba sp. YM-4 contained nine and seven same size fragments, respectively, from a total of 67 and 69 fragments observed in A. culbertsoni and A. polyphaga. An estimate of the genetic divergence was 10.1% between Acanthamoeba sp. YM-4 and A. culbertsoni, and 9.9% between Acanthamoeba sp. YM-4 and A. polyphaga.

  13. Biochemical characterization and novel inhibitor identification ofMycobacterium tuberculosisEndonuclease VIII 2 (Rv3297).

    Science.gov (United States)

    Lata, Kiran; Afsar, Mohammad; Ramachandran, Ravishankar

    2017-12-01

    Nei2 (Rv3297) is a DNA Base Excision Repair (BER) glycosylase that is essential for survival of Mycobacterium tuberculosis in primates. We show that MtbNei2 is a bifunctional glycosylase that specifically acts on oxidized pyrimidine-containing single-stranded, double-stranded, 5'/3' fork and bubble DNA substrates. MtbNei2 possesses Uracil DNA glycosylase activity unlike E. coli Nei. Mutational studies demonstrate that Pro2 and Glu3 located in the active site are essential for glycosylase activity of MtbNei2. Mutational analysis demonstrated that an unstructured C-terminal zinc finger domain that was important for activity in E. coli Nei and Fpg, was not required for the glycosylase activity of MtbNei2. Lastly, we screened the NCI natural product compound database and identified three natural product inhibitors with IC50 values ranging between 41.8 μM-92.7 μM against MtbNei2 in in vitro inhibition assays. Surface Plasmon Resonance (SPR) experiments showed that the binding affinity of the best inhibitor, NSC31867, was 74 nM. The present results set the stage for exploiting this important target in developing new therapeutic strategies that target Mycobacterial BER.

  14. Biochemical characterization and novel inhibitor identification of Mycobacterium tuberculosis Endonuclease VIII 2 (Rv3297

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    Kiran Lata

    2017-12-01

    Full Text Available Nei2 (Rv3297 is a DNA Base Excision Repair (BER glycosylase that is essential for survival of Mycobacterium tuberculosis in primates. We show that MtbNei2 is a bifunctional glycosylase that specifically acts on oxidized pyrimidine-containing single-stranded, double-stranded, 5’/3’ fork and bubble DNA substrates. MtbNei2 possesses Uracil DNA glycosylase activity unlike E. coli Nei. Mutational studies demonstrate that Pro2 and Glu3 located in the active site are essential for glycosylase activity of MtbNei2. Mutational analysis demonstrated that an unstructured C-terminal zinc finger domain that was important for activity in E. coli Nei and Fpg, was not required for the glycosylase activity of MtbNei2. Lastly, we screened the NCI natural product compound database and identified three natural product inhibitors with IC50 values ranging between 41.8 μM-92.7 μM against MtbNei2 in in vitro inhibition assays. Surface Plasmon Resonance (SPR experiments showed that the binding affinity of the best inhibitor, NSC31867, was 74 nM. The present results set the stage for exploiting this important target in developing new therapeutic strategies that target Mycobacterial BER.

  15. Diverse small molecule inhibitors of human apurinic/apyrimidinic endonuclease APE1 identified from a screen of a large public collection.

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    Dorjbal Dorjsuren

    Full Text Available The major human apurinic/apyrimidinic endonuclease APE1 plays a pivotal role in the repair of base damage via participation in the DNA base excision repair (BER pathway. Increased activity of APE1, often observed in tumor cells, is thought to contribute to resistance to various anticancer drugs, whereas down-regulation of APE1 sensitizes cells to DNA damaging agents. Thus, inhibiting APE1 repair endonuclease function in cancer cells is considered a promising strategy to overcome therapeutic agent resistance. Despite ongoing efforts, inhibitors of APE1 with adequate drug-like properties have yet to be discovered. Using a kinetic fluorescence assay, we conducted a fully-automated high-throughput screen (HTS of the NIH Molecular Libraries Small Molecule Repository (MLSMR, as well as additional public collections, with each compound tested as a 7-concentration series in a 4 µL reaction volume. Actives identified from the screen were subjected to a panel of confirmatory and counterscreen tests. Several active molecules were identified that inhibited APE1 in two independent assay formats and exhibited potentiation of the genotoxic effect of methyl methanesulfonate with a concomitant increase in AP sites, a hallmark of intracellular APE1 inhibition; a number of these chemotypes could be good starting points for further medicinal chemistry optimization. To our knowledge, this represents the largest-scale HTS to identify inhibitors of APE1, and provides a key first step in the development of novel agents targeting BER for cancer treatment.

  16. Host Factors Influencing the Retrohoming Pathway of Group II Intron RmInt1, Which Has an Intron-Encoded Protein Naturally Devoid of Endonuclease Activity.

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    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.

  17. Binding affinity of the L-742,001 inhibitor to the endonuclease domain of A/H1N1/PA influenza virus variants: Molecular simulation approaches

    Science.gov (United States)

    Nguyen, Hung; Nguyen, Hoang Linh; Linh, Huynh Quang; Nguyen, Minh Tho

    2018-01-01

    The steered molecular dynamics (SMD), molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) and free energy perturbation (FEP) methods were used to determine the binding affinity of the L-742,001 inhibitor to the endonuclease domain of the A/H1N1/PA influenza viruses (including wild type (WT) and three mutations I79L, E119D and F105S for both pH1N1 PA and PR8 PA viruses). Calculated results showed that the L-742,001 inhibitor not only binds to the PR8 PAs (1934 A influenza virus) better than to the pH1N1 PAs (2009 A influenza virus) but also more strongly interacts with the WT endonuclease domain than with three mutant variants for both pH1N1 PA and PR8 PA viruses. The binding affinities obtained by the SMD, MM-PBSA and FEP methods attain high correlation with available experimental data. Here the FEP method appears to provide a more accurate determination of the binding affinity than the SMD and MM-PBSA counterparts.

  18. Structure-specific endonucleases

    DEFF Research Database (Denmark)

    Minocherhomji, Sheroy; Hickson, Ian D

    2014-01-01

    Fragile sites are conserved loci predisposed to form breaks in metaphase chromosomes. The inherent instability of these loci is associated with chromosomal rearrangements in cancers and is a feature of cells from patients with chromosomal instability syndromes. One class of fragile sites, the com...

  19. Type II restriction endonuclease R.Hpy188I belongs to the GIY-YIG nuclease superfamily, but exhibits an unusual active site.

    Science.gov (United States)

    Kaminska, Katarzyna H; Kawai, Mikihiko; Boniecki, Michal; Kobayashi, Ichizo; Bujnicki, Janusz M

    2008-11-14

    Catalytic domains of Type II restriction endonucleases (REases) belong to a few unrelated three-dimensional folds. While the PD-(D/E)XK fold is most common among these enzymes, crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI). Bioinformatics analyses supported by mutagenesis experiments suggested that some REases belong to the HNH fold (e.g. R.KpnI), and that a small group represented by R.Eco29kI belongs to the GIY-YIG fold. However, for a large fraction of REases with known sequences, the three-dimensional fold and the architecture of the active site remain unknown, mostly due to extreme sequence divergence that hampers detection of homology to enzymes with known folds. R.Hpy188I is a Type II REase with unknown structure. PSI-BLAST searches of the non-redundant protein sequence database reveal only 1 homolog (R.HpyF17I, with nearly identical amino acid sequence and the same DNA sequence specificity). Standard application of state-of-the-art protein fold-recognition methods failed to predict the relationship of R.Hpy188I to proteins with known structure or to other protein families. In order to increase the amount of evolutionary information in the multiple sequence alignment, we have expanded our sequence database searches to include sequences from metagenomics projects. This search resulted in identification of 23 further members of R.Hpy188I family, both from metagenomics and the non-redundant database. Moreover, fold-recognition analysis of the extended R.Hpy188I family revealed its relationship to the GIY-YIG domain and allowed for computational modeling of the R.Hpy188I structure. Analysis of the R.Hpy188I model in the light of sequence conservation among its homologs revealed an unusual variant of the active site, in which the typical Tyr residue of the YIG half-motif had been substituted by a Lys residue. Moreover, some of its homologs have the otherwise invariant Arg residue in

  20. Type II restriction endonuclease R.Hpy188I belongs to the GIY-YIG nuclease superfamily, but exhibits an unusual active site

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    Kobayashi Ichizo

    2008-11-01

    Full Text Available Abstract Background Catalytic domains of Type II restriction endonucleases (REases belong to a few unrelated three-dimensional folds. While the PD-(D/EXK fold is most common among these enzymes, crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI and half-pipe (R.PabI. Bioinformatics analyses supported by mutagenesis experiments suggested that some REases belong to the HNH fold (e.g. R.KpnI, and that a small group represented by R.Eco29kI belongs to the GIY-YIG fold. However, for a large fraction of REases with known sequences, the three-dimensional fold and the architecture of the active site remain unknown, mostly due to extreme sequence divergence that hampers detection of homology to enzymes with known folds. Results R.Hpy188I is a Type II REase with unknown structure. PSI-BLAST searches of the non-redundant protein sequence database reveal only 1 homolog (R.HpyF17I, with nearly identical amino acid sequence and the same DNA sequence specificity. Standard application of state-of-the-art protein fold-recognition methods failed to predict the relationship of R.Hpy188I to proteins with known structure or to other protein families. In order to increase the amount of evolutionary information in the multiple sequence alignment, we have expanded our sequence database searches to include sequences from metagenomics projects. This search resulted in identification of 23 further members of R.Hpy188I family, both from metagenomics and the non-redundant database. Moreover, fold-recognition analysis of the extended R.Hpy188I family revealed its relationship to the GIY-YIG domain and allowed for computational modeling of the R.Hpy188I structure. Analysis of the R.Hpy188I model in the light of sequence conservation among its homologs revealed an unusual variant of the active site, in which the typical Tyr residue of the YIG half-motif had been substituted by a Lys residue. Moreover, some of its homologs

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

    Science.gov (United States)

    Zylicz-Stachula, Agnieszka; Bujnicki, Janusz M; Skowron, Piotr M

    2009-05-29

    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. 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. 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/E)XK nuclease domain related to the corresponding domains in HsdR subunits, but lacks the ATP-dependent translocase module of the HsdR subunit and the additional domains that

  2. 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

  3. Purification, crystallization and preliminary X-ray analysis of the HsdR subunit of the EcoR124I endonuclease from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Lapkouski, Mikalai [Institute of Physical Biology, University of South Bohemia in Ceske Budejovice, Zamek 136, CZ-373 33 Nove Hrady (Czech Republic); Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic, Zamek 136, CZ-373 33 Nove Hrady (Czech Republic); Panjikar, Santosh [EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg (Germany); Kuta Smatanova, Ivana; Csefalvay, Eva, E-mail: jindrova@greentech.cz [Institute of Physical Biology, University of South Bohemia in Ceske Budejovice, Zamek 136, CZ-373 33 Nove Hrady (Czech Republic); Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic, Zamek 136, CZ-373 33 Nove Hrady (Czech Republic)

    2007-07-01

    The purification, crystallization and preliminary diffraction analysis of the HsdR subunit of the EcoR124I endonuclease are described. EcoR124I is a multicomplex enzyme belonging to the type I restriction-modification system from Escherichia coli. Although EcoR124I has been extensively characterized biochemically, there is no direct structural information available about particular subunits. HsdR is a motor subunit that is responsible for ATP hydrolysis, DNA translocation and cleavage of the DNA substrate recognized by the complex. Recombinant HsdR subunit was crystallized using the sitting-drop vapour-diffusion method. Crystals belong to the primitive monoclinic space group, with unit-cell parameters a = 85.75, b = 124.71, c = 128.37 Å, β = 108.14°. Native data were collected to 2.6 Å resolution at the X12 beamline of EMBL Hamburg.

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

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    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.

  5. Identification and quantification of DNA repair protein apurinic/apyrimidinic endonuclease 1 (APE1 in human cells by liquid chromatography/isotope-dilution tandem mass spectrometry.

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    Güldal Kirkali

    Full Text Available Unless repaired, DNA damage can drive mutagenesis or cell death. DNA repair proteins may therefore be used as biomarkers in disease etiology or therapeutic response prediction. Thus, the accurate determination of DNA repair protein expression and genotype is of fundamental importance. Among DNA repair proteins involved in base excision repair, apurinic/apyrimidinic endonuclease 1 (APE1 is the major endonuclease in mammals and plays important roles in transcriptional regulation and modulating stress responses. Here, we present a novel approach involving LC-MS/MS with isotope-dilution to positively identify and accurately quantify APE1 in human cells and mouse tissue. A completely (15N-labeled full-length human APE1 was produced and used as an internal standard. Fourteen tryptic peptides of both human APE1 (hAPE1 and (15N-labeled hAPE1 were identified following trypsin digestion. These peptides matched the theoretical peptides expected from trypsin digestion and provided a statistically significant protein score that would unequivocally identify hAPE1. Using the developed methodology, APE1 was positively identified and quantified in nuclear and cytoplasmic extracts of multiple human cell lines and mouse liver using selected-reaction monitoring of typical mass transitions of the tryptic peptides. We also show that the methodology can be applied to the identification of hAPE1 variants found in the human population. The results describe a novel approach for the accurate measurement of wild-type and variant forms of hAPE1 in vivo, and ultimately for defining the role of this protein in disease development and treatment responses.

  6. Fluorescence quenching of graphene oxide combined with the site-specific cleavage of restriction endonuclease for deoxyribonucleic acid demethylase activity assay

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Lijuan; Qian, Yingdan; Wu, Ping; Zhang, Hui; Cai, Chenxin, E-mail: cxcai@njnu.edu.cn

    2015-04-15

    Highlights: • An approach for sensitive and selective DNA demethylase activity assay is reported. • This assay is based on the fluorescence quenching of GO and site-specific cleavage of endonuclease. • It can determine as low as 0.05 ng mL{sup −1} of MBD2 with a linear range of 0.2–300 ng mL{sup −1}. • It has an ability to recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. • It can avoid false signals, requiring no bisulfite conversion, PCR amplification, radioisotope-labeling. - Abstract: We report on the development of a sensitive and selective deoxyribonucleic acid (DNA) demethylase (using MBD2 as an example) activity assay by coupling the fluorescence quenching of graphene oxide (GO) with the site-specific cleavage of HpaII endonuclease to improve the selectivity. This approach was developed by designing a single-stranded probe (P1) that carries a binding region to facilitate the interaction with GO, which induces fluorescence quenching of the labeled fluorophore (FAM, 6-carboxyfluorescein), and a sensing region, which contains a hemi-methylated site of 5′-CmCGG-3′, to specifically recognize the target (T1, a 32-mer DNA from the promoter region of p53 gene) and hybridize with it to form a P1/T1 duplex. After demethylation with MBD2, the duplex can be specifically cleaved using HpaII, which releases the labeled FAM from the GO surface and results in the recovery of fluorescence. However, this cleavage is blocked by the hemi-methylation of this site. Thus, the magnitude of the recovered fluorescence signal is related to the MBD2 activity, which establishes the basis of the DNA demethylase activity assay. This assay can determine as low as ∼(0.05 ± 0.01) ng mL{sup −1} (at a signal/noise of 3) of MBD2 with a linear range of 0.2–300 ng mL{sup −1} and recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. The advantage of this assay is its ability to avoid false signals and no

  7. The novel influenza A virus protein PA-X and its naturally deleted variant show different enzymatic properties in comparison to the viral endonuclease PA.

    Science.gov (United States)

    Bavagnoli, Laura; Cucuzza, Stefano; Campanini, Giulia; Rovida, Francesca; Paolucci, Stefania; Baldanti, Fausto; Maga, Giovanni

    2015-10-30

    The PA protein of Influenza A virus (IAV) encoded by segment 3 acts as a specialized RNA endonuclease in the transcription of the viral genome. The same genomic segment encodes for a second shorter protein, termed PA-X, with the first 191 N-terminal aminoacids (aa) identical to PA, but with a completely different C-ter domain of 61 aa, due to a ribosomal frameshifting. In addition, it has been shown that several IAV isolates encode for a naturally truncated PA-X variant, PAXΔC20, missing the last 20 aa. The biochemical properties of PA-X and PAXΔC20 have been poorly investigated so far. Here, we have carried out an enzymatic characterization of PA-X and its naturally deleted form, in comparison with PA from the human IAV strain A/WSN/33 (H1N1). Our results showed, to the best of our knowledge for the first time, that PA-X possesses an endonucleolytic activity. Both PA and PA-X preferentially cut single stranded RNA regions, but with some differences. In addition, we showed that PAXΔC20 has severely reduced nuclease activity. These results point to a previously undetected role of the last C-ter 20 aa for the catalytic activity of PA-X and support distinct roles for these proteins in the viral life cycle. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Construction of a high-efficiency cloning system using the Golden Gate method and I-SceI endonuclease for targeted gene replacement in Bacillus anthracis.

    Science.gov (United States)

    Wang, Tiantian; Wang, Dongshu; Lyu, Yufei; Feng, Erling; Zhu, Li; Liu, Chunjie; Wang, Yanchun; Liu, Xiankai; Wang, Hengliang

    2018-02-10

    To investigate gene function in Bacillus anthracis, a high-efficiency cloning system is required with an increased rate of allelic exchange. Golden Gate cloning is a molecular cloning strategy allowing researchers to simultaneously and directionally assemble multiple DNA fragments to construct target plasmids using type IIs restriction enzymes and T4 DNA ligase in the same reaction system. Here, a B. anthracis S-layer protein EA1 allelic exchange vector was successfully constructed using the Golden Gate method. No new restriction sites were introduced into this knockout vector, and seamless assembly of the DNA fragments was achieved. To elevate the efficiency of homologous recombination between the allelic exchange vector and chromosomal DNA, we introduced an I-SceI site into the allelic exchange vector. The eag gene was successfully knocked out in B. anthracis using this vector. Simultaneously, the allelic exchange vector construction method was developed into a system for generating B. anthracis allelic exchange vectors. To verify the effectiveness of this system, some other allelic exchange vectors were constructed and gene replacements were performed in B. anthracis. It is speculated that this gene knockout vector construction system and high-efficiency targeted gene replacement using I-SceI endonuclease can be applied to other Bacillus spp. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Mycobacterium tuberculosis class II apurinic/apyrimidinic-endonuclease/3'-5' exonuclease III exhibits DNA regulated modes of interaction with the sliding DNA β-clamp.

    Science.gov (United States)

    Khanam, Taran; Rai, Niyati; Ramachandran, Ravishankar

    2015-10-01

    The class-II AP-endonuclease (XthA) acts on abasic sites of damaged DNA in bacterial base excision repair. We identified that the sliding DNA β-clamp forms in vivo and in vitro complexes with XthA in Mycobacterium tuberculosis. A novel 239 QLRFPKK245 motif in the DNA-binding domain of XthA was found to be important for the interactions. Likewise, the peptide binding-groove (PBG) and the C-terminal of β-clamp located on different domains interact with XthA. The β-clamp-XthA complex can be disrupted by clamp binding peptides and also by a specific bacterial clamp inhibitor that binds at the PBG. We also identified that β-clamp stimulates the activities of XthA primarily by increasing its affinity for the substrate and its processivity. Additionally, loading of the β-clamp onto DNA is required for activity stimulation. A reduction in XthA activity stimulation was observed in the presence of β-clamp binding peptides supporting that direct interactions between the proteins are necessary to cause stimulation. Finally, we found that in the absence of DNA, the PBG located on the second domain of the β-clamp is important for interactions with XthA, while the C-terminal domain predominantly mediates functional interactions in the substrate's presence. © 2015 John Wiley & Sons Ltd.

  10. A chronocoulometric LNA sensor for amplified detection of K-ras mutation based on site-specific DNA cleavage of restriction endonuclease.

    Science.gov (United States)

    Lin, Liqing; Liu, Ailin; Zhao, Chengfei; Weng, Shaohuang; Lei, Yun; Liu, Qicai; Lin, Xinhua; Chen, Yuanzhong

    2013-04-15

    An amplified chronocoulometric Locked nucleic acid (LNA) sensor (CLS) for selective electrochemical detection of K-ras mutation was developed based on site-specific DNA cleavage of restriction endonuclease EcoRI. Thiolated-hairpin LNA probe with palindrome structure of stem was immobilized on the gold nanoparticles modified gold electrode (NG/AuE). It can be cleaved by EcoRI in the absence of K-ras mutation-type DNA (complementary with the loop part of hairpin probe), but cannot be cleaved in the presence of mutation-type DNA. The difference before and after enzymatic cleavage was then monitored by chronocoulometric biosensor. Electrochemical signals are generated by chronocoulometric interrogation of Hexaammineruthenium (III) chloride (RuHex) that quantitatively binds to surface-confined hairpin LNA probe via electrostatic interactions. The results suggested this CLS had a good specificity to distinguish the K-ras mutation-type, wild-type and non-complementary sequence. There was a good linear relationship between the charge and the logarithmic function of K-ras mutation-type DNA concentration. The detection limit had been estimated as 0.5 fM. It is possible to qualitatively and quantitatively detect K-ras point mutation in pancreatic cancer. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Crystal structure and mutational analysis of Mycobacterium smegmatis FenA highlight active site amino acids and three metal ions essential for flap endonuclease and 5' exonuclease activities.

    Science.gov (United States)

    Uson, Maria Loressa; Carl, Ayala; Goldgur, Yehuda; Shuman, Stewart

    2018-04-09

    Mycobacterium smegmatis FenA is a nucleic acid phosphodiesterase with flap endonuclease and 5' exonuclease activities. The 1.8 Å crystal structure of FenA reported here highlights as its closest homologs bacterial FEN-family enzymes ExoIX, the Pol1 exonuclease domain and phage T5 Fen. Mycobacterial FenA assimilates three active site manganese ions (M1, M2, M3) that are coordinated, directly and via waters, to a constellation of eight carboxylate side chains. We find via mutagenesis that the carboxylate contacts to all three manganese ions are essential for FenA's activities. Structures of nuclease-dead FenA mutants D125N, D148N and D208N reveal how they fail to bind one of the three active site Mn2+ ions, in a distinctive fashion for each Asn change. The structure of FenA D208N with a phosphate anion engaged by M1 and M2 in a state mimetic of a product complex suggests a mechanism for metal-catalyzed phosphodiester hydrolysis similar to that proposed for human Exo1. A distinctive feature of FenA is that it does not have the helical arch module found in many other FEN/FEN-like enzymes. Instead, this segment of FenA adopts a unique structure comprising a short 310 helix and surface β-loop that coordinates a fourth manganese ion (M4).

  12. Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases.

    Science.gov (United States)

    Yang, Diane; Scavuzzo, Marissa A; Chmielowiec, Jolanta; Sharp, Robert; Bajic, Aleksandar; Borowiak, Malgorzata

    2016-02-18

    Efficient gene editing is essential to fully utilize human pluripotent stem cells (hPSCs) in regenerative medicine. Custom endonuclease-based gene targeting involves two mechanisms of DNA repair: homology directed repair (HDR) and non-homologous end joining (NHEJ). HDR is the preferred mechanism for common applications such knock-in, knock-out or precise mutagenesis, but remains inefficient in hPSCs. Here, we demonstrate that synchronizing synchronizing hPSCs in G2/M with ABT phase increases on-target gene editing, defined as correct targeting cassette integration, 3 to 6 fold. We observed improved efficiency using ZFNs, TALENs, two CRISPR/Cas9, and CRISPR/Cas9 nickase to target five genes in three hPSC lines: three human embryonic stem cell lines, neural progenitors and diabetic iPSCs. neural progenitors and diabetic iPSCs. Reversible synchronization has no effect on pluripotency or differentiation. The increase in on-target gene editing is locus-independent and specific to the cell cycle phase as G2/M phase enriched cells show a 6-fold increase in targeting efficiency compared to cells in G1 phase. Concurrently inhibiting NHEJ with SCR7 does not increase HDR or improve gene targeting efficiency further, indicating that HR is the major DNA repair mechanism after G2/M phase arrest. The approach outlined here makes gene editing in hPSCs a more viable tool for disease modeling, regenerative medicine and cell-based therapies.

  13. Human DNA contains sequences homologous to the 5'-non-coding region of hepatits C virus: characterization with restriction endonucleases reveals individual varieties.

    Science.gov (United States)

    Dennin, Reinhard H; Wo, Jianer

    2003-07-01

    To investigate a 272 base pair section of the 5'-non-coding region of genomic DNA from the peripheral blood monounuclear cells of healthy hepatitis virus C (HCV)-negative human subjects (not patients). This sequence section bears interest because (1) it harbors several potential methylation (Cp-rich) sites, and (2) it represents the largest part of its internal ribosomal entry site. A pre-PCR digestion protocol was established making consistent use of four restriction endonucleases selected for certain features: SmaI, XmaCI, MspI, and HpaII are inhibited if methylation(s) are present at certain cytosines within their cutting sequences. The suspected HCV-specific sequence was found in the DNA of each subject tested. The pre-PCR digestion assay reveals individual differences in their pattern of methylation, which may be due to possible epigenetic phenomena. The results provide formal proof that these HCV-specific sequences are contained in the genomic or extra chromosomal target DNA, and probably belong to a new class of endogenous sequences.

  14. Human apurinic/apyrimidinic endonuclease (APE1) is a prognostic factor in ovarian, gastro-oesophageal and pancreatico-biliary cancers.

    Science.gov (United States)

    Al-Attar, A; Gossage, L; Fareed, K R; Shehata, M; Mohammed, M; Zaitoun, A M; Soomro, I; Lobo, D N; Abbotts, R; Chan, S; Madhusudan, S

    2010-02-16

    Altered DNA repair may be associated with aggressive tumour biology and impact upon response to chemotherapy and radiotherapy. We investigated whether expression of human AP endonuclease (APE1), a key multifunctional protein involved in DNA BER, would impact on clinicopathological outcomes in ovarian, gastro-oesophageal, and pancreatico-biliary cancer. Formalin-fixed human ovarian, gastro-oesophageal, and pancreatico-biliary cancers were constructed into TMAs. Expression of APE1 was analysed by IHC and correlated to clinicopathological variables. In ovarian cancer, nuclear APE1 expression was seen in 71.9% (97 out of 135) of tumours and correlated with tumour type (P=0.006), optimal debulking (P=0.009), and overall survival (P=0.05). In gastro-oesophageal cancers previously exposed to neoadjuvant chemotherapy, 34.8% (16 out of 46) of tumours were positive in the nucleus and this correlated with shorter overall survival (P=0.005), whereas cytoplasmic localisation correlated with tumour dedifferentiation (P=0.034). In pancreatico-biliary cancer, nuclear staining was seen in 44% (32 out of 72) of tumours. Absence of cytoplasmic staining was associated with perineural invasion (P=0.007), vascular invasion (P=0.05), and poorly differentiated tumours (P=0.068). A trend was noticed with advanced stage (P=0.077). Positive clinicopathological correlations of APE1 expression suggest that APE1 is a potential drug target in ovarian, gastro-oesophageal, and pancreatico-biliary cancers.

  15. A putative Type IIS restriction endonuclease GeoICI from Geobacillus sp.--A robust, thermostable alternative to mezophilic prototype BbvI.

    Science.gov (United States)

    Zebrowska, Joanna; Zolnierkiewicz, Olga; Skowron, Marta A; Zylicz-Stachula, Agnieszka; Jezewska-Frackowiak, Joanna; Skowron, Piotr M

    2016-03-01

    Screening of extreme environments in search for novel microorganisms may lead to the discovery of robust enzymes with either new substrate specificities or thermostable equivalents of those already found in mesophiles, better suited for biotechnology applications. Isolates from Iceland geysers' biofilms, exposed to a broad range of temperatures, from ambient to close to water boiling point, were analysed for the presence of DNA-interacting proteins, including restriction endonucleases (REases). GeoICI, a member of atypical Type IIS REases, is the most thermostable isoschizomer of the prototype BbvI, recognizing/cleaving 5'-GCAGC(N8/12)-3'DNA sequences. 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, (iii) run-off sequencing of GeoICI cleavage products and (iv) shotgun cloning and sequencing of Λ DNA fragmented with GeoICI. Geobacillus sp. genomic DNA was PCR-screened for the presence of other specialized REases-MTases and as a result, another putative REase- MTase, GeoICII, related to the Thermus sp. family of bifunctional REases-methyltransferases (MTases) was detected.

  16. In vivo expression of the nucleolar group I intron-encoded I-dirI homing endonuclease involves the removal of a spliceosomal intron

    DEFF Research Database (Denmark)

    Vader, A; Nielsen, Henrik; Johansen, S

    1999-01-01

    as truncation and polyadenylation downstream of the ORF 3' end. A spliceosomal intron, the first to be reported within a group I intron and the rDNA, is removed before the I-DirI mRNA associates with the polysomes. Taken together, our results imply that DiSSU1 uses a unique combination of intron......The Didymium iridis DiSSU1 intron is located in the nuclear SSU rDNA and has an unusual twin-ribozyme organization. One of the ribozymes (DiGIR2) catalyses intron excision and exon ligation. The other ribozyme (DiGIR1), which along with the endonuclease-encoding I-DirI open reading frame (ORF......) is inserted in DiGIR2, carries out hydrolysis at internal processing sites (IPS1 and IPS2) located at its 3' end. Examination of the in vivo expression of DiSSU1 shows that after excision, DiSSU1 is matured further into the I-DirI mRNA by internal DiGIR1-catalysed cleavage upstream of the ORF 5' end, as well...

  17. The herpesvirus alkaline exonuclease belongs to the restriction endonuclease PD-(D/E)XK superfamily: insight from molecular modeling and phylogenetic analysis.

    Science.gov (United States)

    Bujnicki, J M; Rychlewski, L

    2001-03-01

    The PD-(D/E)XK superfamily of deoxyribonucleases (ENases) comprises restriction endonucleases, exonucleases and nicking enzymes, which share a common fold and the architecture of the active site. Their extreme divergence generally hampers identification of novel members based solely on sequence comparisons. Here we report a remote similarity between the phage lambda exonuclease (lambda-exo), branching out early in the evolutionary history of ENases (3), with the family of alkaline exonucleases (AE) encoded by various viruses infecting higher Eukaryota. The predicted structural compatibility and the conservation of the functionally important residues between AE and ENases strongly suggest a distant evolutionary relationship between these proteins. According to the results of extensive sequence database mining, sequence/structure threading and molecular modeling it is plausible that the AE proteins with lambda-exo and some other putative phage-encoded exonucleases form a distinct subfamily of PD-(D/E)XK ENases. The phylogenetic history of this subfamily is inferred using sequence alignment and distance matrix methods.

  18. Rapid and Sensitive Detection of Shigella spp. and Salmonella spp. by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique

    Science.gov (United States)

    Wang, Yi; Wang, Yan; Luo, Lijuan; Liu, Dongxin; Luo, Xia; Xu, Yanmei; Hu, Shoukui; Niu, Lina; Xu, Jianguo; Ye, Changyun

    2015-01-01

    Shigella and Salmonella are frequently isolated from various food samples and can cause human gastroenteritis. Here, a novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully established and validated for simultaneous detection of Shigella strains and Salmonella strains in only a single reaction. Two sets of MERT-LAMP primers for 2 kinds of pathogens were designed from ipaH gene of Shigella spp. and invA gene of Salmonella spp., respectively. Under the constant condition at 63°C, the positive results were yielded in as short as 12 min with the genomic DNA extracted from the 19 Shigella strains and 14 Salmonella strains, and the target pathogens present in a sample could be simultaneously identified based on distinct fluorescence curves in real-time format. Accordingly, the multiplex detection assay significantly reduced effort, materials and reagents used, and amplification and differentiation were conducted at the same time, obviating the use of postdetection procedures. The analytical sensitivity of MERT-LAMP was found to be 62.5 and 125 fg DNA/reaction with genomic templates of Shigella strains and Salmonella strains, which was consist with normal LAMP assay, and at least 10- and 100-fold more sensitive than that of qPCR and conventional PCR approaches. The limit of detection of MERT-LAMP for Shigella strains and Salmonella strains detection in artificially contaminated milk samples was 5.8 and 6.4 CFU per vessel. In conclusion, the MERT-LAMP methodology described here demonstrated a potential and valuable means for simultaneous screening of Shigella and Salmonella in a wide variety of samples. PMID:26697000

  19. A new prototype IIS/IIC/IIG endonuclease-methyltransferase TsoI from the thermophile Thermus scotoductus, recognising 5'-TARCCA(N11/9)-3' sequences.

    Science.gov (United States)

    Jezewska-Frackowiak, Joanna; Lubys, Arvydas; Vitkute, Jolanta; Zakareviciene, Laimute; Zebrowska, Joanna; Krefft, Daria; Skowron, Marta A; Zylicz-Stachula, Agnieszka; Skowron, Piotr M

    2015-01-20

    The Thermus sp. family of IIS/IIG/IIC enzymes includes the thermostable, bifunctional, fused restriction endonuclease (REase)-methyltransferases (MTase): TaqII, Tth111II/TthHB27I, TspGWI, TspDTI and TsoI. The enzymes are large proteins (approximately 120kDa), their enzymatic activities are affected by S-adenosylmethionine (SAM), they recognise similar asymmetric cognate sites and cleave at a distance of 11/9 nucleotides (nt). The enzymes exhibit similarities of their amino acid (aa) sequences and DNA catalytic motifs. Thermus sp. enzymes are an example of functional aa sequence homologies among REases recognising different, yet related DNA sequences. The family consists of TspGWI- and TspDTI-subfamilies. TsoI appears to be a non-identical 'triplet', related to TspDTI and Tth111II/TthHB27I. The discovery of TsoI, purified from Thermus scotoductus, is described. This prototype, displaying a novel specificity, which was determined by: (i) cleavage of a reference plasmid and bacteriophage DNA, (ii) cleavage of custom PCR DNA substrates, (iii) run-off sequencing of cleavage products and (iv) shotgun cloning and sequencing of bacteriophage lambda (λ) DNA digested with TsoI. The enzyme recognises a degenerated 5'-TARCCA-3' sequence, whereas DNA strands are cut 11/9 nt downstream. The discovery of the TsoI prototype is of practical importance in biotechnology, as it extends the palette of cleavage specificities for gene cloning. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Smith, Maria W; Ghindilis, Andrei L; Seoudi, Ihab A; Smith, Kenneth; Billharz, Rosalind; Simon, Holly M

    2014-01-01

    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 unpurified

  1. Fluorescence quenching of graphene oxide combined with the site-specific cleavage of restriction endonuclease for deoxyribonucleic acid demethylase activity assay.

    Science.gov (United States)

    Ji, Lijuan; Qian, Yingdan; Wu, Ping; Zhang, Hui; Cai, Chenxin

    2015-04-15

    We report on the development of a sensitive and selective deoxyribonucleic acid (DNA) demethylase (using MBD2 as an example) activity assay by coupling the fluorescence quenching of graphene oxide (GO) with the site-specific cleavage of HpaII endonuclease to improve the selectivity. This approach was developed by designing a single-stranded probe (P1) that carries a binding region to facilitate the interaction with GO, which induces fluorescence quenching of the labeled fluorophore (FAM, 6-carboxyfluorescein), and a sensing region, which contains a hemi-methylated site of 5'-CmCGG-3', to specifically recognize the target (T1, a 32-mer DNA from the promoter region of p53 gene) and hybridize with it to form a P1/T1 duplex. After demethylation with MBD2, the duplex can be specifically cleaved using HpaII, which releases the labeled FAM from the GO surface and results in the recovery of fluorescence. However, this cleavage is blocked by the hemi-methylation of this site. Thus, the magnitude of the recovered fluorescence signal is related to the MBD2 activity, which establishes the basis of the DNA demethylase activity assay. This assay can determine as low as ∼(0.05±0.01) ng mL(-1) (at a signal/noise of 3) of MBD2 with a linear range of 0.2-300 ng mL(-1) and recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. The advantage of this assay is its ability to avoid false signals and no requirement of bisulfite conversion, PCR amplification, radioisotope labeling, or separation. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Insights into the Origin of Clostridium botulinum Strains: Evolution of Distinct Restriction Endonuclease Sites in rrs (16S rRNA gene).

    Science.gov (United States)

    Bhushan, Ashish; Mukherjee, Tanmoy; Joshi, Jayadev; Shankar, Pratap; Kalia, Vipin Chandra

    2015-06-01

    Diversity analysis of Clostridium botulinum strains is complicated by high microheterogeneity caused by the presence of 9-22 copies of rrs (16S rRNA gene). The need is to mine genetic markers to identify very closely related strains. Multiple alignments of the nucleotide sequences of the 212 rrs of 13 C. botulinum strains revealed intra- and inter-genomic heterogeneity. Low intragenomic heterogeneity in rrs was evident in strains 230613, Alaska E43, Okra, Eklund 17B, Langeland, 657, Kyoto, BKT015925, and Loch Maree. The most heterogenous rrs sequences were those of C. botulinum strains ATCC 19397, Hall, H04402065, and ATCC 3502. In silico restriction mapping of these rrs sequences was observable with 137 type II Restriction endonucleases (REs). Nucleotide changes (NC) at these RE sites resulted in appearance of distinct and additional sites, and loss in certain others. De novo appearances of RE sites due to NC were recorded at different positions in rrs gene. A nucleotide transition A>G in rrs of C. botulinum Loch Maree and 657 resulted in the generation of 4 and 10 distinct RE sites, respectively. Transitions A>G, G>A, and T>C led to the loss of RE sites. A perusal of the entire NC and in silico RE mapping of rrs of C. botulinum strains provided insights into their evolution. Segregation of strains on the basis of RE digestion patterns of rrs was validated by the cladistic analysis involving six house keeping genes: dnaN, gyrB, metG, prfA, pyrG, and Rho.

  3. 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

  4. Related bifunctional restriction endonuclease-methyltransferase triplets: TspDTI, Tth111II/TthHB27I and TsoI with distinct specificities.

    Science.gov (United States)

    Zylicz-Stachula, Agnieszka; Zolnierkiewicz, Olga; Lubys, Arvydas; Ramanauskaite, Danute; Mitkaite, Goda; Bujnicki, Janusz M; Skowron, Piotr M

    2012-04-10

    We previously defined a family of restriction endonucleases (REases) from Thermus sp., which share common biochemical and biophysical features, such as the fusion of both the nuclease and methyltransferase (MTase) activities in a single polypeptide, cleavage at a distance from the recognition site, large molecular size, modulation of activity by S-adenosylmethionine (SAM), and incomplete cleavage of the substrate DNA. Members include related thermophilic REases with five distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI. TspDTI, TsoI and isoschizomers Tth111II/TthHB27I recognize different, but related sequences: 5'-ATGAA-3', 5'-TARCCA-3' and 5'-CAARCA-3' respectively. Their amino acid sequences are similar, which is unusual among REases of different specificity. To gain insight into this group of REases, TspDTI, the prototype member of the Thermus sp. enzyme family, was cloned and characterized using a recently developed method for partially cleaving REases. TspDTI, TsoI and isoschizomers Tth111II/TthHB27I are closely related bifunctional enzymes. They comprise a tandem arrangement of Type I-like domains, like other Type IIC enzymes (those with a fusion of a REase and MTase domains), e.g. TspGWI, TaqII and MmeI, but their sequences are only remotely similar to these previously characterized enzymes. The characterization of TspDTI, a prototype member of this group, extends our understanding of sequence-function relationships among multifunctional restriction-modification enzymes.

  5. A Qualitative and Quantitative Assay to Study DNA/Drug Interaction ...

    African Journals Online (AJOL)

    Purpose: To explore the use of restriction inhibition assay (RIA) to study the binding specificity of some anticancer drugs. Methods: A 448 bp DNA fragment derived from pBCKS+ plasmid (harboring the polylinker region with multiple restriction endonuclease sites) was used as a template for sequence selective inhibition of ...

  6. A Qualitative and Quantitative Assay to Study DNA/Drug Interaction ...

    African Journals Online (AJOL)

    Research Article. A Qualitative and Quantitative Assay to Study. DNA/Drug Interaction Based on Sequence Selective. Inhibition of Restriction Endonucleases. Syed A Hassan1*, Lata Chauhan2, Ritu Barthwal2 and Aparna Dixit3. 1 Faculty of Computing and Information Technology, King Abdul Aziz University, Rabigh-21911 ...

  7. Amplification of Hsp 65 gene and usage of restriction endonuclease for identification of non tuberculous rapid grower mycobacterium.

    Science.gov (United States)

    Verma, Ajoy Kumar; Sarin, Rohit; Arora, Vijay Kumar; Kumar, Gavish; Arora, Jyoti; Singh, Paras; Myneedu, Vithal Prasad

    2018-01-01

    The rapid grower mycobacteria have emerged as significant group of human pathogen amongst the Runyon group IV organisms that are capable of causing infection in both the healthy and immunocompromised hosts. Study aimed to identification of species amongst rapid grower non tuberculous mycobacterial isolates by polymerase chain reaction - restriction enzyme analysis (PRA). Analysis and comparison of results with standard biochemical tests. Rapid grower non tuberculous mycobacteria had been collected from liquid culture section during the study period. All isolates were identified by conventional biochemical tests. A 441bp fragment of hsp65 genes was amplified and digested by two restriction enzymes, BstEII and HaeIII. Digested products were analyzed using polyacrilamid gel electrophoresis (PAGE). During study, 121 rapid grower mycobacterial isolates were subjected for species identification. Isolates were obtained from pulmonary samples (72) and extrapulmonary samples (49). In the PRA test 8 different types of rapid grower mycobacteria were identified after analyzing the fragments generated through restriction enzymes. Mycobacterium chelonae (57/121) was the most common isolate in pulmonary and extrapulmonary samples. Mycobacterium fortuitum (42), Mycobacterium abscessus (11), Mycobacterium immunogen (06), Mycobacterium peregrinum (02), Mycobacterium smegmatis (01), Mycobacterium wolinskyi (01), Mycobacterium goodii (01) were identified as other species of rapid grower non tuberculous mycobacteria. PRA is a rapid and accurate system for the identification of species of non tuberculous mycobacteria. Results of PRA and biochemical tests are concordant up to 98%. Copyright © 2017 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.

  8. Two-stage gene assembly/cloning of a member of the TspDTI subfamily of bifunctional restriction endonucleases, TthHB27I.

    Science.gov (United States)

    Krefft, Daria; Zylicz-Stachula, Agnieszka; Mulkiewicz, Ewa; Papkov, Aliaksei; Jezewska-Frackowiak, Joanna; Skowron, Piotr M

    2015-01-20

    The Thermus sp. family of bifunctional type IIS/IIG/IIC restriction endonucleases (REase)-methyltransferases (MTase) comprises thermo-stable TaqII, TspGWI, TspDTI, TsoI, Tth111II/TthHB27I enzymes as well as a number of putative enzymes/open reading frames (ORFs). All of the family members share properties including a large protein size (ca. 120kDa), amino acid (aa) sequence homologies, enzymatic activity modulation by S-adenosylmethionine (SAM), recognition of similar asymmetric cognate DNA sites and cleavage at a distance of 11/9 nt. Analysis of the enzyme aa sequences and domain/motif organisation led to further Thermus sp. family division into the TspDTI and TspGWI subfamilies. The latter exhibits an unprecedented phenomenon of DNA recognition change upon substitution of SAM by its analogue, sinefungin (SIN), towards a very frequent DNA cleavage. We report cloning in Escherichia coli (E. coli), using a two-stage procedure and a putative tthHB27IRM gene, detected by bioinformatics analysis of the Thermus thermophilus HB27 (T. thermophilus) genome. The functionality of a 3366 base pair (bp)-/1121 aa-long, high GC content ORF was validated experimentally through the expression in E. coli. Protein features corroborated with the reclassification of TthHB27I into the TspDTI subfamily, which manifested in terms of aa-sequence/motif homologies and insensitivity to SIN-induced specificity shift. However, both SAM and SIN stimulated the REase DNA cleavage activity by at least 16-32 times; the highest was observed for the Thermus sp. family. The availability of TthHB27I and the need to include SAM or SIN in the reaction in order to convert the enzyme from "hibernation" status to efficient DNA cleavage is of practical significance in molecular biotechnology, extending the palette of available REase specificities. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Ellen S. Motta

    2010-01-01

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

  10. Endonuclease from Gram-Negative Bacteria Serratia marcescens Is as Effective as Pulmozyme in the Hydrolysis of DNA in Sputum

    Science.gov (United States)

    Vafina, Gulnaz; Zainutdinova, Elmira; Bulatov, Emil; Filimonova, Maria N.

    2018-01-01

    One of the approaches to effective airway cleansing is the degradation of DNA into smaller fragments. For this purpose Pulmozyme® is used with high efficacy because it contains recombinant DNase I as its active component. The aim of the study was to comparatively analyze DNase activity of Pulmozyme® and the nuclease from gram-negative bacteria Serratia marcescens, because at optimal conditions the catalytic efficiency of the nuclease is much higher than the efficiency of DNase I. Highly polymerized DNA and purulent-mucous sputum were used as substrates. The examination showed that both S. marcescens nuclease and Pulmozyme® hydrolyzed DNA in sputum. Also S. marcescens nuclease was found capable of hydrolyzing DNA in conditions that are standard for Pulmozyme® and suitable for its therapeutic application. For manifesting the similar hydrolytic activity the nuclease amount in the assay mixture containing highly polymerized DNA or the sonicated sputum and NaCl together with calcium- or magnesium- cations can be about 10- time lower than that of the recombinant DNase I. In the presence of magnesium cations the DNase activity of both S. marcescens nuclease and Pulmozyme® was higher than in the presence of calcium cations. PMID:29503617

  11. High efficiency of targeted mutagenesis in arabidopsis via meiotic promoter-driven expression of Cas9 endonuclease

    KAUST Repository

    Eid, Ayman

    2016-05-28

    Key message: The use of a meiosis I-specific promoter increased the efficiency of targeted mutagenesis and will facilitate the manipulation of homologous recombination. Abstract: The CRISPR/Cas9 system has been harnessed for targeted engineering of eukaryotic genomes, including plants; however, CRISPR/Cas9 efficiency varies considerably in different plant tissues and species. In Arabidopsis, the generation of homozygous or bi-allelic mutants in the first (T1) generation is inefficient. Here, we used specific promoters to drive the expression of Cas9 during meiosis to maximize the efficiency of recovering heritable mutants in T1 plants. Our data reveal that the use of a promoter active in meiosis I resulted in high-efficiency (28 %) recovery of targeted mutants in the T1 generation. Moreover, this method enabled efficient simultaneous targeting of three genes for mutagenesis. Taken together, our results show that the use of meiosis-specific promoters will improve methods for functional genomic analysis and studying the molecular underpinnings of homologous recombination. © 2016, Springer-Verlag Berlin Heidelberg.

  12. Three-stage biochemical selection: cloning of prototype class IIS/IIC/IIG restriction endonuclease-methyltransferase TsoI from the thermophile Thermus scotoductus.

    Science.gov (United States)

    Skowron, Piotr M; Vitkute, Jolanta; Ramanauskaite, Danute; Mitkaite, Goda; Jezewska-Frackowiak, Joanna; Zebrowska, Joanna; Zylicz-Stachula, Agnieszka; Lubys, Arvydas

    2013-08-06

    In continuing our research into the new family of bifunctional restriction endonucleases (REases), we describe the cloning of the tsoIRM gene. Currently, the family includes six thermostable enzymes: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI, TsoI, isolated from various Thermus sp. and two thermolabile enzymes: RpaI and CchII, isolated from mesophilic bacteria Rhodopseudomonas palustris and Chlorobium chlorochromatii, respectively. The enzymes have several properties in common. They are large proteins (molecular size app. 120 kDa), coded by fused genes, with the REase and methyltransferase (MTase) in a single polypeptide, where both activities are affected by S-adenosylmethionine (SAM). They recognize similar asymmetric cognate sites and cleave at a distance of 11/9 nt from the recognition site. Thus far, we have cloned and characterised TaqII, Tth111II, TthHB27I, TspGWI and TspDTI. TsoI REase, which originate from thermophilic Thermus scotoductus RFL4 (T. scotoductus), was cloned in Escherichia coli (E. coli) using two rounds of biochemical selection of the T. scotoductus genomic library for the TsoI methylation phenotype. DNA sequencing of restriction-resistant clones revealed the common open reading frame (ORF) of 3348 bp, coding for a large polypeptide of 1116 aminoacid (aa) residues, which exhibited a high level of similarity to Tth111II (50% identity, 60% similarity). The ORF was PCR-amplified, subcloned into a pET21 derivative under the control of a T7 promoter and was subjected to the third round of biochemical selection in order to isolate error-free clones. Induction experiments resulted in synthesis of an app. 125 kDa protein, exhibiting TsoI-specific DNA cleavage. Also, the wild-type (wt) protein was purified and reaction optima were determined. Previously we identified and cloned the Thermus family RM genes using a specially developed method based on partial proteolysis of thermostable REases. In the case of TsoI the classic biochemical selection

  13. Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5'-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi.

    Science.gov (United States)

    Ishikawa, Ken; Watanabe, Miki; Kuroita, Toshihiro; Uchiyama, Ikuo; Bujnicki, Janusz M; Kawakami, Bunsei; Tanokura, Masaru; Kobayashi, Ichizo

    2005-07-21

    To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction-modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restriction genes were identified and then amplified in such a way that they were removed from their own translation signal. During their cloning into a plasmid, the genes became connected with a plant translation signal. After in vitro transcription by T7 RNA polymerase, the mRNAs were separated from the template DNA and translated in a wheat-germ-based cell-free protein synthesis system. The resulting solution could be directly assayed for restriction activity. We identified two deoxyribonucleases. The novel enzyme was denoted as PabI, purified and found to recognize 5'-GTAC and leave a 3'-TA overhang (5'-GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90 degrees C and optimally active at a pH of around 6 and in NaCl concentrations ranging from 100 to 200 mM. We predict that it has a novel 3D structure.

  14. Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5′-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi

    Science.gov (United States)

    Ishikawa, Ken; Watanabe, Miki; Kuroita, Toshihiro; Uchiyama, Ikuo; Bujnicki, Janusz M.; Kawakami, Bunsei; Tanokura, Masaru; Kobayashi, Ichizo

    2005-01-01

    To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction–modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restriction genes were identified and then amplified in such a way that they were removed from their own translation signal. During their cloning into a plasmid, the genes became connected with a plant translation signal. After in vitro transcription by T7 RNA polymerase, the mRNAs were separated from the template DNA and translated in a wheat-germ-based cell-free protein synthesis system. The resulting solution could be directly assayed for restriction activity. We identified two deoxyribonucleases. The novel enzyme was denoted as PabI, purified and found to recognize 5′-GTAC and leave a 3′-TA overhang (5′-GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90°C and optimally active at a pH of around 6 and in NaCl concentrations ranging from 100 to 200 mM. We predict that it has a novel 3D structure. PMID:16040595

  15. The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2 T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells.

    Science.gov (United States)

    Duan, Yajian; Ma, Gaoen; Huang, Xionggao; D'Amore, Patricia A; Zhang, Feng; Lei, Hetian

    2016-07-29

    The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T. The next-generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Physico-chemical and biological study of excision-repair of UV-irradiated PHIX 174 RF DNA in vitro

    International Nuclear Information System (INIS)

    Heijneker, H.L.

    1975-01-01

    A study is presented on the excision repair of ultraviolet-irradiated PHIX 174 RFI DNA in vitro with UV-specific endonuclease from micrococcus luteus, DNA polymerase I from E. coli and DNA ligase from phage T 4 infected E. coli. Excision repair was measured by physico-chemical and by biological methods. It is shown that more than 90% of the pyrimidine dimers can be repaired in vitro and that the repaired molecules have regained full biological activity. Endonuclease III was not essential for excision repair in vitro and did not stimulate repair; from this it was concluded that UV-endo generates 3' OH endgroups. The usefulness of the methods with regard to the study of excision repair is discussed

  17. Studies of Genetic Variation in the AIDS Virus: Relevance to Disease Pathogenesis, Anti-Viral Therapy, and Vaccine Development

    Science.gov (United States)

    1989-03-15

    1 sequences from uncultured patient tissues into M13 phage for sequence analysis. This will allow, for the first time, direct analysis of HIV-1...the H9/HTLV-Illb isolate after digestion with 7 restriction endonucleases (Sstl, Pvull, Bglll, Hindlll, EcoRI, Cvnl, Kpnl). Recombinant . phage ...and classical phage cloning techniques to fully characterize HIV variation in vivo and to study its biologic relevance. Isolation and Genetic and

  18. Application of genome editing technologies to the study and treatment of hematological disease.

    Science.gov (United States)

    Pellagatti, Andrea; Dolatshad, Hamid; Yip, Bon Ham; Valletta, Simona; Boultwood, Jacqueline

    2016-01-01

    Genome editing technologies have advanced significantly over the past few years, providing a fast and effective tool to precisely manipulate the genome at specific locations. The three commonly used genome editing technologies are Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas9 (CRISPR/Cas9) system. ZFNs and TALENs consist of endonucleases fused to a DNA-binding domain, while the CRISPR/Cas9 system uses guide RNAs to target the bacterial Cas9 endonuclease to the desired genomic location. The double-strand breaks made by these endonucleases are repaired in the cells either by non-homologous end joining, resulting in the introduction of insertions/deletions, or, if a repair template is provided, by homology directed repair. The ZFNs, TALENs and CRISPR/Cas9 systems take advantage of these repair mechanisms for targeted genome modification and have been successfully used to manipulate the genome in human cells. These genome editing tools can be used to investigate gene function, to discover new therapeutic targets, and to develop disease models. Moreover, these genome editing technologies have great potential in gene therapy. Here, we review the latest advances in the application of genome editing technology to the study and treatment of hematological disorders. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Dual-probe electrochemical DNA biosensor based on the "Y" junction structure and restriction endonuclease assisted cyclic enzymatic amplification for detection of double-strand DNA of PML/RARα related fusion gene.

    Science.gov (United States)

    Wang, Kun; Lei, Yun; Zhong, Guang-Xian; Zheng, Yan-Jie; Sun, Zhou-Liang; Peng, Hua-Ping; Chen, Wei; Liu, Ai-Lin; Chen, Yuan-Zhong; Lin, Xin-Hua

    2015-09-15

    Taking advantage of "Y" junction structure and restriction endonuclease assisted cyclic enzymatic amplification, a dual-probe electrochemical DNA (DE-DNA) biosensor was designed to detect double-stranded DNA (dsDNA) of acute promyelocytic leukemia (APL) related gene. Two groups of detection probes were designed, and each group was composed of a biotinylated capture probe and an assisted probe. They were separately complementary with two strands of target dsDNA in order to prevent the reannealing of the two separate strands from target dsDNA. First, thiol functionalized capture probes (C1 and C2) were severally assembled onto two different gold electrodes, followed by hybridizing with target dsDNA (S1a-S1b) and assistant probes to form two Y-junction-structure ternary complexes. Subsequently, restriction sites on the ternary complexes were digested by Rsa I, which can release S1a, S1b and biotins from the electrode surfaces. Meanwhile, the released S1a and S1b can further hybridize with the unhybridized corresponding detection probes and then initiate another new hybridization-cleavage-separation cycle. Finally, the current signals were produced by the enzyme-catalyzed reaction of streptavidin-horse reddish peroxidase (streptavidin-HRP). The distinct difference in current signals between different sequences allowed detection of target dsDNA down to a low detection limit of 47 fM and presented excellent specificity with discriminating only a single-base mismatched dsDNA sequence. Moreover, this biosensor was also used for assay of polymerase chain reaction (PCR) samples with satisfactory results. According to the results, the power of the DE-DNA biosensor as a promising tool for the detection of APL and other diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Characterized mechanism of alpha-mangostin-induced cell death: caspase-independent apoptosis with release of endonuclease-G from mitochondria and increased miR-143 expression in human colorectal cancer DLD-1 cells.

    Science.gov (United States)

    Nakagawa, Yoshihito; Iinuma, Munekazu; Naoe, Tomoki; Nozawa, Yoshinori; Akao, Yukihiro

    2007-08-15

    alpha-Mangostin, a xanthone from the pericarps of mangosteen (Garcinia mangostana Linn.), was evaluated for in vitro cytotoxicity against human colon cancer DLD-1 cells. The number of viable cells was consistently decreased by the treatment with alpha-mangostin at more than 20 microM. The cytotoxic effect of 20 microM alpha-mangostin was found to be mainly due to apoptosis, as indicated by morphological findings. Western blotting, the results of an apoptosis inhibition assay using caspase inhibitors, and the examination of caspase activity did not demonstrate the activation of any of the caspases tested. However, endonuclease-G released from mitochondria with the decreased mitochondrial membrane potential was shown. The levels of phospho-Erk1/2 were increased in the early phase until 1h after the start of treatment and thereafter decreased, and increased again in the late phase. On the other hand, the level of phospho-Akt was sharply reduced with the process of apoptosis after 6h of treatment. Interestingly, the level of microRNA-143, which negatively regulates Erk5 at translation, gradually increased until 24h following the start of treatment. We also examined the synergistic growth suppression in DLD-1 cells by the combined treatment of the cells with alpha-mangostin and 5-FU which is one of the most effective chemotherapeutic agents for colorectal adenocarcinoma. The co-treatment with alpha-mangostin and 5-FU, both at 2.5 microM, augmented growth inhibition compared with the treatment with 5 microM of alpha-mangostin or 5 microM 5-FU alone. These findings indicate unique mechanisms of alpha-mangostin-induced apoptosis and its action as an effective chemosensitizer.

  1. Combination of restriction endonuclease digestion with the ΔΔCt method in real-time PCR to monitor etoxazole resistance allele frequency in the two-spotted spider mite.

    Science.gov (United States)

    Osakabe, Masahiro; Imamura, Tsuyoshi; Nakano, Ryohei; Kamikawa, Satoshi; Tadatsu, Misono; Kunimoto, Yoshinori; Doi, Makoto

    2017-06-01

    Monitoring resistance allele frequency at the early stage of resistance development is important for the successful acaricide resistance management. Etoxazole is a mite growth inhibitor to which resistance is conferred by an amino acid substitution in the chitin synthase 1 (CHS1; I1017F) in T. urticae. If the susceptible allele can be specifically digested by restriction endonuclease, the ΔΔCt method using real-time PCR for genomic DNA (RED-ΔΔCt method) may be available for monitoring the resistance allele frequency. We tested whether the etoxazole resistance allele frequency in a pooled sample was accurately measured by the RED-ΔΔCt method and validated whether the resistance variant frequency was correlated with etoxazole resistance phenotype in a bioassay. Finally, we performed a pilot test using field populations. Strong linearity of the measures by the RED-ΔΔCt method with practical resistance allele frequencies; resistance allele frequency in the range between 0.5% to at least 0.75% was strictly represented. The strong linear relationship between hatchability of haploid male eggs after the etoxazole treatments (phenotype) and resistance allele frequencies in their mothers provided direct evidence that I1017F is a primary resistance factor to etoxazole in the strains used for experiments. The pilot test revealed a significant correlation between egg hatchability (including both diploid female eggs and haploid male eggs) and estimators in field populations. Consequently, we concluded that the RED-ΔΔCt method is a powerful tool for monitoring a resistance allele in a pooled sample. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. CHROMOSOME BANDING IN CRUSTACEA. I. KARYOTYPE, Ag-NORs, C BANDING AND TREATMENT WITH EcoRI, PstI and KpnI RESTRICTION ENDONUCLEASES IN Artemia franciscana

    Directory of Open Access Journals (Sweden)

    Ingrid Vilar Accioly

    2014-08-01

    Full Text Available Características cariotípicas do microcrustáceo Artemia franciscana Kellog, 1906, introduzida nas salinas do litoral nordeste do Brasil, na década de 70, foram investigadas através de coloração convencional, bandamento C, endonucleases de restrição (EcoRI, PstI e KpnI e Ag-NORs. O cariótipo consiste de 42 cromossomos, onde se individualiza sobre alguns pares a presença de constrições secundárias. Grandes blocos heterocromáticos encontram-se distribuídos nas porções teloméricas da maioria dos cromossomos. A digestão com PstI e KpnI revelou um padrão similar ao obtido pelo bandamento C. Preparações tratadas com EcoRI apresentam digestão das regiões heterocromáticas indicando a presença de sítios de restrição nestas regiões. Ag-NORs múltiplas estão associadas a blocos heterocromáticos. Os dados apresentados representam passo inicial para identificação de possíveis modificações ocorridas após o isolamento geográfico desta amostra, assim como no entendimento das modificações evolutivas ocorridas no cariótipo deste grupo. Palavras-chave: bandamento cromossômico, camarão de água salgada, citogenética de crustáceos. DOI: http://dx.doi.org/10.18561/2179-5746/biotaamazonia.v4n2p15-19

  3. 16S rRNA PCR followed by restriction endonuclease digestion: a rapid approach for genus level identification of important enteric bacterial pathogens.

    Science.gov (United States)

    Vergis, J; Negi, M; Poharkar, K; Das, D P; Malik, S V S; Kumar, A; Doijad, S P; Barbuddhe, S B; Rawool, D B

    2013-12-01

    The study describes a rapid approach for detection of common enteric bacterial pathogens, which involves partial amplification of the 16S rRNA gene by PCR using a colony from selective medium followed by restriction enzyme (RE) digestion using the EcoRI, HindIII and SalI enzymes. On the basis of RE digestion analysis different genera namely, Escherichia, Salmonella, Shigella, Vibrio, Campylobacter, Arcobacter, Yesinia and Listeria were differentiated. © 2013.

  4. Thermostable proteins bioprocesses: The activity of restriction endonuclease-methyltransferase from Thermus thermophilus (RM.TthHB27I cloned in Escherichia coli is critically affected by the codon composition of the synthetic gene.

    Directory of Open Access Journals (Sweden)

    Daria Krefft

    Full Text Available Obtaining thermostable enzymes (thermozymes is an important aspect of biotechnology. As thermophiles have adapted their genomes to high temperatures, their cloned genes' expression in mesophiles is problematic. This is mainly due to their high GC content, which leads to the formation of unfavorable secondary mRNA structures and codon usage in Escherichia coli (E. coli. RM.TthHB27I is a member of a family of bifunctional thermozymes, containing a restriction endonuclease (REase and a methyltransferase (MTase in a single polypeptide. Thermus thermophilus HB27 (T. thermophilus produces low amounts of RM.TthHB27I with a unique DNA cleavage specificity. We have previously cloned the wild type (wt gene into E. coli, which increased the production of RM.TthHB27I over 100-fold. However, its enzymatic activities were extremely low for an ORF expressed under a T7 promoter. We have designed and cloned a fully synthetic tthHB27IRM gene, using a modified 'codon randomization' strategy. Codons with a high GC content and of low occurrence in E. coli were eliminated. We incorporated a stem-loop circuit, devised to negatively control the expression of this highly toxic gene by partially hiding the ribosome-binding site (RBS and START codon in mRNA secondary structures. Despite having optimized 59% of codons, the amount of produced RM.TthHB27I protein was similar for both recombinant tthHB27IRM gene variants. Moreover, the recombinant wt RM.TthHB27I is very unstable, while the RM.TthHB27I resulting from the expression of the synthetic gene exhibited enzymatic activities and stability equal to the native thermozyme isolated from T. thermophilus. Thus, we have developed an efficient purification protocol using the synthetic tthHB27IRM gene variant only. This suggests the effect of co-translational folding kinetics, possibly affected by the frequency of translational errors. The availability of active RM.TthHB27I is of practical importance in molecular biotechnology

  5. Thermostable proteins bioprocesses: The activity of restriction endonuclease-methyltransferase from Thermus thermophilus (RM.TthHB27I) cloned in Escherichia coli is critically affected by the codon composition of the synthetic gene.

    Science.gov (United States)

    Krefft, Daria; Papkov, Aliaksei; Zylicz-Stachula, Agnieszka; Skowron, Piotr M

    2017-01-01

    Obtaining thermostable enzymes (thermozymes) is an important aspect of biotechnology. As thermophiles have adapted their genomes to high temperatures, their cloned genes' expression in mesophiles is problematic. This is mainly due to their high GC content, which leads to the formation of unfavorable secondary mRNA structures and codon usage in Escherichia coli (E. coli). RM.TthHB27I is a member of a family of bifunctional thermozymes, containing a restriction endonuclease (REase) and a methyltransferase (MTase) in a single polypeptide. Thermus thermophilus HB27 (T. thermophilus) produces low amounts of RM.TthHB27I with a unique DNA cleavage specificity. We have previously cloned the wild type (wt) gene into E. coli, which increased the production of RM.TthHB27I over 100-fold. However, its enzymatic activities were extremely low for an ORF expressed under a T7 promoter. We have designed and cloned a fully synthetic tthHB27IRM gene, using a modified 'codon randomization' strategy. Codons with a high GC content and of low occurrence in E. coli were eliminated. We incorporated a stem-loop circuit, devised to negatively control the expression of this highly toxic gene by partially hiding the ribosome-binding site (RBS) and START codon in mRNA secondary structures. Despite having optimized 59% of codons, the amount of produced RM.TthHB27I protein was similar for both recombinant tthHB27IRM gene variants. Moreover, the recombinant wt RM.TthHB27I is very unstable, while the RM.TthHB27I resulting from the expression of the synthetic gene exhibited enzymatic activities and stability equal to the native thermozyme isolated from T. thermophilus. Thus, we have developed an efficient purification protocol using the synthetic tthHB27IRM gene variant only. This suggests the effect of co-translational folding kinetics, possibly affected by the frequency of translational errors. The availability of active RM.TthHB27I is of practical importance in molecular biotechnology, extending

  6. Defective mitochondrial respiration, altered dNTP pools and reduced AP endonuclease 1 activity in peripheral blood mononuclear cells of Alzheimer's disease patients

    DEFF Research Database (Denmark)

    Maynard, Scott; Hejl, Anne-Mette; Dinh, Tran Thuan Son

    2015-01-01

    AIMS: Accurate biomarkers for early diagnosis of Alzheimer's disease (AD) are badly needed. Recent reports suggest that dysfunctional mitochondria and DNA damage are associated with AD development. In this report, we measured various cellular parameters, related to mitochondrial bioenergetics...... and DNA damage, in peripheral blood mononuclear cells (PBMCs) of AD and control participants, for biomarker discovery. METHODS: PBMCs were isolated from 53 patients with AD of mild to moderate degree and 30 age-matched healthy controls. Tests were performed on the PBMCs from as many of these participants...... on adjustments for gender and/or age. CONCLUSIONS: This study reveals impaired mitochondrial respiration, altered dNTP pools and reduced DNA repair activity in PBMCs of AD patients, thus suggesting that these biochemical activities may be useful as biomarkers for AD....

  7. Crystal Structure of a Monomeric Form of Severe Acute Respiratory Syndrome Coronavirus Endonuclease Nsp15 Suggests a Role for Hexamerization As An Allosteric Switch

    Energy Technology Data Exchange (ETDEWEB)

    Joseph, J.S.; Saikatendu, K.S.; Subramanian, V.; Neuman, B.W.; Buchmeier, M.J.; Stevens, R.C.; Kuhn, P.; /Scripps Res. Inst.

    2007-07-09

    Mature nonstructural protein-15 (nsp15) from the severe acute respiratory syndrome coronavirus (SARS-CoV) contains a novel uridylate-specific Mn{sup 2+}-dependent endoribonuclease (NendoU). Structure studies of the full-length form of the obligate hexameric enzyme from two CoVs, SARS-CoV and murine hepatitis virus, and its monomeric homologue, XendoU from Xenopus laevis, combined with mutagenesis studies have implicated several residues in enzymatic activity and the N-terminal domain as the major determinant of hexamerization. However, the tight link between hexamerization and enzyme activity in NendoUs has remained an enigma. Here, we report the structure of a trimmed, monomeric form of SARS-CoV nsp15 (residues 28 to 335) determined to a resolution of 2.9 Angstroms. The catalytic loop (residues 234 to 249) with its two reactive histidines (His 234 and His 249) is dramatically flipped by {approx}120 degrees into the active site cleft. Furthermore, the catalytic nucleophile Lys 289 points in a diametrically opposite direction, a consequence of an outward displacement of the supporting loop (residues 276 to 295). In the full-length hexameric forms, these two loops are packed against each other and are stabilized by intimate intersubunit interactions. Our results support the hypothesis that absence of an adjacent monomer due to deletion of the hexamerization domain is the most likely cause for disruption of the active site, offering a structural basis for why only the hexameric form of this enzyme is active.

  8. Monitoring the effects of exposure to lead and cadmium in working and living environment through standard biochemical blood parameters and liver endonucleases activity

    Directory of Open Access Journals (Sweden)

    Nikolić Ružica S.

    2011-01-01

    Full Text Available Heavy metals as pollutants in the working and living environment are a serious health and environmental problem because they are toxic, non-biodegradable, accumulate in living systems and have a long half-life in soil. Sources of lead contamination are combustion products in the chemical industry and metallurgy, industrial waste water, landfills, traffic etc. Lead enters into the body via the food chain and drinking water. In the body lead is deposited in the liver, kidneys, brain and mineral tissues. Excretion of lead causes damage to the epithelial cells of certain organs. High level exposure to cadmium is usually the result of environmental pollution by human activities. Exposure to cadmium can lead to acute and chronic tissue damage of various organs, including liver and kidneys in humans and in animals. In this paper we analyzed the effects of lead and cadmium exposure, in working and living environment, on the model system of experimental animals, particularly the activity of certain liver enzymes, acid and alkaline DNase, and standard biochemical blood parameters. The study showed that lead and cadmium significantly affect the protein content, red blood cells, hemoglobin and hematocrit, and the activity of liver enzymes. This harmful effect of this toxic metal can be reduced by the supplements.

  9. [PCR-RFLP and sequencing studies of malaria vectors (Diptera, Culicidae, Anopheles) in Kyrgyzstan].

    Science.gov (United States)

    Goriacheva, I I; Zvantsov, A B; Gordeev, M I; Bezzhonova, O V; Usenbaev, N T; Ezhov, M N

    2011-01-01

    The results of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, obtained after treatment of PCR-products with restriction endonuclease CfoI, could identify two members of the Anopheles maculipennis complex: An. maculipennis and An. artemievi. Treatment of amplification products with restriction endonuclease BsuI gave rise to fragment lengths of 192 and 218 bp, characteristic of An. artemievi, in the populations of the Talas (settlement of Kizil-Adyr, Kara-Bura District), Dzhelalabad (towns of Tashkumyr and Kara-Kul), and Osh (town of Gulcha, Alai District; village of Langar, Kara-Suisky District) Regions. After treatment of PCR-products with restriction endonuclease BstACI, fragment lengths of 292 and 150 bp, characteristic of An. messeae, were obtained for the mosquitoes of Issyk-Kul (town of Balykchi) and Naryn (settlement of Kochkorka, Kochkor District) Regions. To identify the molecular forms of An. superpictus, the investigators sequenced the amplification products obtained by PCR with 5.8S and 28S rRNA gene-specific primers. Analysis of the primary structure of the second internal transcribed spacer, by using the international databases, has indicated that molecular form X is prevalent in the study districts of Kyrgyzstan. The COI-COII region of the mitochondrial genome of the vector also underwent PCR-RFLP analysis. Three new haplotypes with restriction patterns of about 540, 420, 200, 150, 140 bp, about 540, 360, 280, 150, 140 bp, and about 580, 540, and 150, 140 bp have been identified along with the previously described haplotype X characterized by restriction products of 540, 420, 260, 150, and 140 bp in length.

  10. The use of recombinant DNA techniques to study radiation-induced damage, repair and genetic change in mammalian cells

    International Nuclear Information System (INIS)

    Thacker, J.

    1986-01-01

    A brief introduction is given to appropriate elements of recombinant DNA techniques and applications to problems in radiobiology are reviewed with illustrative detail. Examples are included of studies with both 254 nm ultraviolet light and ionizing radiation and the review progresses from the molecular analysis of DNA damage in vitro through to the nature of consequent cellular responses. The review is dealt with under the following headings: Molecular distribution of DNA damage, The use of DNA-mediated gene transfer to assess damage and repair, The DNA double strand break: use of restriction endonucleases to model radiation damage, Identification and cloning of DNA repair genes, Analysis of radiation-induced genetic change. (UK)

  11. GlaI digestion of mouse γ-satellite DNA: study of primary structure and ACGT sites methylation

    Directory of Open Access Journals (Sweden)

    Gonchar Danila A

    2009-07-01

    Full Text Available Abstract Background Patterns of mouse DNA hydrolysis with restriction enzymes are coincided with calculated diagrams of genomic DNA digestion in silico, except presence of additional bright bands, which correspond to monomer and dimer of γ-satellite DNA. Only small portion of mouse γ-satellite DNA sequences are presented in databases. Methyl-directed endonuclease GlaI cleaves mouse DNA and may be useful for a detailed study of primary structure and CG dinucleotides methylation in γ-satellite DNA. Results We have constructed a physical map and produced experimental patterns of mouse γ-satellite DNA hydrolysis with unique site-specific methyl-directed endonuclease GlaI and several restriction endonucleases. Fifty two DNA fragments of γ-satellite DNA have been cloned and sequenced. We have not observed any mutations of CG dinucleotide in position 208 of monomeric γ-satellite DNA and confirmed 50% methylation of this CG dinucleoitide. A comparison of consensus sequences of arrayed γ-satellite DNA and small blocks of satellite DNA (140 monomers and less has shown a higher level of mutations and an absence of conserved CG dinucleotide in last ones. A replacement of CG dinucleotide by CA-dinucleotide in positions 178 and 17 in chromosomes 9 and 3, respectively, has been observed in blocks of monomers. Conclusion Arrayed γ-satellite DNA from mouse has at least one conservative CG-dinucleotide. Consensus sequences of this DNA and γ-satellite DNA in small blocks of monomers are differing. The last one displays a higher level of CG dinucleotides mutations and an absence of conservative CG-dinucleotide. Presence of conservative and half-methylated CG-dinucleotide supports an idea of importance of this CG dinucleotide methylation/demethylation in arrayed γ-satellite DNA functioning.

  12. In vitro enzymatic studies on the nature and repair of x-ray-induced damages in DNA. Final report

    International Nuclear Information System (INIS)

    Wallace, S.S.

    1981-03-01

    An enzyme has been purified some 4000 fold from Escherichia coli which recognizes alkali stable base damages in x-irradiated DNA. The enzyme has broad specificity incising: DNA damaged by OsO 4 which produces thymine glycols, DNA treated with heat and acid which produces apurinic sites, and DNA uv-irradiated with high fluences which produces a variety of damages including the above. These activities co-chromatograph through Fraction VII the most purified form; however, the optimum reaction parameters differ among the various substrates suggesting the presence of more than one active site. Similar studies have been done with Saccharomyces cerevisiae. Several apurinic activities have been elucidated in this organism, one of which, Endonuclease E, has been purified over 1000 fold. Endonuclease E has been characterized with respect to various reaction parameters as well as by gel electrophoresis. Both the E. coli and yeast enzymes have been used to quantify DNA damage. Apurinic PM2 DNA and OsO 4 -treated PM2 DNA have also been used in a transfection system to estimate the inactivation efficiencies of AP sites and thymine glycols. AP sites have a relatively high inactivation efficiency and contribute about 15% to the inactivation of x-irradiated PM2 phage while thymine glycols contribute significantly less

  13. A putative Type IIS restriction endonuclease GeoICI from ...

    Indian Academy of Sciences (India)

    2016-02-15

    38] DOI 10.1007/ s12038-016-9595-z. 1. Introduction. Soil from geothermal locations harbours a wide variety of thermophilic microorganisms, which comprise a rich source of compounds for industrial and scientific applications.

  14. Partial Purification and Characterization of Restriction Endonuclease from Neisseria meningitidis.

    Science.gov (United States)

    1983-12-01

    applicable, further diseminleation of the documet , other than those impose by seuity csefation. using standeed statements such as; ?h GROUP tie tftua #ty...reiinbot ty which the documet will be iotseisftial iftlf conrtillstl hy the origlinating activity. This noinbai m "bost be unie to this documet .

  15. 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, ...

  16. A putative Type IIS restriction endonuclease GeoICI from ...

    Indian Academy of Sciences (India)

    2016-02-15

    Feb 15, 2016 ... Screening of extreme environments in search for novel microorganisms may lead to the discovery of robust enzymes with either new substrate specificities or thermostable equivalents of those already found in mesophiles, better suited for biotechnology applications. Isolates from Iceland geysers' biofilms, ...

  17. Class 2 CRISPR-Cas RNA-guided endonucleases

    DEFF Research Database (Denmark)

    Stella, Stefano; Alcón, Pablo; Montoya, Guillermo

    2017-01-01

    CRISPR-Cas is a bacterial defense system against phage infection and nucleic acid invasion. Class 2 type II CRISPR-Cas9 has also been widely used for genome engineering. Here, we review novel insights into the CRISPR class 2 type V enzymes, specifically Cpf1 and C2c1, which display different DNA...

  18. Structure-function relationships of E-type endonuclease colicins

    NARCIS (Netherlands)

    Bremer, Ewald Theodorus Johannes van den

    2004-01-01

    Colicin cytotoxicity can take various guises, the most remarkable being degradation of bacterial DNA, requiring the 60 kDa toxin to translocate its C-terminal, 15 kDa enzymatic domain across two membrane boundaries in order to reach the cytoplasm. Current theories concerning colicin translocation

  19. Endonuclease specificity and sequence dependence of type IIS restriction enzymes.

    Science.gov (United States)

    Lundin, Sverker; Jemt, Anders; Terje-Hegge, Finn; Foam, Napoleon; Pettersson, Erik; Käller, Max; Wirta, Valtteri; Lexow, Preben; Lundeberg, Joakim

    2015-01-01

    Restriction enzymes that recognize specific sequences but cleave unknown sequence outside the recognition site are extensively utilized tools in molecular biology. Despite this, systematic functional categorization of cleavage performance has largely been lacking. We established a simple and automatable model system to assay cleavage distance variation (termed slippage) and the sequence dependence thereof. We coupled this to massively parallel sequencing in order to provide sensitive and accurate measurement. With this system 14 enzymes were assayed (AcuI, BbvI, BpmI, BpuEI, BseRI, BsgI, Eco57I, Eco57MI, EcoP15I, FauI, FokI, GsuI, MmeI and SmuI). We report significant variation of slippage ranging from 1-54%, variations in sequence context dependence, as well as variation between isoschizomers. We believe this largely overlooked property of enzymes with shifted cleavage would benefit from further large scale classification and engineering efforts seeking to improve performance. The gained insights of in-vitro performance may also aid the in-vivo understanding of these enzymes.

  20. Endonuclease specificity and sequence dependence of type IIS restriction enzymes.

    Directory of Open Access Journals (Sweden)

    Sverker Lundin

    Full Text Available Restriction enzymes that recognize specific sequences but cleave unknown sequence outside the recognition site are extensively utilized tools in molecular biology. Despite this, systematic functional categorization of cleavage performance has largely been lacking. We established a simple and automatable model system to assay cleavage distance variation (termed slippage and the sequence dependence thereof. We coupled this to massively parallel sequencing in order to provide sensitive and accurate measurement. With this system 14 enzymes were assayed (AcuI, BbvI, BpmI, BpuEI, BseRI, BsgI, Eco57I, Eco57MI, EcoP15I, FauI, FokI, GsuI, MmeI and SmuI. We report significant variation of slippage ranging from 1-54%, variations in sequence context dependence, as well as variation between isoschizomers. We believe this largely overlooked property of enzymes with shifted cleavage would benefit from further large scale classification and engineering efforts seeking to improve performance. The gained insights of in-vitro performance may also aid the in-vivo understanding of these enzymes.

  1. A putative Type IIS restriction endonuclease GeoICI from ...

    Indian Academy of Sciences (India)

    ... Piotr M Skowron1. Department of Molecular Biotechnology, Institute for Environment and Human Health Protection, Faculty of Chemistry,University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland; Department of Molecular Biology, Faculty of Biology, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland ...

  2. In vitro studies on organophosphate pesticides induced oxidative DNA damage in rat lymphocytes.

    Science.gov (United States)

    Ojha, A; Srivastava, N

    2014-02-01

    Organophosphate (OP) pesticides are widely used for agricultural and household pest control. We studied the genotoxicity of the commonly used OP pesticides chlorpyrifos (CPF), methyl parathion (MPT), and malathion (MLT), individually and in combination, in Wistar rat peripheral blood lymphocytes in vitro. DNA single-strand and double-strand breaks were measured by single cell gel electrophoresis (SCGE; comet assay). To test whether the DNA lesions were caused by oxidative stress, the DNA repair enzymes formamidoaminopyrimidineglycosylase (Fpg) and endonuclease (Endo III), which convert base damages to strand breaks, were used. Significant increases in strand breaks and in levels of the reactive oxygen species (ROS) superoxide anion and hydrogen peroxide were observed in lymphocytes treated with pesticides. MPT exposure caused the greatest DNA damage and ROS production, followed by CPF and ML. Our results demonstrate genotoxic potential of these OP pesticides. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Design, Synthesis and Structure-activity Studies of Rhodanine Derivatives as HIV-1 Integrase Inhibitors

    Directory of Open Access Journals (Sweden)

    Kavya Ramkumar

    2010-06-01

    Full Text Available Raltegravir was the first HIV-1 integrase inhibitor that gained FDA approval for use in the treatment of HIV-1 infection. Because of the emergence of IN inhibitor-resistant viral strains, there is a need to identify innovative second-generation IN inhibitors. Previously, we identified 2-thioxo-4-thiazolidinone (rhodanine-containing compounds as IN inhibitors. Herein, we report the design, synthesis and docking studies of a series of novel rhodanine derivatives as IN inhibitors. All these compounds were further tested against human apurinic/apyrimidinic endonuclease 1 (APE1 to determine their selectivity. Two compounds showed significant cytotoxicity in a panel of human cancer cell lines. Taken together, our results show that rhodanines are a promising class of compounds for developing drugs with antiviral and anticancer properties.

  4. Comparative Study of Genome Divergence in Salmonids with Various Rates of Genetic Isolation

    Directory of Open Access Journals (Sweden)

    Elena A. Shubina

    2013-01-01

    Full Text Available The aim of the study is a comparative investigation of changes that certain genome parts undergo during speciation. The research was focused on divergence of coding and noncoding sequences in different groups of salmonid fishes of the Salmonidae (Salmo, Parasalmo, Oncorhynchus, and Salvelinus genera and the Coregonidae families under different levels of reproductive isolation. Two basic approaches were used: (1 PCR-RAPD with a 20–22 nt primer design with subsequent cloning and sequencing of the products and (2 a modified endonuclease restriction analysis. The restriction fragments were shown with sequencing to represent satellite DNA. Effects of speciation are found in repetitive sequences. The revelation of expressed sequences in the majority of the employed anonymous loci allows for assuming the adaptive selection during allopatric speciation in isolated char forms.

  5. BIOTECHNOLOGY IN THE STUDY OF MOLLUSCS

    Directory of Open Access Journals (Sweden)

    Christopher J. Bayne

    2012-09-01

    Full Text Available The optimization of conditions and media for the in vitro culture of molluscan cells have yielded advances such that primary cultures can now be maintained for weeks or months. With one exception, molluscan cells have proven refractory to the establishment of continous cell lines. Knowledge derived from these studies has helped investigators maintain and grow intramolluscan parasites in vitro.Recent advances in cell fusion, cell cloning and transfection of oncogenes have yet to be applied in molluscan cell culture. Once established, future cell lines will permit a great variety of studies including the control of gene expression, post-transcriptional processing including glycosylation, and the produc­tion in quantity of molluscan factors such as lectins, hormoned, immunogenic macromolecules and others of interest. In addition these lines will also facilitate screening for molluscan viruses and other cellular pathogens, and the propagation of these putative agents of biological control. Such cell lines would also permit the study of molecular mechanisms of drug and hormone actions, and the rational design of drugs.Molecular genetic protocols have been restricted so far to studies of evolution and systematics within molluscan taxa. Both RNA and DNA probes are being developed from ribosomal, mitochondrial and nuclear compartments. Both direct sequencing and restriction length polymorphisms detected using endonucleases are yielding useful data.

  6. COX-2 Gene Promoter Polymorphism and Coronary Artery Disease in Middle-Aged Men: The Helsinki Sudden Death Study

    Directory of Open Access Journals (Sweden)

    Kati H. Huuskonen

    2008-01-01

    Full Text Available Cyclooxygenase (COX catalyzes formation of prostaglandins that contribute to the inflammation in atherosclerosis. Our objective was to study whether the functional C variant of the −765G→C polymorphism in the human COX-2 gene associates with the severity of coronary atherosclerosis measured at the coronary artery level. The Helsinki sudden death study autopsy material (n = 300 comprised of Finnish men who died suddenly. The area of atherosclerotic lesions in the coronary arteries was quantitated, and coronary narrowing was measured. The occurrence of myocardial infarction (MI was assessed. Genotyping was by restriction endonuclease analysis. Men carrying the minor C allele had larger areas of complicated lesions (P = .024 and a higher number of coronary arteries that had over 50% stenosis (P = .036 compared to men representing the common GG genotype. The COX-2 polymorphism was not associated with MI. Our data suggest that COX-2 may be involved in plaque growth.

  7. P450XXI (steroid 21-hydroxylase) gene deletions are not found in family studies of congenital adrenal hyperplasia

    International Nuclear Information System (INIS)

    Matteson, K.J.; Phillips, J.A. III; Miller, W.L.; Chung, B.C.; Orlando, P.J.; Frisch, H.; Ferrandez, A.; Burr, I.M.

    1987-01-01

    Congenital adrenal hyperplasia (CAH) is a common genetic disorder due to defective 21-hydroxylation of steroid hormones. The human P450XXIA2 gene encodes cytochrome P450c21 [steroid 21-monooxygenase (steroid 21-hydroxylase)], which mediates 21-hydroxylation. The P450XXIA2 gene may be distinguished from the duplicated P450XXIA1 pseudogene by cleavage with the restriction endonuclease Taq I, with the XXIA2 gene characterized by a 3.7-kilobase (kb) fragment and the XXIA1 pseudogene characterized by a 3.2-kb fragment. Restriction endonuclease mapping by several laboratories has suggested that deletion of the P450XXIA2 gene occurs in about 25% of patients with CAH, as their genomic DNA lacks detectable 3.7-kb Taq I fragments. The authors have cloned human P450c21 cDNA and used it to study genomic DNA prepared from 51 persons in 10 families, each of which includes 2 or more persons with CAH. After Taq I digestion, apparent deletions are seen in 7 of the 20 alleles of the probands; using EcoRI, apparent deletions are seen in 9 of the 20 alleles. However, the apparently deleted alleles seen with Taq I do not coincide with those seen with EcoRI. Furthermore, studies with Bgl II, EcoRI, Kpn I, and Xba I yield normal patterns with at least two enzymes in all cases. Since all probands yielded normal patterns with at least two of the five enzymes used, they conclude that the P450XXIA2 gene deletions widely reported in CAH patients probably represent gene conversions, unequal crossovers,or polymorphisms rather than simple gene deletions

  8. Mobile DNA elements in T4 and related phages.

    Science.gov (United States)

    Edgell, David R; Gibb, Ewan A; Belfort, Marlene

    2010-10-28

    Mobile genetic elements are common inhabitants of virtually every genome where they can exert profound influences on genome structure and function in addition to promoting their own spread within and between genomes. Phage T4 and related phage have long served as a model system for understanding the molecular mechanisms by which a certain class of mobile DNA, homing endonucleases, promote their spread. Homing endonucleases are site-specific DNA endonucleases that initiate mobility by introducing double-strand breaks at defined positions in genomes lacking the endonuclease gene, stimulating repair and recombination pathways that mobilize the endonuclease coding region. In phage T4, homing endonucleases were first discovered as encoded within the self-splicing td, nrdB and nrdD introns of T4. Genomic data has revealed that homing endonucleases are extremely widespread in T-even-like phage, as evidenced by the astounding fact that ~11% of the T4 genome encodes homing endonuclease genes, with most of them located outside of self-splicing introns. Detailed studies of the mobile td intron and its encoded endonuclease, I-TevI, have laid the foundation for genetic, biochemical and structural aspects that regulate the mobility process, and more recently have provided insights into regulation of homing endonuclease function. Here, we summarize the current state of knowledge regarding T4-encoded homing endonucleases, with particular emphasis on the td/I-TevI model system. We also discuss recent progress in the biology of free-standing endonucleases, and present areas of future research for this fascinating class of mobile genetic elements.

  9. Mobile DNA elements in T4 and related phages

    Directory of Open Access Journals (Sweden)

    Belfort Marlene

    2010-10-01

    Full Text Available Abstract Mobile genetic elements are common inhabitants of virtually every genome where they can exert profound influences on genome structure and function in addition to promoting their own spread within and between genomes. Phage T4 and related phage have long served as a model system for understanding the molecular mechanisms by which a certain class of mobile DNA, homing endonucleases, promote their spread. Homing endonucleases are site-specific DNA endonucleases that initiate mobility by introducing double-strand breaks at defined positions in genomes lacking the endonuclease gene, stimulating repair and recombination pathways that mobilize the endonuclease coding region. In phage T4, homing endonucleases were first discovered as encoded within the self-splicing td, nrdB and nrdD introns of T4. Genomic data has revealed that homing endonucleases are extremely widespread in T-even-like phage, as evidenced by the astounding fact that ~11% of the T4 genome encodes homing endonuclease genes, with most of them located outside of self-splicing introns. Detailed studies of the mobile td intron and its encoded endonuclease, I-TevI, have laid the foundation for genetic, biochemical and structural aspects that regulate the mobility process, and more recently have provided insights into regulation of homing endonuclease function. Here, we summarize the current state of knowledge regarding T4-encoded homing endonucleases, with particular emphasis on the td/I-TevI model system. We also discuss recent progress in the biology of free-standing endonucleases, and present areas of future research for this fascinating class of mobile genetic elements.

  10. DNA Riboprinting and Biochemical Studies on The Effect of Exposure to Pulsed Electromagnetic Field on Rats

    International Nuclear Information System (INIS)

    Marzook, E.A.; EL-Abiad, N.M.; Awwad, M.H.; El-Aragi, G.M.

    2006-01-01

    In the present study pulsed electromagnetic field (PEMF) was used to evaluate the effect of exposure on some molecular and biochemical aspects in male albino rats (Rattus rattus). Three groups of rats were exposed to PEMF (10, 15, 20 pulses/day three times per week for three weeks) with frequency of 8-12 GHz. An unexposed group was considered the control group. At the end of experiment, serum levels of total protein, albumin, globulin, A/G ratio, testosterone, triglycerides and cholesterol were determined. The results revealed that exposure to electromagnetic field induced significant increases in scrum total protein, globulin, triglycerides and testosterone hormone on the second exposed group only. Non significant changes were found in serum levels of albumin and cholesterol in the same group with decreased A/G ratio. All the tested parameters were not affected on the other two exposed groups. An indicative hypothesis for spotting the effect of electromagnetic spectrum on the liver of albino rats was proposed based on the examination of total DNA genome by using PacI, Sfil, Notl and SWal restriction endonucleases. The results achieved have established the value of the physical map as an additional complement to the standard biodosimetric methods

  11. Contribution to the study of markers in lungs carcinogenesis and analysis of factors predicting the benefit of chemotherapy

    International Nuclear Information System (INIS)

    Olaussen, K.A.

    2006-06-01

    A better definition of early bio markers in lung carcinogenesis should enhance the development of molecular means to perform screening, diagnostic, and chemo-prevention of patients at high risk of lung cancer. We studied epigenetic deregulation of multiple promoters (p16(INK4a), HOX A9, MAGE A 1 et MAGE B2) in sputum samples from smokers at high risk and from patients with non-small cell lung cancer (N.S.C.L.C.). This molecular test, based on easily accessible sample,s can be modulated according to the aims of the investigator (e.g. screening or confirmation of diagnosis). Secondly, we have studied two candidate proteins as predictive markers of the benefit of adjuvant chemotherapy in patients with resected lung cancer. The multivariate analysis shows that tumor expression of the catalytic sub-unit of telomerase is not able to predict survival in patients included in the lAL T study' of adjuvant chemotherapy. However, tumor expression of the DNA repair protein ERCC1 identifies a sub-group of patients (ERCC1 negative) whose chances of survival are increased by 35% by means of cisplatin-based adjuvant chemotherapy. Further, tumor ERCC1 expression has a positive prognostic value in the non-treated control group. The need for a deeper understanding in cancerology of the physiological role of the ERCC1 endonuclease is discussed in this thesis. (author)

  12. Studying disorders of vertebrate iron and heme metabolism using zebrafish.

    Science.gov (United States)

    van der Vorm, Lisa N; Paw, Barry H

    2017-01-01

    Iron is a crucial component of heme- and iron-sulfur clusters, involved in vital cellular functions such as oxygen transport, DNA synthesis, and respiration. Both excess and insufficient levels of iron and heme-precursors cause human disease, such as iron-deficiency anemia, hemochromatosis, and porphyrias. Hence, their levels must be tightly regulated, requiring a complex network of transporters and feedback mechanisms. The use of zebrafish to study these pathways and the underlying genetics offers many advantages, among others their optical transparency, ex-vivo development and high genetic and physiological conservations. This chapter first reviews well-established methods, such as large-scale mutagenesis screens that have led to the initial identification of a series of iron and heme transporters and the generation of a variety of mutant lines. Other widely used techniques are based on injection of RNA, including complementary morpholino knockdown and gene overexpression. In addition, we highlight several recently developed approaches, most notably endonuclease-based gene knockouts such as TALENs or the CRISPR/Cas9 system that have been used to study how loss of function can induce human disease phenocopies in zebrafish. Rescue by chemical complementation with iron-based compounds or small molecules can subsequently be used to confirm causality of the genetic defect for the observed phenotype. All together, zebrafish have proven to be - and will continue to serve as an ideal model to advance our understanding of the pathogenesis of human iron and heme-related diseases and to develop novel therapies to treat these conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Homology modeling, molecular docking and DNA binding studies of nucleotide excision repair UvrC protein from M. tuberculosis.

    Science.gov (United States)

    Parulekar, Rishikesh S; Barage, Sagar H; Jalkute, Chidambar B; Dhanavade, Maruti J; Fandilolu, Prayagraj M; Sonawane, Kailas D

    2013-08-01

    Mycobacterium tuberculosis is a Gram positive, acid-fast bacteria belonging to genus Mycobacterium, is the leading causative agent of most cases of tuberculosis. The pathogenicity of the bacteria is enhanced by its developed DNA repair mechanism which consists of machineries such as nucleotide excision repair. Nucleotide excision repair consists of excinuclease protein UvrABC endonuclease, multi-enzymatic complex which carries out repair of damaged DNA in sequential manner. UvrC protein is a part of this complex and thus helps to repair the damaged DNA of M. tuberculosis. Hence, structural bioinformatics study of UvrC protein from M. tuberculosis was carried out using homology modeling and molecular docking techniques. Assessment of the reliability of the homology model was carried out by predicting its secondary structure along with its model validation. The predicted structure was docked with the ATP and the interacting amino acid residues of UvrC protein with the ATP were found to be TRP539, PHE89, GLU536, ILE402 and ARG575. The binding of UvrC protein with the DNA showed two different domains. The residues from domain I of the protein VAL526, THR524 and LEU521 interact with the DNA whereas, amino acids interacting from the domain II of the UvrC protein included ARG597, GLU595, GLY594 and GLY592 residues. This predicted model could be useful to design new inhibitors of UvrC enzyme to prevent pathogenesis of Mycobacterium and so the tuberculosis.

  14. Association between Genetic Polymorphisms of DNA Repair Genes and Chromosomal Damage for 1,3-Butadiene-Exposed Workers in a Matched Study in China.

    Science.gov (United States)

    Xiang, Menglong; Sun, Lei; Dong, Xiaomei; Yang, Huan; Liu, Wen-bin; Zhou, Niya; Han, Xue; Zhou, Ziyuan; Cui, Zhihong; Liu, Jing-yi; Cao, Jia; Ao, Lin

    2015-01-01

    The aim of the study was to examine the association between polymorphisms of DNA repair genes and chromosomal damage of 1,3-butadiene- (BD-) exposed workers. The study was conducted in 45 pairs of occupationally exposed workers in a BD product workshop and matched control workers in an administrative office and a circulatory water workshop in China. Newly developed biomarkers (micronuclei, MNi; nucleoplasmic bridges, NPBs; nuclear buds, NBUDs) in the cytokinesis-blocked micronucleus (CBMN) cytome assay were adopted to detect chromosomal damage. PCR and PCR-restriction fragment length polymorphism (RFLP) are adopted to analyze polymorphisms of DNA repair genes, such as X-ray repair cross-complementing Group 1 (XRCC1), O6-methylguanine-DNA methyltransferase (MGMT), poly (adenosine diphosphate-ribose) polymerases (ADPRT), and apurinic/apyrimidinic endonucleases (APE1). The BD-exposed workers exhibited increased frequencies of MNi and NPBs when compared to subjects in the control group. The results also show that the BD-exposed workers carrying XRCC1 diplotypes TCGA-CCGG (4.25 ± 2.06 ‰) (FR = 2.10, 95% CI: 1.03-4.28) and TCGG-TCGA (5.80 ± 3.56 ‰) (FR = 2.75, 95% CI: 0.76-2.65) had statistically higher NBUD frequencies than those who carried diplotype TCGG-TCGG (1.89 ± 1.27 ‰). Our study suggests that polymorphisms of XRCC1 gene may influence chromosomal damage in BD-exposed workers.

  15. Direct Monitoring of Allosteric Recognition of Type IIE Restriction Endonuclease EcoRII*

    Science.gov (United States)

    Takahashi, Shuntaro; Matsuno, Hisao; Furusawa, Hiroyuki; Okahata, Yoshio

    2008-01-01

    EcoRII is a homodimer with two domains consisting of a DNA-binding N terminus and a catalytic C terminus and recognizes two specific sequences on DNA. It shows a relatively complicated cleavage reaction in bulk solution. After binding to either recognition site, EcoRII cleaves the other recognition site of the same DNA (cis-binding) strand and/or the recognition site of the other DNA (trans-binding) strand. Although it is difficult to separate these two reactions in bulk solution, we could simply obtain the binding and cleavage kinetics of only the cis-binding by following the frequency (mass) changes of a DNA-immobilized quartz-crystal microbalance (QCM) responding to the addition of EcoRII in aqueous solution. We obtained the maximum binding amounts (Δmmax), the dissociation constants (Kd), the binding and dissociation rate constants (kon and koff), and the catalytic cleavage reaction rate constants (kcat) for wild-type EcoRII, the N-terminal-truncated form (EcoRII N-domain), and the mutant derivatives in its C-terminal domain (K263A and R330A). It was determined from the kinetic analyses that the N-domain, which covers the catalytic C-domain in the absence of DNA, preferentially binds to the one DNA recognition site while transforming EcoRII into an active form allosterically, and then the secondary C-domain binds to and cleaves the other recognition site of the DNA strand. PMID:18367450

  16. Control of Transcriptional Fidelity by Active Center Tuning as Derived from RNA Polymerase Endonuclease Reaction*

    Science.gov (United States)

    Sosunova, Ekaterina; Sosunov, Vasily; Epshtein, Vitaly; Nikiforov, Vadim; Mustaev, Arkady

    2013-01-01

    Precise transcription by cellular RNA polymerase requires the efficient removal of noncognate nucleotide residues that are occasionally incorporated. Mis-incorporation causes the transcription elongation complex to backtrack, releasing a single strand 3′-RNA segment bearing a noncognate residue, which is hydrolyzed by the active center that carries two Mg2+ ions. However, in most x-ray structures only one Mg2+ is present. This Mg2+ is tightly bound to the active center aspartates, creating an inactive stable state. The first residue of the single strand RNA segment in the backtracked transcription elongation complex strongly promotes transcript hydrolytic cleavage by establishing a network of interactions that force a shift of stably bound Mg2+ to release some of its aspartate coordination valences for binding to the second Mg2+ thus enabling catalysis. Such a rearrangement that we call active center tuning (ACT) occurs when all recognition contacts of the active center-bound RNA segment are established and verified by tolerance to stress. Transcription factor Gre builds on the ACT mechanism in the same reaction by increasing the retention of the second Mg2+ and by activating the attacking water, causing 3000–4000-fold reaction acceleration and strongly reinforcing proofreading. The unified mechanism for RNA synthesis and degradation by RNA polymerase predicts that ACT also executes NTP selection thereby contributing to high transcription fidelity. PMID:23283976

  17. Control of transcriptional fidelity by active center tuning as derived from RNA polymerase endonuclease reaction.

    Science.gov (United States)

    Sosunova, Ekaterina; Sosunov, Vasily; Epshtein, Vitaly; Nikiforov, Vadim; Mustaev, Arkady

    2013-03-01

    Precise transcription by cellular RNA polymerase requires the efficient removal of noncognate nucleotide residues that are occasionally incorporated. Mis-incorporation causes the transcription elongation complex to backtrack, releasing a single strand 3'-RNA segment bearing a noncognate residue, which is hydrolyzed by the active center that carries two Mg(2+) ions. However, in most x-ray structures only one Mg(2+) is present. This Mg(2+) is tightly bound to the active center aspartates, creating an inactive stable state. The first residue of the single strand RNA segment in the backtracked transcription elongation complex strongly promotes transcript hydrolytic cleavage by establishing a network of interactions that force a shift of stably bound Mg(2+) to release some of its aspartate coordination valences for binding to the second Mg(2+) thus enabling catalysis. Such a rearrangement that we call active center tuning (ACT) occurs when all recognition contacts of the active center-bound RNA segment are established and verified by tolerance to stress. Transcription factor Gre builds on the ACT mechanism in the same reaction by increasing the retention of the second Mg(2+) and by activating the attacking water, causing 3000-4000-fold reaction acceleration and strongly reinforcing proofreading. The unified mechanism for RNA synthesis and degradation by RNA polymerase predicts that ACT also executes NTP selection thereby contributing to high transcription fidelity.

  18. Polyphosphate present in DNA preparations from fungal species of Collectotrichum inhibits restriction endonucleases and other enzymes

    Science.gov (United States)

    Rodriguez, R.J.

    1993-01-01

    During the development of a procedure for the isolation of total genomic DNA from filamentous fungi (Rodriguez, R. J., and Yoder, 0. C., Exp. Mycol. 15, 232-242, 1991) a cell fraction was isolated which inhibited the digestion of DNA by restriction enzymes. After elimination of DNA, RNA, proteins, and lipids, the active compound was purified by gel filtration to yield a single fraction capable of complete inhibition of restriction enzyme activity. The inhibitor did not absorb uv light above 220 nm, and was resistant to alkali and acid at 25°C and to temperatures as high as 100°C. More extensive analyses demonstrated that the inhibitor was also capable of inhibiting T4 DNA ligase and TaqI DNA polymerase, but not DNase or RNase. Chemical analyses indicated that the inhibitor was devoid of carbohydrates, proteins, lipids, and nucleic acids but rich in phosphorus. A combination of nuclear magnetic resonance, metachromatic shift of toluidine blue, and gel filtration indicated that the inhibitor was a polyphosphate (polyP) containing approximately 60 phosphate molecules. The mechanism of inhibition appeared to involve complexing of polyP to the enzymatic proteins. All species of Colletotrichum analyzed produced polyP equivalent in chain length and concentration. A modification to the original DNA extraction procedure is described which eliminates polyP and reduces the time necessary to obtain DNA of sufficient purity for restriction enzyme digestion and TaqI polymerase amplification.

  19. Polymerase Synthesis of Photocaged DNA Resistant against Cleavage by Restriction Endonucleases

    Czech Academy of Sciences Publication Activity Database

    Vaníková, Zuzana; Hocek, Michal

    2014-01-01

    Roč. 53, č. 26 (2014), s. 6734-6737 ISSN 1433-7851 R&D Projects: GA ČR GA14-04289S Institutional support: RVO:61388963 Keywords : DNA * DNA polymerase * nucleotides * photocaging * protective groups Subject RIV: CC - Organic Chemistry Impact factor: 11.261, year: 2014

  20. Demonstration of the Principles of Restriction Endonuclease Cleavage Reactions Using Thermostable Bfl I from "Anoxybacillus Flavithermus"

    Science.gov (United States)

    Sharma, Prince; D'Souza, David R.; Bhandari, Deepali; Parashar, Vijay; Capalash, Neena

    2003-01-01

    Restriction enzymes are basic tools in recombinant DNA technology. To shape the molecular biology experiments, the students must know how to work with these molecular scissors. Here, we describe an integrated set of experiments, introduced in the "Advances in Molecular Biology and Biotechnology" postgraduate course, which covers the important…

  1. Restriction endonuclease analysis of mitochondrial DNA as an aid in the taxonomy of Naegleria and Vahlkampfia.

    Science.gov (United States)

    Milligan, S M; Band, R N

    1988-05-01

    Using restriction enzyme analysis, mitochondrial DNA fragment patterns from seven strains of pathogenic and nonpathogenic Naegleria and one strain of Vahlkampfia were compared to estimate nucleotide sequence divergence. Significantly high levels of estimated genetic variation between strains of N. gruberi, N. fowleri, and N. jadini support the current taxonomic level of the individual Naegleria species and suggest a distinct phylogeny for each group. Naegleria lovaniensis, strain TS, was shown to have significant nucleotide sequence homology with N. gruberi, strain EGs, suggesting that the two groups share a close taxonomic relationship. The pathogenic strain MB-41 of N. fowleri exhibited distinct genetic divergence from the highly homologous, pathogenic strain Nf66 and the drug-cured strain 6088. Morphologically distinct strains EGs and 1518/la of N. gruberi exhibited significantly large sequence divergence consistent with a more distant taxonomic relationship. Amoebae from the genus Vahlkampfia expressed genetic similarity with strains of N. gruberi.

  2. Archaeal rRNA operons, intron splicing and homing endonucleases, RNA polymerase operons and phylogeny

    DEFF Research Database (Denmark)

    Garrett, Roger Antony; Aagaard, Claus Sindbjerg; Andersen, Morten

    1994-01-01

    Over the past decade our laboratory has had a strong interest in defining the phylogenetic status of the archaea. This has involved determining and analysing the sequences of operons of both rRNAs and RNA polymerases and it led to the discovery of the first archaeal rRNA intron. What follows...

  3. Engineering a Nickase on the Homing Endonuclease I-DmoI Scaffold

    DEFF Research Database (Denmark)

    Molina, Rafael; Marcaida, María José; Redondo, Pilar

    2015-01-01

    developed a new variant that is able to cut preferentially the coding DNA strand, generating a nicked DNA target. Our structural and biochemical analysis shows that by decoupling the action of the catalytic residues acting on each strand we can inhibit one of them while keeping the other functional....

  4. Mitochondrial DNA differentiation in the Japanese brown frog Rana japonica as revealed by restriction endonuclease analysis.

    Science.gov (United States)

    Sumida, M

    1997-04-01

    To elucidate mtDNA differentiation in the Japanese brown frog Rana japonica, and compare it with results from allozyme analysis and crossing experiments, RFLP analysis was conducted on 78 frogs from 16 populations in Honshu. Purified mtDNA was digested with eight six-base recognizing restriction enzymes and analyzed by 1% agarose-slab gel electrophoresis. Cleavage patterns of the mtDNA showed three distinct genome size classes: small (18.5 kb), middle (20.0 kb) and large (21.5 kb). Ten haplotypes (I approximately X) were observed among the 16 populations. The expected nucleotide divergences within populations ranged from 0 to 0.47% with a mean of 0.08%. The net nucleotide divergences among 16 populations ranged from 0 to 7.74% with a mean of 3.49%. The UPGMA dendrogram and NJ tree, which were constructed based on the net nucleotide divergences, showed that R. japonica diverged first into the eastern and western groups. The eastern group subsequently differentiated into a subgroup containing six populations and the Akita population, and the western group divided into several subgroups. These results, as well as the results of allozyme analysis and crossing experiments, suggest the the eastern and western groups have experienced secondary contact, and introgression has occurred in the Akita population.

  5. Assembly of Francisella novicida Cpf1 endonuclease in complex with guide RNA and target DNA

    DEFF Research Database (Denmark)

    Alcón, Pablo; Montoya, Guillermo; Stella, Stefano

    2017-01-01

    Bacteria and archaea use the CRISPR-Cas system as an adaptive response against infection by foreign nucleic acids. Owing to its remarkable flexibility, this mechanism has been harnessed and adopted as a powerful tool for genome editing. The CRISPR-Cas system includes two classes that are subdivid...

  6. Cucurbiturils as supramolecular inhibitors of DNA restriction by type II endonucleases.

    Science.gov (United States)

    Parente Carvalho, Cátia; Norouzy, Amir; Ribeiro, Vera; Nau, Werner M; Pischel, Uwe

    2015-03-14

    Cucurbiturils (CB6 and CB7) were shown to inhibit the enzymatically catalyzed restriction of plasmids and linear DNA. This effect can be inverted by supramolecular masking of the macrocycles through competitive complexation with polyamines. These experiments provide supramolecular control of biocatalytic processes.

  7. Asymmetric DNA recognition by the OkrAI endonuclease, an isoschizomer of BamHI

    Energy Technology Data Exchange (ETDEWEB)

    Vanamee, Éva Scheuring; Viadiu, Hector; Chan, Siu-Hong; Ummat, Ajay; Hartline, Adrian M.; Xu, Shuang-yong; Aggarwal, Aneel K. (Mount Sinai Hospital); (NEB)

    2011-11-18

    Restriction enzymes share little or no sequence homology with the exception of isoschizomers, or enzymes that recognize and cleave the same DNA sequence. We present here the structure of a BamHI isoschizomer, OkrAI, bound to the same DNA sequence (TATGGATCCATA) as that cocrystallized with BamHI. We show that OkrAI is a more minimal version of BamHI, lacking not only the N- and C-terminal helices but also an internal 310 helix and containing {beta}-strands that are shorter than those in BamHI. Despite these structural differences, OkrAI recognizes the DNA in a remarkably similar manner to BamHI, including asymmetric contacts via C-terminal 'arms' that appear to 'compete' for the minor groove. However, the arms are shorter than in BamHI. We observe similar DNA-binding affinities between OkrAI and BamHI but OkrAI has higher star activity (at 37 C) compared to BamHI. Together, the OkrAI and BamHI structures offer a rare opportunity to compare two restriction enzymes that work on exactly the same DNA substrate.

  8. Shape-selective recognition of DNA abasic sites by metallohelices: inhibition of human AP endonuclease 1

    Czech Academy of Sciences Publication Activity Database

    Malina, Jaroslav; Scott, P.; Brabec, Viktor

    2015-01-01

    Roč. 43, č. 11 (2015), s. 5297-5306 ISSN 1362-4962 R&D Projects: GA ČR(CZ) GAP205/11/0856 Institutional support: RVO:68081707 Keywords : BASE EXCISION-REPAIR * CYTOSINE OPPOSITE * BINDING Subject RIV: BO - Biophysics

  9. 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...... into the double-stranded DNA. Unzipping of the double-stranded DNA occurs in a cleft arranged by acidic and hydrophobic residues facilitating the crRNA-DNA hybrid formation. The PAM single-stranded DNA is funnelled towards the nuclease site through a mixed hydrophobic and basic cavity. In this catalytic...... conformation, the PAM-interacting domain and the helix-loop-helix motif in the REC1 domain adopt a 'rail' shape and 'flap-on' conformations, respectively, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms the 'septum', separating the displaced PAM strand...

  10. Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix.

    Science.gov (United States)

    Horton, John R; Wang, Hua; Mabuchi, Megumu Yamada; Zhang, Xing; Roberts, Richard J; Zheng, Yu; Wilson, Geoffrey G; Cheng, Xiaodong

    2014-10-29

    MspJI belongs to a family of restriction enzymes that cleave DNA containing 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC). MspJI is specific for the sequence 5(h)mC-N-N-G or A and cleaves with some variability 9/13 nucleotides downstream. Earlier, we reported the crystal structure of MspJI without DNA and proposed how it might recognize this sequence and catalyze cleavage. Here we report its co-crystal structure with a 27-base pair oligonucleotide containing 5mC. This structure confirms that MspJI acts as a homotetramer and that the modified cytosine is flipped from the DNA helix into an SRA-like-binding pocket. We expected the structure to reveal two DNA molecules bound specifically to the tetramer and engaged with the enzyme's two DNA-cleavage sites. A coincidence of crystal packing precluded this organization, however. We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically. The latter interaction, which prevented cleavage-site engagement, also involved base flipping and might represent the sequence-interrogation phase that precedes specific recognition. MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits. Such interchange of function might explain how other complex multimeric restriction enzymes act. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases

    Directory of Open Access Journals (Sweden)

    Carmen F Bjurström

    2016-01-01

    Full Text Available We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs, transcriptional activator-like effector nucleases (TALENs, and clustered regularly interspaced short palindromic repeat (CRISPR/Cas9 systems designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34+ progenitor cells. ZFNs and TALENs were delivered as in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA (pU6.g1 or in vitro transcribed gRNA (gR.1. Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34+ cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34+ cells had the capacity to engraft in NOD-PrkdcSCID-IL2Rγnull mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34+ cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs, highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs.

  12. Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication

    NARCIS (Netherlands)

    Kindler, Eveline; Gil-Cruz, Cristina; Spanier, Julia; Li, Yize; Wilhelm, Jochen; Rabouw, Huib H; Züst, Roland; Hwang, Mihyun; V'kovski, Philip; Stalder, Hanspeter; Marti, Sabrina; Habjan, Matthias; Cervantes-Barragan, Luisa; Elliot, Ruth; Karl, Nadja; Gaughan, Christina; van Kuppeveld, Frank J M; Silverman, Robert H; Keller, Markus; Ludewig, Burkhard; Bergmann, Cornelia C; Ziebuhr, John; Weiss, Susan R; Kalinke, Ulrich; Thiel, Volker

    2017-01-01

    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

  13. Pulsed Field Gel Electrophoresis (PFGE: a DNA finger printing technique to study the genetic diversity of blood disease bacterium of banana

    Directory of Open Access Journals (Sweden)

    HADIWIYONO

    2011-01-01

    Full Text Available Hadiwiyono, Widada J, Subandiyah S, Fegan F (2011 Pulsed Field Gel Electrophoresis (PFGE: a DNA finger printing technique to study the genetic diversity of blood disease bacterium of banana. Biodiversitas 12: 12-16. Blood disease bacterium (BDB is the most important pathogen of bananas in Indonesia. In some field, the disease incidence reaches over 80%. Epidemiologically, the disease is similar to moko disease in South America and bugtok disease in the Philippines caused by Ralstonia solanacearum race 2. However, BDB is different in phenotype and genotype from the two diseases. Previously BDB was limited in South Sulawesi since 1920s – 1980s and recently was reported in 27 of 30 provinces in Indonesia. Pulsed-Field Gel Electrophoresis (PFGE is a genomic DNA fingerprinting method, which employs rare cutting restriction endonucleases to digest genome prior to electrophoresis using specialized condition to separate of large DNA fragments. The results showed that PFGE analysis was a discriminative tool to study the genetic diversity of BDB. Based on the PFGE analysis, BDB isolates obtained from different localities in Yogyakarta and Central Java were quit diverse.

  14. Epidemiology of Clostridium difficile: a hospital-based descriptive study in Argentina and Mexico

    Directory of Open Access Journals (Sweden)

    Gustavo Lopardo

    2015-01-01

    Full Text Available A prospective study was conducted in four tertiary hospitals in Argentina and Mexico in order to describe the occurrence of Clostridium difficile infection (CDI in these settings. The objective was to evaluate the incidence of CDI in at-risk populations in Argentina (one center and Mexico (three centers and to further explore potential study sites for vaccine development in this region. A prospective, descriptive, CDI surveillance study was conducted among hospitalized patients aged ≥40 years who had received ≥48 h of antibiotic treatment. Stool samples were collected from those with diarrhea within 30 days after starting antibiotics and analyzed for toxins A and B by ELISA, and positive samples were further tested by toxinogenic culture and restriction endonuclease analysis type assay. Overall, 466 patients were enrolled (193 in Argentina and 273 in Mexico of whom 414 completed the follow-up. Of these, 15/414 (3.6% experienced CDI episodes occurring on average 18.1 days after admission to hospital and 15.9 days after the end of antibiotics treatment. The incidence rate of CDI was 3.1 (95% CI 1.7–5.2 per 1000 patient-days during hospitalization, and 1.1 (95% CI 0.6–1.8 per 1000 patient-days during the 30-day follow-up period. This study highlighted the need for further evaluation of the burden of CDI in both countries, including the cases occurring after discharge from hospital.

  15. Differentiation of Shigella flexneri strains by rRNA gene restriction patterns.

    OpenAIRE

    Faruque, S M; Haider, K; Rahman, M M; Abdul Alim, A R; Ahmad, Q S; Albert, M J; Sack, R B

    1992-01-01

    We studied the restriction endonuclease cleavage patterns of rRNA genes (ribotypes) of 72 clinical isolates of Shigella flexneri representing eight serotypes to determine whether ribotyping could be used to distinguish S. flexneri strains and to compare the discriminating ability of the method with that of serotyping. By using a cloned Escherichia coli rRNA operon as the probe, Southern blot hybridization of restriction endonuclease-digested total DNA was carried out. Ribotyping of the isolat...

  16. Viruses of eukaryotic green algae. Final technical report, June 1, 1989--February 1, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Van Etten, J.L.

    1992-12-31

    We have isolated and partially characterized many large, polyhedral, DNA containing, plaque forming viruses which infect certain unicellular, eukaryotic, chlorella-like green algae. These viruses have several unique features, including the fact that they code for DNA site-specific endonucleases and DNA methyltransferases. The primary objectives of this study were to identify, clone, and characterize some of the virus-encoded DNA methyltransferases and DNA restriction endonucleases in order to understand their biological function.

  17. Repair of DNA treated with γ-irradiation and chemical carcinogens. Progress report, February 1, 1976--February 28, 1977

    International Nuclear Information System (INIS)

    Goldthwait, D.A.

    1977-01-01

    Enzymes from E. coli which recognize damage in DNA due to chemical carcinogens and γ-irradiation are under study. Endonuclease II, which recognizes DNA treated with methylmethane sulfonate, methylnitrosourea, 7-bromomethyl-12-methylbenz[a]anthracene and γ-irradiation. Apurinic endonuclease (endonuclease VI) recognizes apurinic and apyrimidinic acid sites. These enzymes have now been purified to homogeneity and separated from exonuclease III which other groups have thought to be identical with one or both of the endonucleases. A series of mutants of E. coli were found to lack one, two, or all of these enzymes. The genetic loci for the two endonucleases are very close to the Exonuclease III locus at 38.2 minutes. An attempt to relate in vivo sensitivity to MMS to lack of specific enzymes has been made. Substrates for endonuclease II have been examined. β-propiolactone treated DNA has been found to be substrate and details of this are being examined. The nature of the lesion in γ-irradiated DNA is under investigation. A search for mammalian enzymes which are N-glycosidases is underway

  18. Studies on the transmission of malignant catarrhal fever in experimental animals: A serial infection of cattle and buffalo by means of whole blood inoculation

    Directory of Open Access Journals (Sweden)

    Agus Wiyono

    1999-12-01

    Full Text Available Malignant catarrhal fever (MCF is a fatal disease especially affecting cattle and buffaloes. A study on the serial blood transmission of MCF was conducted by injecting whole blood of MCF animals into 9 experimental animals. Diagnosis of MCF was based on the clinico-pathological fmdings and polymerase chain reaction (PCR test. The disease has successfully, been achieved in six animals of three Bali cattle and three buffaloes but not in a Bali-cross breed and two Bos indicus (Ongole cattle. Wide range of clinical signs and gross-pathological features were observed. The study showed the degree of susceptibility of experimental animals: Bali cattle and buffalo were highly susceptible (3 out of 3 affected with MCF, Bali-cross breed and Bos indicus (Ongole cattle seemed not susceptible to whole blood experimental transmission. It shows that when Bali cattle acted as inoculum donor, buffalo tended to be clinically more severe than Bali cattle. On the other hand, when buffalo acted as inoculum donor, Bali cattle suffered from MCF more severe than buffalo. The diagnosis of MCF by histopathological examination and the PCR test bad positive correlation (100% in the first experiment, while in the second experiment the PCR test tends to be more sensitive. Based on the restriction endonuclease (RE test, the MCF causal agent in this study appeared to be genetically similar in each case. It is concluded that the serial experimental transmission of MCF by means of whole blood inoculation has been successfully achieved in Bali cattle and buffalo but not in Bali-cross breed and Ongole cattle, and there is a positive correlation between the PCR test and histopathological examination with the PCR test tends to be more sensitive.

  19. Studies with the human cohesin establishment factor, ChlR1. Association of ChlR1 with Ctf18-RFC and Fen1.

    Science.gov (United States)

    Farina, Andrea; Shin, Jae-Ho; Kim, Do-Hyung; Bermudez, Vladimir P; Kelman, Zvi; Seo, Yeon-Soo; Hurwitz, Jerard

    2008-07-25

    Human ChlR1 (hChlR1), a member of the DEAD/DEAH subfamily of helicases, was shown to interact with components of the cohesin complex and play a role in sister chromatid cohesion. In order to study the biochemical and biological properties of hChlR1, we purified the protein from 293 cells and demonstrated that hChlR1 possesses DNA-dependent ATPase and helicase activities. This helicase translocates on single-stranded DNA in the 5' to 3' direction in the presence of ATP and, to a lesser extent, dATP. Its unwinding activity requires a 5'-singlestranded region for helicase loading, since flush-ended duplex structures do not support unwinding. The helicase activity of hChlR1 is capable of displacing duplex regions up to 100 bp, which can be extended to 500 bp by RPA or the cohesion establishment factor, the Ctf18-RFC (replication factor C) complex. We show that hChlR1 interacts with the hCtf18-RFC complex, human proliferating cell nuclear antigen, and hFen1. The interactions between Fen1 and hChlR1 stimulate the flap endonuclease activity of Fen1. Selective depletion of either hChlR1 or Fen1 by targeted small interfering RNA treatment results in the precocious separation of sister chromatids. These findings are consistent with a role of hChlR1 in the establishment of sister chromatid cohesion and suggest that its action may contribute to lagging strand processing events important in cohesion.

  20. Determinants for association and gRNA-directed endonuclease cleavage by purified RNA editing complexes from Trypanosoma brucei

    OpenAIRE

    Hernandez, Alfredo; Panigrahi, Aswini; Cifuentes-Rojas, Catherine; Sacharidou, Anastasia; Stuart, Kenneth; Cruz-Reyes, Jorge

    2008-01-01

    U-insertion/deletion RNA editing in the single mitochondrion of ancient kinetoplastids is a unique mRNA maturation process needed for translation. Multi-subunit editing complexes recognize many pre-mRNA sites and modify them via cycles of three catalytic steps: guide-RNA (gRNA) mediated cleavage, insertion or deletion of uridylates at the 3’ terminus of the upstream cleaved piece, and ligation of the two mRNA pieces. While catalytic and many structural protein subunits of these complexes have...

  1. Interdomain communication in the endonuclease/motor subunit of type I restriction-modification enzyme EcoR124I

    Czech Academy of Sciences Publication Activity Database

    Sinha, Dhiraj; Shamayeva, Katerina; Ramasubramani, V.; Řeha, David; Bialevich, V.; Khabiri, Morteza; Guzanová, Alena; Milbar, N.; Weiserová, Marie; Cséfalvay, Eva; Carey, J.; Ettrich, Rüdiger

    2014-01-01

    Roč. 20, č. 7 (2014), s. 2334 ISSN 1610-2940 R&D Projects: GA ČR GAP207/12/2323 Institutional support: RVO:67179843 ; RVO:61388971 Keywords : DNA restriction enzymes * Molecular modeling * QM/MM calculations * principal components analysis * E. coli * Multisubunit enzyme complex * Correlated loop motions Subject RIV: EH - Ecology, Behaviour; EE - Microbiology, Virology (MBU-M) Impact factor: 1.736, year: 2014

  2. Staphylococcal pathogenicity island DNA packaging system involving cos-site packaging and phage-encoded HNH endonucleases.

    Science.gov (United States)

    Quiles-Puchalt, Nuria; Carpena, Nuria; Alonso, Juan C; Novick, Richard P; Marina, Alberto; Penadés, José R

    2014-04-22

    Staphylococcal pathogenicity islands (SaPIs) are the prototypical members of a widespread family of chromosomally located mobile genetic elements that contribute substantially to intra- and interspecies gene transfer, host adaptation, and virulence. The key feature of their mobility is the induction of SaPI excision and replication by certain helper phages and their efficient encapsidation into phage-like infectious particles. Most SaPIs use the headful packaging mechanism and encode small terminase subunit (TerS) homologs that recognize the SaPI-specific pac site and determine SaPI packaging specificity. Several of the known SaPIs do not encode a recognizable TerS homolog but are nevertheless packaged efficiently by helper phages and transferred at high frequencies. In this report, we have characterized one of the non-terS-coding SaPIs, SaPIbov5, and found that it uses two different, undescribed packaging strategies. SaPIbov5 is packaged in full-sized phage-like particles either by typical pac-type helper phages, or by cos-type phages--i.e., it has both pac and cos sites--a configuration that has not hitherto been described for any mobile element, phages included--and uses the two different phage-coded TerSs. To our knowledge, this is the first example of SaPI packaging by a cos phage, and in this, it resembles the P4 plasmid of Escherichia coli. Cos-site packaging in Staphylococcus aureus is additionally unique in that it requires the HNH nuclease, carried only by cos phages, in addition to the large terminase subunit, for cos-site cleavage and melting.

  3. Distribution of the ribosome associated endonuclease Rae1 and the potential role of conserved amino acids in codon recognition.

    Science.gov (United States)

    Condon, Ciarán; Piton, Jéremie; Braun, Frédérique

    2018-03-20

    We recently identified a novel ribonuclease in Bacillus subtilis called Rae1 that cleaves mRNAs in a translation-dependent manner. Rae1 is a member of the NYN/PIN family of ribonucleases and is highly conserved in the Firmicutes, the Cyanobacteria and the chloroplasts of photosynthetic algae and plants. We have proposed a model in which Rae1 enters the A-site of ribosomes that are paused following translation of certain sequences that are still ill-defined. In the only case identified thus far, Rae1 cleaves between a conserved glutamate and lysine codon during translation of a short peptide called S1025. Certain other codons are also tolerated on either side of the cleavage site, but these are recognized less efficiently. The model of Rae1 docked in the A-site allows us to make predictions about which conserved residues may be important for recognition of mRNA, the tRNA in the adjacent P-site and binding to the 50S ribosome subunit.

  4. Multiplex-endonuclease genotyping approach (MEGA): a tool for the fine-scale detection of unlinked polymorphic DNA markers.

    NARCIS (Netherlands)

    Agbo, E.C.; Duim, B.; Majiwa, P.A.O.; Buscher, P.; Claassen, E.; Pas, te M.F.W.

    2003-01-01

    Restriction enzyme-detectable polymorphisms have been used for assessing genetic differences and generating informative genetic markers. The most detailed fingerprinting analyses have been obtained using the AFLP (amplified fragment length polymorphism) technique, which accesses subsets of

  5. Highly efficient one-step scarless protein tagging by type IIS restriction endonuclease-mediated precision cloning.

    Science.gov (United States)

    Xu, Zhen; Rui, Yan-Ning; Balzeau, Julien; Menezes, Miriam R; Niu, Airu; Hagan, John P; Kim, Dong H

    2017-08-12

    Protein tagging with a wide variety of epitopes and/or fusion partners is used routinely to dissect protein function molecularly. Frequently, the required DNA subcloning is inefficient, especially in cases where multiple constructs are desired for a given protein with unique tags. Additionally, the generated clones have unwanted junction sequences introduced. To add versatile tags into the extracellular domain of the transmembrane protein THSD1, we developed a protein tagging technique that utilizes non-classical type IIS restriction enzymes that recognize non-palindromic DNA sequences and cleave outside of their recognition sites. Our results demonstrate that this method is highly efficient and can precisely fuse any tag into any position of a protein in a scarless manner. Moreover, this method is cost-efficient and adaptable because it uses commercially available type IIS restriction enzymes and is compatible with the traditional cloning system used by many labs. Therefore, precision tagging technology will benefit a number of researchers by providing an alternate method to integrate an array of tags into protein expression constructs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Multiplex-endonuclease genotyping approach (MEGA): a tool for the fine-scale detection of unlinked polymorphic DNA markers

    NARCIS (Netherlands)

    Agbo, Eddy Chukwura; Duim, Birgitta; Majiwa, Phelix A. O.; Büscher, Philippe; Claassen, Eric; te Pas, Marinus F. W.

    2003-01-01

    Restriction enzyme-detectable polymorphisms have been used for assessing genetic differences and generating informative genetic markers. The most detailed fingerprinting analyses have been obtained using the AFLP (amplified fragment length polymorphism) technique, which accesses subsets of

  7. 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

  8. 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

    2012-08-01

    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').

  9. Fragmentation of bacteriophage S13 replicative from DNA by restriction endonucleases from Hemophilus influenzae and Hemophilus aegyptius.

    NARCIS (Netherlands)

    F.G. Grosveld (Frank); K.M. Ojamaa; J.H. Spencer

    1976-01-01

    textabstractThe restriction enzymes Hind from Hemophilus influenzae and HaeIII from Hemophilus aegyptius cleave bacteriophage S13 replicative form (RF) DNA into 13 and 10 specific fragments, respectively. The sizes of these fragments were estimated by gel electrophoresis, electron microscopy, and

  10. Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System

    NARCIS (Netherlands)

    Zetsche, Bernd; Gootenberg, Jonathan S.; Abudayyeh, Omar O.; Slaymaker, Ian M.; Makarova, Kira S.; Essletzbichler, Patrick; Volz, Sara E.; Joung, Julia; Oost, van der John; Regev, Aviv; Koonin, Eugene V.; Zhang, Feng

    2015-01-01

    The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized

  11. Anti-influenza activity of marchantins, macrocyclic bisbibenzyls contained in liverworts.

    Directory of Open Access Journals (Sweden)

    Yuma Iwai

    Full Text Available The H1N1 influenza A virus of swine-origin caused pandemics throughout the world in 2009 and the highly pathogenic H5N1 avian influenza virus has also caused epidemics in Southeast Asia in recent years. The threat of influenza A thus remains a serious global health issue and novel drugs that target these viruses are highly desirable. Influenza A possesses an endonuclease within its RNA polymerase which comprises PA, PB1 and PB2 subunits. To identify potential new anti-influenza compounds in our current study, we screened 33 different types of phytochemicals using a PA endonuclease inhibition assay in vitro and an anti-influenza A virus assay. The marchantins are macrocyclic bisbibenzyls found in liverworts, and plagiochin A and perrottetin F are marchantin-related phytochemicals. We found from our screen that marchantin A, B, E, plagiochin A and perrottetin F inhibit influenza PA endonuclease activity in vitro. These compounds have a 3,4-dihydroxyphenethyl group in common, indicating the importance of this moiety for the inhibition of PA endonuclease. Docking simulations of marchantin E with PA endonuclease suggest a putative "fitting and chelating model" as the mechanism underlying PA endonuclease inhibition. The docking amino acids are well conserved between influenza A and B. In a cultured cell system, marchantin E was further found to inhibit the growth of both H3N2 and H1N1 influenza A viruses, and marchantin A, E and perrotein F showed inhibitory properties towards the growth of influenza B. These marchantins also decreased the viral infectivity titer, with marchantin E showing the strongest activity in this assay. We additionally identified a chemical group that is conserved among different anti-influenza chemicals including marchantins, green tea catechins and dihydroxy phenethylphenylphthalimides. Our present results indicate that marchantins are candidate anti-influenza drugs and demonstrate the utility of the PA endonuclease assay in

  12. 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

  13. Shigella sonnei biotype g carrying class 2 integrons in southern Italy: a retrospective typing study by pulsed field gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Romani Cristina

    2006-07-01

    Full Text Available Abstract Background Emergence and global dissemination of multiresistant strains of enteric pathogens is a very concerning problem from both epidemiological and Public Health points of view. Shigella sonnei is the serogroup of Shigella most frequently responsible for sporadic and epidemic enteritis in developed countries. The dissemination is associated most often to human to human transmission, but foodborne episodes have also been described. In recent years the circulation of multiresistant strains of S. sonnei biotype g carrying a class 2 integron has been reported in many countries worldwide. In southern Italy a strain with similar properties has been responsible for a large community outbreak occurred in 2003 in Palermo, Sicily. The objective of this study was to date the emergence of the biotype g strain carrying the class 2 integron in southern Italy and to evaluate the genetic heterogeneity of biotype g S. sonnei isolated throughout an extended interval of time. Methods A total of 31 clinical isolates of S. sonnei biotype g identified in southern Italy during the years 1971–2000 were studied. The strains were identified at the serogroup level, characterized by biochemical tests and submitted to antimicrobial susceptibility testing. Molecular typing was performed by pulsed field gel electrophoresis (PFGE after digestion of DNA by XbaI. Carriage of class 2 integrons was investigated by polymerase chain reaction (PCR with specific primers and confirmed by restriction endonuclease analysis of amplicons. Results The 15 isolates of S. sonnei biotype g identified in the decade 1971–1980 showed highly heterogeneous drug resistance profiles and pulsotypes. None of the isolates was simultaneous resistant to streptomycin and trimethoprim and none was class 2 integron positive. On the contrary, this resistance phenotype and class 2 integron carriage were very common among the 16 strains of biotype g identified in the following two decades

  14. Molecular diagnostic of Philadelphia chromosome in patients affected by mieloid leukemia

    International Nuclear Information System (INIS)

    Campos Rudin, M.E.

    1996-01-01

    The main objective of this study, was to contribute with new elements, to the clinical diagnostic and the monitoring of hematologic diseases, through molecular techniques. Such technique is known as S outhern Blot . Non radioactive and radioactive methods were used, to sift the presence or absence of Ph chromosome. The sound denominated Transprobe-1 and the Endonuclease Bgl II were used. 41 samples proceeding from pacients diagnosed with LLMC and 9 patients grouped with myloproliferatives sindromes or myeloplast sindromes, which were getting treatment at Hospital San Juan de Dios or at Hospital Mexico, were analyzed. The studies detected the presence of rearrangements between Mocr/ABL and (Ph +), and in the remaining; the results were negative Ph . The author found that the application of one single endonuclease is usefull to make a first general sift of the patients; but the application of another endonuclease is required, to confirm the cases that resulted Ph negative. (S. Grainger)

  15. Interactions of radiation repair systems in escherichia colt with antineoplastic anthracyclines

    International Nuclear Information System (INIS)

    Kacinski, B.M.; Rupp, W.D.

    1984-01-01

    The authors have studied the interactions of several anthracylines with the UVR DN*a repair system of E. coli. The authors found that doxorubicin is quite toxic for uvr- but not for uvr+strains. They also found that exposure of cells carrying a multicopy plasmid to this agent yielded plasmid DNA molecules with lesions which were recognized and cleaved by purified E. coli UVRABC DNA repair endonuclease. A derivative of doxorubicin was not measurably toxic for either uvr+ or uvr- strains but nevertheless produced lesions which were substrates for the UVRABC endonuclease. The authors propose that anthracycline toxicity in uvr- E. coli may correlate with clinical toxicity while anthracylcine antineopolastic activity may correlate with their ability to produce UVRABC endonuclease-sensitive DNA damage

  16. The use of molecular biology techniques for the diagnosis and epidemiological study of foot-and-mouth disease virus in Thailand

    International Nuclear Information System (INIS)

    Linchongsubongkoch, W.; Janukit, T.; Romlumdoan, S.; Phusirimongkol, A.

    2000-01-01

    The detection of foot-and-mouth disease (FMD) virus from various kinds of field samples (tissue extract and cell culture isolate) was studied using the polymerase chain reaction (PCR) technique. The gene selected for diagnosis was the polymerase gene and an amplification target product of 454 bp in length was produced using AP5/AP6 primer sets. The PCR product was further examined by NcoI endonuclease digestion. The presence of the internal restriction site was confirmed by demonstration of two small fragments of 330 bp and 124 bp in length. Forty-nine samples that gave positive and negative results by ELISA typing and were positive by the PCR test were tested by NcoI digestion to confirm the results. About 10% of PCR products could not be confirmed by the method. Furthermore the FMD RNA polymerase gene could be detected by the PCR method in samples negative in both ELISA typing and the virus isolation test. A total of 23 samples were examined and compared after each stage of the testing process. At the end of the extraction for ELISA the amplification product band at 454 bp was detected in 74% of the negative tissue extract samples, and in 48% at the end of the virus isolation procedure. The PCR technique was shown to rapidly and sensitively detect FMD viral genome, when compared with virus titration by tissue culture infectious dose 50% (TCID 50 ) method. The PCR was about 10 times more sensitive than the virus titration technique in detection of virus. Therefore, the PCR technique can be used in conjunction with current procedures for FMD diagnosis, to support the routine standard ELISA typing and virus isolation test on clinical samples. The first step of the nucleotide sequencing technique was introduced with a view to study genomic differentiation of FMD outbreak viruses. The appropriate primer sets for each of the three endemic sero-types were optimized and used to detect the PCR products from field isolate viruses. The PCR products of FMDV type O, A and

  17. The entire β-globin gene cluster is deleted in a form of τδβ-thalassemia.

    NARCIS (Netherlands)

    E.R. Fearon; H.H.Jr. Kazazian; P.G. Waber (Pamela); J.I. Lee (Joseph); S.E. Antonarakis; S.H. Orkin (Stuart); E.F. Vanin; P.S. Henthorn; F.G. Grosveld (Frank); A.F. Scott; G.R. Buchanan

    1983-01-01

    textabstractWe have used restriction endonuclease mapping to study a deletion involving the beta-globin gene cluster in a Mexican-American family with gamma delta beta-thalassemia. Analysis of DNA polymorphisms demonstrated deletion of the beta-globin gene from the affected chromosome. Using a DNA

  18. Molecular phylogeny of Fusarium species by AFLP fingerprint ...

    African Journals Online (AJOL)

    The high-resolution genotyping method of amplified fragment length polymorphism (AFLP) analysis was used to study the genetic relationships within and between natural populations of five Fusarium spp. AFLP templates were prepared by the digestion of Fusarium DNA with EcoRI and MseI restriction endonucleases and ...

  19. 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

  20. Structure-based insights into the repair of UV-damaged DNA

    NARCIS (Netherlands)

    Meulenbroek, Elisabeth Maria

    2012-01-01

    Repair of damage in the DNA is essential for an organism. Therefore, several repair mechanisms have evolved. In this thesis, the mechanism of Transcription-Coupled Nucleotide Excision Repair (TC-NER) and the UV Damage Endonuclease repair pathway (UVDE) have been studied. Central to TC-NER is the

  1. Molecular basis of the mutagenic and lethal effects of ultraviolet irradiation

    International Nuclear Information System (INIS)

    Grossman, L.

    1982-01-01

    Using bacteria as a model, the molecular basis of the mutagenic and lethal effects of uv radiation is being studied. Attention is focused on the mechanism of action of uv-1 specific endonucleases in the repair of damaged DNA. The isolation and identification of similar enzymes in human cells are being conducted concurrently

  2. Evaluation of insulin-like growth factor-I gene polymorphism in ...

    African Journals Online (AJOL)

    This study aimed to detect the genetic polymorphism of IGF-1 in different Egyptian sheep and goat breeds. The amplified fragments at 320-bp were digested with HaeIII endonuclease and the results show the presence of three different genotypes: CC (15.71%), CG (29.29%) and GG (55.0%). The nucleotide sequence ...

  3. FnCpf1: a novel and efficient genome editing tool for Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Swiat, M.A.; Dashko, S.; den Ridder, M.J.; Wijsman, M.; van der Oost, John; Daran, J.G.; Daran-Lapujade, P.A.S.

    2017-01-01

    Cpf1 is a new class II family of CRISPR-Cas RNA-programmable endonucleases with unique features that make it a very attractive alternative or complement to Cas9 for genome engineering. Using constitutively expressed Cpf1 from Francisella novicida, the present study demonstrates that FnCpf1 can

  4. FnCpf1: a novel and efficient genome editing tool for Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Swiat, Michal A.; Dashko, Sofia; Ridder, den Maxime; Wijsman, Melanie; Oost, van der John; Daran, Jean Marc; Daran-Lapujade, Pascale

    2017-01-01

    Cpf1 is a new class II family of CRISPR-Cas RNA-programmable endonucleases with unique features that make it a very attractive alternative or complement to Cas9 for genome engineering. Using constitutively expressed Cpf1 from Francisella novicida, the present study demonstrates that FnCpf1 can

  5. Genetic variability of cultivated cowpea in Benin assessed by ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-12-17

    Dec 17, 2008 ... Res. Crop Evol. 48: 559-566. Mignouna HD, Ng NQ, Ikea J, Thottapilly G (1998). Genetic diversity in cowpea as revealed by random amplified polymorphic DNA. J. Gen. Breed. 52: 151-159. Nei M, Li WH (1979). Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc.

  6. Studying Genes

    Science.gov (United States)

    ... NIGMS NIGMS Home > Science Education > Studying Genes Studying Genes Tagline (Optional) Middle/Main Content Area PDF Version (382 KB) Other Fact Sheets What are genes? Genes are segments of DNA that contain instructions ...

  7. Study Drugs

    Science.gov (United States)

    ... What Are Study Drugs? Doctors prescribe medicines like Adderall and Ritalin to treat conditions like attention deficit ... stimulants are used as study drugs: amphetamines like Adderall, Dexedrine, or Vyvanse methylphenidates like Ritalin or Concerta ...

  8. Game Studies

    NARCIS (Netherlands)

    Raessens, J.F.F.

    2016-01-01

    This entry describes game studies as a dynamic interdisciplinary field of academic study and research that focuses on digital games and play in a wide variety of social and cultural contexts. It examines the history of game studies from its prehistory, when games were looked at as part of other

  9. HTS Teologiese Studies / Theological Studies

    African Journals Online (AJOL)

    HTS Teologiese Studies/Theological Studies is an acclaimed Open Access journal with broad coverage that promotes multidisciplinary, religious, and biblical aspects of studies in the international theological arena. The journal's publication criteria are based on high ethical standards and the rigor of the methodology and ...

  10. Purification, crystallization and preliminary X-ray analysis of the HsdR subunit of the EcoR124I endonuclease from Escherichia coli

    Czech Academy of Sciences Publication Activity Database

    Lapkouski, Mikalai

    2007-01-01

    Roč. 63, č. 7 (2007), s. 582-585 ISSN 1744-3091 R&D Projects: GA MŠk(CZ) LC06010 Institutional research plan: CEZ:AV0Z60870520 Keywords : DNA translocation Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 0.645, year: 2007

  11. Differentiation of Moraxella bovoculi sp. nov. from other coccoid moraxellae by the use of polymerase chain reaction and restriction endonuclease analysis of amplified DNA.

    Science.gov (United States)

    Angelos, John A; Ball, Louise M

    2007-09-01

    Moraxella ovis was historically the only coccoid Moraxella identified in cultures of ocular fluid from cattle with infectious bovine keratoconjunctivitis (IBK) and could be morphologically and biochemically differentiated from Moraxella bovis. Moraxella bovoculi sp. nov. is a recently characterized Moraxella isolated from ulcerated eyes of calves with IBK in northern California in 2002. Like Moraxella ovis, M. bovoculi sp. nov. is a gram-negative coccus/diplococcus. All 18 original isolates of M. bovoculi sp. nov. possessed phenylalanine deaminase (PADase) activity and could therefore be differentiated from M. ovis and M. bovis. During the characterization of 44 additional isolates of hemolytic gram-negative cocci that were cultured from ulcerated eyes of IBK-affected calves, 2 PADase-negative isolates were identified that could not be differentiated biochemically from M. ovis; however, the DNA sequence of the 16S-23S intergenic spacer region (ISR) of the isolates matched the 16S-23S ISR DNA sequence of M. bovoculi sp. nov. To facilitate the identification of PADase-negative moraxellae, a polymerase chain reaction (PCR) coupled with restriction enzyme digestion analysis of amplified DNA was developed. Amplification of the 16S-23S ISR followed by AfaI digestion of amplified DNA could differentiate M. bovoculi sp. nov. from M. ovis and other moraxellae. The DNA sequence analysis of the amplified 16S-23S ISR from the 42 PADase-positive isolates of hemolytic gram-negative cocci indicated that all were M. bovoculi sp. nov. and all possessed an AfaI site. A PCR coupled with restriction analysis of amplified DNA can aid in identifying M. bovoculi sp. nov.

  12. Translocation, switching and gating: potential roles for ATP in long-range communication on DNA by Type III restriction endonucleases.

    Science.gov (United States)

    Szczelkun, Mark D

    2011-04-01

    To cleave DNA, the Type III RM (restriction-modification) enzymes must communicate the relative orientation of two recognition sequences, which may be separated by many thousands of base pairs. This long-range interaction requires ATP hydrolysis by a helicase domain, and both active (DNA translocation) and passive (DNA sliding) modes of motion along DNA have been proposed. Potential roles for ATP binding and hydrolysis by the helicase domains are discussed, with a focus on bipartite ATPases that act as molecular switches.

  13. Identification of restriction endonuclease with potential ability to cleave the HSV-2 genome: Inherent potential for biosynthetic versus live recombinant microbicides

    Directory of Open Access Journals (Sweden)

    Wayengera Misaki

    2008-08-01

    Full Text Available Abstract Background Herpes Simplex virus types 1 and 2 are enveloped viruses with a linear dsDNA genome of ~120–200 kb. Genital infection with HSV-2 has been denoted as a major risk factor for acquisition and transmission of HIV-1. Developing biomedical strategies for HSV-2 prevention is thus a central strategy in reducing global HIV-1 prevalence. This paper details the protocol for the isolation of restriction endunucleases (REases with potent activity against the HSV-2 genome and models two biomedical interventions for preventing HSV-2. Methods and Results Using the whole genome of HSV-2, 289 REases and the bioinformatics software Webcutter2; we searched for potential recognition sites by way of genome wide palindromics. REase application in HSV-2 biomedical therapy was modeled concomitantly. Of the 289 enzymes analyzed; 77(26.6% had potential to cleave the HSV-2 genome in > 100 but 400 but Conclusion Viral genome slicing by way of these bacterially- derived R-M enzymatic peptides may have therapeutic potential in HSV-2 infection; a cofactor for HIV-1 acquisition and transmission.

  14. Identification of restriction endonuclease with potential ability to cleave the HSV-2 genome: inherent potential for biosynthetic versus live recombinant microbicides.

    Science.gov (United States)

    Wayengera, Misaki; Kajumbula, Henry; Byarugaba, Wilson

    2008-08-07

    Herpes Simplex virus types 1 and 2 are enveloped viruses with a linear dsDNA genome of approximately 120-200 kb. Genital infection with HSV-2 has been denoted as a major risk factor for acquisition and transmission of HIV-1. Developing biomedical strategies for HSV-2 prevention is thus a central strategy in reducing global HIV-1 prevalence. This paper details the protocol for the isolation of restriction endunucleases (REases) with potent activity against the HSV-2 genome and models two biomedical interventions for preventing HSV-2. Using the whole genome of HSV-2, 289 REases and the bioinformatics software Webcutter2; we searched for potential recognition sites by way of genome wide palindromics. REase application in HSV-2 biomedical therapy was modeled concomitantly. Of the 289 enzymes analyzed; 77(26.6%) had potential to cleave the HSV-2 genome in > 100 but 400 but enzymes: BmyI, Bsp1286I, Bst2UI, BstNI, BstOI, EcoRII, HgaI, MvaI, and SduI cleaved in more than 700 sites. But for the 4: PacI, PmeI, SmiI, SwaI that had no sign of activity on HSV-2 genomic DNA, all 130(45%) other enzymes cleaved < 100 times. In silico palindromics has a PPV of 99.5% for in situ REase activity (2) Two models detailing how the REase EcoRII may be applied in developing interventions against HSV-2 are presented: a nanoparticle for microbicide development and a "recombinant lactobacillus" expressing cell wall anchored receptor (truncated nectin-1) for HSV-2 plus EcoRII. Viral genome slicing by way of these bacterially- derived R-M enzymatic peptides may have therapeutic potential in HSV-2 infection; a cofactor for HIV-1 acquisition and transmission.

  15. Quantification of DNA by Agarose Gel Electrophoresis and Analysis of the Topoisomers of Plasmid and M13 DNA Following Treatment with a Restriction Endonuclease or DNA Topoisomerase I

    Science.gov (United States)

    Tweedie, John W.; Stowell, Kathryn M.

    2005-01-01

    A two-session laboratory exercise for advanced undergraduate students in biochemistry and molecular biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves treatment of various topological forms of…

  16. Development of microsatellite markers and a restriction endonuclease digest assay for non-invasive sampling of endangered white-rumped, slender-billed and red-headed vultues

    Science.gov (United States)

    Y.A. Kapetanakos; I.J. Lovette; T.E. Katzner

    2014-01-01

    Southeast Asian vultures have been greatly reduced in range and population numbers, but it is challenging to use traditional tagging and monitoring techniques to track changes in their populations. Genotypes derived from non-invasively collected feather samples provide an alternative and effective means to 'capture' individual vultures for mark-recapture...

  17. Decrease in Abundance of Apurinic/Apyrimidinic Endonuclease Causes Failure of Base Excision Repair in Culture-Adapted Human Embryonic Stem Cells

    Czech Academy of Sciences Publication Activity Database

    Krutá, M.; Bálek, L.; Hejnová, R.; Dobšáková, Z.; Eiselleová, L.; Matulka, K.; Bárta, T.; Fojtík, P.; Fajkus, Jiří; Hampl, A.; Dvořák, P.; Rotrekl, V.

    2013-01-01

    Roč. 31, č. 4 (2013), s. 693-702 ISSN 1066-5099 R&D Projects: GA ČR(CZ) GBP302/12/G157 Grant - others:GA MŠk(CZ) ED1.100/02/0123 Institutional support: RVO:68081707 Keywords : DNA -DAMAGE * GENOMIC INSTABILITY * HETEROZYGOUS MICE Subject RIV: BO - Biophysics Impact factor: 7.133, year: 2013

  18. Caspase-independent apoptosis in Friend's erythroleukemia cells: role of mitochondrial ATP synthesis impairment in relocation of apoptosis-inducing factor and endonuclease G.

    Science.gov (United States)

    Comelli, Marina; Genero, Nadia; Mavelli, Irene

    2009-02-01

    Mitochondria have emerged as the central components of both caspase-dependent and independent apoptosis signalling pathways through release of different apoptogenic proteins. We previously documented that parental and differentiated Friend's erythroleukemia cells were induced to apoptosis by oligomycin and H(2)O(2) exposure, showing that the energy impairment occurring in both cases as a consequence of a severe mitochondrial F(0)F(1)ATPsynthase inactivation was a common early feature. Here we provide evidence for AIF and Endo G mitochondrio-nuclear relocation in both cases, as a component of caspase-independent apoptosis pathways. No detectable change in mitochondrial transmembrane potential and no variation in mitochondrial levels of Bcl-2 and Bax are observed. These results point to the osmotic rupture of the mitochondrial outer membrane as occurring in response to cell exposure to the two energy-impairing treatments under conditions preserving the mitochondrial inner membrane. A critical role of the mitochondrial F(0)F(1)ATP synthase inhibition in this process is also suggested.

  19. Studying Sideways

    DEFF Research Database (Denmark)

    Plesner, Ursula

    2011-01-01

    the methodological concern with power related to the ideas of studying up or down and introduce another set of concerns in relation to producing good empirical material when we “study sideways.” The argument is based on analyses of interview situations from a concrete research project, where researcher...

  20. Absorption studies

    International Nuclear Information System (INIS)

    Ganatra, R.D.

    1992-01-01

    Absorption studies were once quite popular but hardly anyone does them these days. It is easier to estimate the blood level of the nutrient directly by radioimmunoassay (RIA). However, the information obtained by estimating the blood levels of the nutrients is not the same that can be obtained from the absorption studies. Absorption studies are primarily done to find out whether some of the essential nutrients are absorbed from the gut or not and if they are absorbed, to determine how much is being absorbed. In the advanced countries, these tests were mostly done to detect pernicious anaemia where vitamin B 12 is not absorbed because of the lack of the intrinsic factor in the stomach. In the tropical countries, ''malabsorption syndrome'' is quire common. In this condition, several nutrients like fat, folic acid and vitamin B 12 are not absorbed. It is possible to study absorption of these nutrients by radioisotopic absorption studies

  1. Initial Study

    DEFF Research Database (Denmark)

    Torp, Kristian

    2009-01-01

    Congestion is a major problem in most cities and the problem is growing (Quiroga, 2000) (Faghri & Hamad, 2002). When the congestion level is increased the drivers notice this as delays in the traffic (Taylor, Woolley, & Zito, 2000), i.e., the travel time for the individual driver is simply...... increased. In the initial study presented here, the time it takes to pass an intersection is studied in details. Two major signal-controlled four-way intersections in the center of the city Aalborg are studied in details to estimate the congestion levels in these intersections, based on the time it takes...

  2. Actuarial Studies

    Data.gov (United States)

    U.S. Department of Health & Human Services — The Office of the Actuary in the Centers for Medicare and Medicaid Services (CMS) from time to time conducts studies on various aspects of the Medicare and Medicaid...

  3. Feasibility study

    International Nuclear Information System (INIS)

    Gibbs, P.; Kalas, P.

    1975-01-01

    The feasibility study itself examines the technical, economic and financial implications of a nuclear power station in depth so as to make sure that nuclear power is the right course to take. This means that it is quite an expensive operation and it is to avoid wasting this money that a pre-feasibility study is carried out. This preliminary study should eliminate cases where the electrical system cannot absorb the capacity of a nuclear station of commercial size, where other sources of power such as hydro-electricity, gas or cheap coal would make nuclear obviously uneconomic or where no suitable sites exist. If this first rather simple survey shows that nuclear power is a credible solution to a utilities need for electricity or heat production plant, then the next stage is a full feasibility study. (orig./TK) [de

  4. INTERHEART STUDY

    Indian Academy of Sciences (India)

    First page Back Continue Last page Overview Graphics. INTERHEART STUDY. About 90% of CHD Risk (“PAR”) can be explained by 9 Risk Factors: Smoking. Dyslipidemia. High BP. Diabetes. Abdominal Obesity. Psychosocial Factors. Fruits & Vegetables. Exercise. Alcohol.

  5. Floodplain Study

    Data.gov (United States)

    Montgomery County of Maryland — The purpose of a floodplain study is to establish the 100-year floodplain limits within or near a development in order to preserve the natural resources within the...

  6. European Studies

    Directory of Open Access Journals (Sweden)

    V. O. Pechatnov

    2014-01-01

    Full Text Available The study of Western countries and teaching courses on the related subjects have longstanding and established tradition at MGIMO-University. The basis of this brilliant research and teaching tradition was laid down by such academicians as E.V. Tarle and V.G. Trukhanovsky, Professor L.I. Clove, Y. Borisov, F.I. Notovitch, G.L. Rozanov. Their work in 1940-1960's at the Department of World History at MGIMO-University progressed in following directions: France studies, German studies, American studies. The work resulted in a number of monographs and textbooks on modern history and foreign policy of the studied countries and regions. The aim of the publications was dictated by the goal of the Institute - to prepare the specialists in international affairs primarily for practical work. A close relationship with the Foreign Ministry was "binding advantage" sometimes limiting researchers in choosing periods and subjects for the study. At the same time the undisputed advantage and quality of regional studies at MGIMO were strengthened by the practical relevance of research, making it a vital and interesting not only for specialists but also for students and researchers from other research centers. Another characteristic of the tradition is the analysis of foreign policy and diplomacy in a close relationship with the socio-economic and political processes. Such an integrated approach to regional geography also formed largely under the influence of institutional profile designed to train highly skilled and versatile specialists in specific countries and regions with a good knowledge of their languages, history, economics, politics, law and culture. Therefore, scientific and educational-methodical work at MGIMO-University has always relied on a wealth of empirical data and has been focused on the analysis of real-world phenomena and processes, acute problems of foreign countries. Scientific research at MGIMO-University traditionally intertwined with

  7. Project studies

    DEFF Research Database (Denmark)

    Geraldi, Joana; Söderlund, Jonas

    2018-01-01

    Project organising is a growing field of scholarly inquiry and management practice. In recent years, two important developments have influenced this field: (1) the study and practice of projects have extended their level of analysis from mainly focussing on individual projects to focussing on micro...... of 'organising' filled the field of organisation studies with new ideas and intellectual challenges. To take advantage of such developments, organisational scholars had to consider different forms of organising as part of 'organisation studies', and continuously adapt their frames of reference and forms...... by emancipatory interest and the pragmatic desire for changes in the status quo through the reorganisation of inherent contradictions, giving voice to minorities while addressing major economic and social problems. We termed them type 1, type 2 and type 3, respectively.The juxtaposition of levels of analysis...

  8. Study Drugs

    OpenAIRE

    Lam, Stephanie Phuong; Roosta, Natalie; Nielsen, Mikkel Fuhr; Meyer, Maria Holmgaard; Friis, Katrine Birk

    2016-01-01

    In recent years, students around the world, started to use preparations as Ritalin and Modafinil,also known as study drugs, to improve their cognitive abilities1. It is a common use among thestudents in United States of America, but it is a new tendency in Denmark. Our main focus is tolocate whether study drugs needs to be legalized in Denmark or not. To investigate this ourstarting point is to understand central ethical arguments in the debate. We have chosen twoarguments from Nick Bostrom a...

  9. Study of the interaction of enzyme Heparanase 1 (HPSE1) active with deoxyribonucleic acids

    International Nuclear Information System (INIS)

    Cid, Gisele da Silva

    2016-01-01

    The human heparanase 1 (HPSE 1) is a protein with multiple functions and has emerged as a promising therapeutic target in the context of antitumor therapy. This fact is due to its clinical relevance in the tumor development and progression, as determined by their enzymatic ability to degrade heparan sulfate (HS), the main constituent of the extracellular matrix, providing a tumor microenvironment to tumor dissemination. In addition, this protein plays a significant role in the increase of tumor cells migration ionizing radiation dose delivery in radiotherapy from the increase in the expression levels of HPSE1. In order to evaluate in more detail the functions of active HPSE1, it has been proposed to characterize the interaction of human heparanase protein 1 with deoxyribonucleic acids. Our results are original and point to a new function of HPSE1 of the endonuclease type. (author)

  10. Binding site of ribosomal proteins on prokaryotic 5S ribonucleic acids: a study with ribonucleases

    DEFF Research Database (Denmark)

    Douthwaite, S; Christensen, A; Garrett, R A

    1982-01-01

    The binding sites of ribosomal proteins L18 and L25 on 5S RNA from Escherichia coli were probed with ribonucleases A, T1, and T2 and a double helix specific cobra venom endonuclease. The results for the protein-RNA complexes, which were compared with those for the free RNA [Douthwaite, S...... stearothermophilus 5S RNA. Several protein-induced changes in the RNA structures were identified; some are possibly allosteric in nature. The two prokaryotic 5S RNAs were also incubated with total 50S subunit proteins from E. coli and B. stearothermophilus ribosomes. Homologous and heterologous reconstitution....... stearothermophilus 5S RNA, which may have been due to a third ribosomal protein L5....

  11. Immunology studies

    International Nuclear Information System (INIS)

    Smith, D.M.; Baron, P.A.; Drake, G.A.; LaBauve, P.M.; London, J.E.; Wilson, J.S.

    1977-01-01

    The following studies were conducted in the field of immunology; a model system to determine toxic effects on the immune system using 3 H-uridine uptake by Feells of rats; and survival in lethally irradiatd mice receiving allogenic fetal liver and thymus

  12. Hydrogeological study

    International Nuclear Information System (INIS)

    Massa, E.; Heinzen, W.; Santana, J.

    1987-01-01

    This work shows the hydrogeological study and well drilling carried out in the Teaching Formation Institute San Jose de Mayo Province Uruguay. It was developed a geological review in the National Directorate of Geology and Mining data base as well as field working, geology and hydrogeology recognition and area well drilling inventory.

  13. Colour Studies

    African Journals Online (AJOL)

    DR Nneka

    2015-04-14

    Apr 14, 2015 ... Technology picked up color study on a serious level and because of that we now have the glowing .... on necessary concepts and theories of colour gleaned from the fields of physics, physiology, and ..... The way our body reacts to colour and light is another important subject the artist and designer should ...

  14. Study Proposal

    African Journals Online (AJOL)

    MATHEMATICS DEPARTMENT

    courses like Mathematics, Physics,. Engineering and Computer Science in. Science and Technology field require various studies of mathematics at graduate level. Now-a-days, it will be difficult to science & Technology courses to solve problems with out mathematics [1]. High school education is very important to.

  15. Counselling Studies.

    Science.gov (United States)

    Feltham, Colin

    2001-01-01

    Proposes that a discipline of "counseling studies" be considered as an extension of counseling and a contribution to social guidance. Suggests features of such a discipline, including: a focus on the individual person; interdisciplinarity; humanistic values; applied and theoretical dialectic; critical attitude; and dynamic nature.…

  16. Invisibility Studies

    DEFF Research Database (Denmark)

    Invisibility Studies explores current changes in the relationship between what we consider visible and what invisible in different areas of contemporary culture. Contributions trace how these changes make their marks on various cultural fields and investigate the cultural significance of these de...

  17. Club studies

    DEFF Research Database (Denmark)

    Demant, Jakob Johan; Ravn, Signe; Harder, Sidsel Kirstine

    2010-01-01

    suggests a mixed-methods approach to club studies that combine quantitative data, qualitative interviews and ethnography conducted in the club space. By introducing the concept of ‘socionautics', this review suggests that the researcher travels into the social landscape of youth, clubs and drugs...

  18. The Restriction Fragment Map of Rat-Liver Mitochondrial DNA : A Reconsideration

    NARCIS (Netherlands)

    Pepe, G.; Bakker, H.; Holtrop, M.; Bollen, J.E.; Bruggen, E.F.J. van; Cantatore, P.; Terpstra, P.; Saccone, C.

    1977-01-01

    1. Rat-liver mitochondrial DNA (mtDNA) contains at least 8 cleavage sites for the restriction endonuclease Eco RI, 6 for the restriction endonuclease Hind III, 2 for the restriction endonuclease Bam HI and 11 for the restriction endonuclease Hap II. 2. The physical map of the restriction fragments

  19. Genetic and biochemical studies of the lipid-containing bacteriophage PR4

    International Nuclear Information System (INIS)

    Vanden Boom, T.J.

    1989-01-01

    Bacteriophage PR4 is a lipid-containing bacterial virus able to infect Escherichia coli and Salmonella typhimurium. The icosahedral virion consists of an external protein capsid layer which surrounds a membrane vesicle enclosed ds DNA genome. The author has analyzed the time course of phage PR4 protein synthesis and have identified at least 34 proteins present in phage infected cells not detected in uninfected control cultures. In addition, he has isolated a more extensive set of conditional-lethal nonsense mutants of this virus. This collection of mutants permitted the identification of seven additional phage PR4 gene products, including the terminal genome protein and an accessory lytic factor. The present collection of phage PR4 mutants has been assigned to 19 distinct genetic groups on the basis of genetic complementation tests and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the proteins produced in mutant-infected UV-irradiated cells. A restriction endonuclease map of the phage PR4 genome was constructed which includes 59 sites for ten restriction endonucleases. In addition, he has constructed a collection of recombinant plasmids containing subgenomic DNA fragments of bacteriophage PR4. He has used this collection of plasmids to generate a physical-genetic map of the PR4 genome. The physical-genetic map localizes mutations in 13 phage PR4 genetic groups on the viral DNA molecule. To investigate the role of phosphatidylglycerol (PG) in phage assembly and infectivity, he propagated PR4 on an E. coli mutant defective in PG synthesis. The PG content of phage PR4 grown on the mutant host accounted for 0.4% of the total viral phospholipids, representing a 90-fold decrease in PG relative to the PG content of phage grown on a wild type host

  20. Study Strategies

    DEFF Research Database (Denmark)

    Nielsen, Camilla Kirketerp; Noer, Vibeke Røn

    module”. The projects differ in terms of both starting point and main focus of research (an alternative educational model and profession-orientated Game-based learning). However, in the on-going process of research, an empirical ´harmony´ on common recurrent themes related to students `study strategies......ID: 1277 / 22 SES 06 B: 2 22. Research in Higher Education Format of Presentation: Paper Alternative EERA Network: 19. Ethnography Topics: NW 22: Teaching, learning and assessment in higher education Keywords: Profession-oriented learning, study strategies, professionalisation processes......-class until graduation and entrance into the nursing field as professional nurses. Case 2: In the veterinary educational environment, a pedagogical development project on development, implementation and evaluation of game-based learning as a possible way of strengthening the interplay between practice...