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Sample records for giy-yig homing endonuclease

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

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    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. Phylogenomic analysis of the GIY-YIG nuclease superfamily

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    Bujnicki Janusz M

    2006-04-01

    Full Text Available Abstract Background The GIY-YIG domain was initially identified in homing endonucleases and later in other selfish mobile genetic elements (including restriction enzymes and non-LTR retrotransposons and in enzymes involved in DNA repair and recombination. However, to date no systematic search for novel members of the GIY-YIG superfamily or comparative analysis of these enzymes has been reported. Results We carried out database searches to identify all members of known GIY-YIG nuclease families. Multiple sequence alignments together with predicted secondary structures of identified families were represented as Hidden Markov Models (HMM and compared by the HHsearch method to the uncharacterized protein families gathered in the COG, KOG, and PFAM databases. This analysis allowed for extending the GIY-YIG superfamily to include members of COG3680 and a number of proteins not classified in COGs and to predict that these proteins may function as nucleases, potentially involved in DNA recombination and/or repair. Finally, all old and new members of the GIY-YIG superfamily were compared and analyzed to infer the phylogenetic tree. Conclusion An evolutionary classification of the GIY-YIG superfamily is presented for the very first time, along with the structural annotation of all (subfamilies. It provides a comprehensive picture of sequence-structure-function relationships in this superfamily of nucleases, which will help to design experiments to study the mechanism of action of known members (especially the uncharacterized ones and will facilitate the prediction of function for the newly discovered ones.

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

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    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

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

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    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

  5. 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

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

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    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.

  7. Home and away- the evolutionary dynamics of homing endonucleases

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    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.

  8. 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

  9. Germline excision of transgenes in Aedes aegypti by homing endonucleases.

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    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.

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

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    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.

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

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    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...

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

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    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...

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

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    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

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

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    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.

  15. 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

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

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    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.

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

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    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.

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

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    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.

  19. 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...

  20. 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...

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

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    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.

  2. 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...

  3. 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.

  4. 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.

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    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

  5. 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

  6. 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

  7. Characterization of SNP and Structural Variations in the Mitochondrial Genomes of Tilletia indica and Its Closely Related Species Formed Basis for a Simple Diagnostic Assay.

    Directory of Open Access Journals (Sweden)

    Mui-Keng Tan

    Full Text Available Tilletia indica causes the disease Karnal bunt in wheat. The disease is under international quarantine regulations. Comparative mitochondrial (mt genome analysis of T. indica (KX394364 and DQ993184 and T. walkeri (EF536375 has found 325 to 328 SNPs, 57 to 60 short InDels (from 1 to 13 nt, two InDels (30 and 61 nt and five (>200 nt presence/absence variations (PAVs between the two species. The mt genomes of both species have identical gene order. The numbers of SNPs and InDels between the mt genomes of the two species are approximately nine times of the corresponding numbers between the two T. indica isolates. There are eight SNPs between T. indica and T. walkeri that resulted in amino acid substitutions in the mt genes of cob, nad2 and nad5. In contrast, there is no amino acid substitution in the mt genes of the T. indica isolates from the SNPs found. The five PAVs present in T. indica (DQ993184 are absent in T. walkeri. Four PAVs are more than 1 kb and are not present in every T. indica isolate. Analysis of their presence and absence separates a collection of T. indica isolates into 11 subgroups. Two PAVs have ORFs for the LAGLIDAG endonuclease and two have ORFs for the GIY-YIG endonuclease family, which are representatives of homing endonuclease genes (HEGs. These intron- encoded HEGs confer intron mobility and account for their fluid distribution in T. indica isolates. The small PAV of 221 bp, present in every T. indica isolate and unique to the species, was used as the genetic fingerprint for the successful development of a rapid, highly sensitive and specific loop mediated isothermal amplification (LAMP assay. The simple procedure of the LAMP assay and the easy detection formats will enable the assay to be automated for high throughput diagnosis.

  8. 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...

  9. 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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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. Problem-Solving Test: Restriction Endonuclease Mapping

    Science.gov (United States)

    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…

  13. Isothermal detection of RNA with restriction endonucleases.

    Science.gov (United States)

    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.

  14. Selection of restriction endonucleases using artificial cells.

    Science.gov (United States)

    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.

  15. 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.

  16. 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...

  17. Home

    Directory of Open Access Journals (Sweden)

    Rokeya Sakhawat Hossain

    2012-11-01

    Full Text Available A fiery feminist piece that argues that Indian women are all homeless; animals have homes but Indian women have none, because they have to depend on the mercy of their "keepers"; therefore, Indian women live a life worse than animals.

  18. 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.

  19. 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)

  20. 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)

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

    Science.gov (United States)

    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.

  2. 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.

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

    Science.gov (United States)

    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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  3. 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.

  4. 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...

  5. 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

  6. 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...

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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...

  13. 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....

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  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. 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. 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

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. Home education

    OpenAIRE

    Beck, Christian W.

    2013-01-01

    Home Education seems to be a successful way to educate. Academic results and socialization processes in home education are promising. Already home education is global, home educators everywhere educate their children themselves without schools. They develop new forms of local and international co-operation. Is home education an impulse to a renewing of modern education? Is home education globalization otherwise?

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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.

  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. 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

  1. 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.

  2. 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

  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. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. [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.

  17. Home, Smart Home

    DEFF Research Database (Denmark)

    Hansen, Ellen Kathrine; Olesen, Gitte Gylling Hammershøj; Mullins, Michael

    2013-01-01

    The article places focus on how smart technologies integrated in a one family- home and particular the window offer unique challenges and opportunities for designing buildings with the best possible environments for people and nature. Toward an interdisciplinary approach, we address the interaction...... between daylight defined in technical terms and daylight defined in aesthetic, architectural terms. Through field-tests of a Danish carbon-neutral home and an analysis of five key design parameters, we explore the contradictions and potentials in smart buildings, using the smart window as example of how...... to the energy design is central. The study illuminates an approach of the design of smart houses as living organisms by connecting technology with the needs of the occupants with the power and beauty of daylight....

  18. 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.

  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. 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.

  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. 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...

  4. 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

  5. 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...

  6. 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.

  7. 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.

  8. 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

  9. 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

  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. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. Home Modifications

    Science.gov (United States)

    ... content Skip Navigation Department of Health and Human Services Your Browser does not support javascript, so the search function on this page is disabled 1-800-677-1116 Home > Resources > Factsheets > Home ...

  17. Home hemodialysis

    DEFF Research Database (Denmark)

    Agar, John W; Perkins, Anthony; Heaf, James G

    2015-01-01

    We describe the infrastructure that is necessary for hemodialysis in the home focusing on physical requirements, the organization of plumbing and water, and the key features that should guide the selection of machines that are suitable for home use.......We describe the infrastructure that is necessary for hemodialysis in the home focusing on physical requirements, the organization of plumbing and water, and the key features that should guide the selection of machines that are suitable for home use....

  18. Home Dissolution

    DEFF Research Database (Denmark)

    Gram-Hanssen, Kirsten; Bech-Danielsen, Claus

    2008-01-01

    Building a home and creating a family are highly inter­connec­ted processes. So what happens with the home when people separate or divorce? In this paper we address this question both from a quantitative and a qualitative approach. Based on an extensive database with socio-economic background data...... problems of dissolving a home. How to decide who should stay, who should move and how was it to stay alone in or to leave the matrimonial home?...

  19. 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.

  20. Dipankar Home

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education. Dipankar Home. Articles written in Resonance – Journal of Science Education. Volume 18 Issue 10 October 2013 pp 905-916 General Article. Bohr's Philosophy of Wave–Particle Complementarity · Dipankar Home · More Details Fulltext PDF ...

  1. Homing oneself

    DEFF Research Database (Denmark)

    Winther, Ida Wentzel

    2009-01-01

    What is home? A building, a physical and mental phenomenon, or a concept?  There are many homes and ways `to home oneself´. Many of us quite often dwell in other places than at home (as professional commuters between two places, as travellers staying in hotels, as children of divorced parents...... living one week with mom and one week with dad). We spend so much time in and between these movements and settings that we may even (have to learn how to) ‘home ourselves'. In other words, we may use different strategies in order to create a homely feeling and a certain sense of belonging. This paper...... expands on the notion that home indicates more than a house, but also responds to the overuse of the concept home. The aim of this article is to examine how home is done, stretched between everyday life, practices, dreams, loss and cultural ideas of home. My intention is not to remove home...

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  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. 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.

  12. 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...

  13. 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.

  14. 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.

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

    Science.gov (United States)

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

    2017-07-14

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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Home Care

    Science.gov (United States)

    ... Aging & Health A to Z › Home Care Font size A A A Print Share Glossary Basic Facts & Information Other Resources Caregiving How To's Tools & Tips Latest Research Getting More Help Related Topics Assisted Living Community-Based Care Nursing Homes Join our e- ...

  7. 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

  8. 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...

  9. 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...

  10. 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...

  11. Nursing Homes

    Science.gov (United States)

    ... changed dramatically over the past several decades. These changes have been driven by government regulations and consumer pressures. Today’s nursing homes are highly regulated, high-quality institutions for ...

  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. Nursing Home

    OpenAIRE

    Allocca Hernandez, Giacomo Antonio

    2016-01-01

    Getting old involves a lot of changes in life. Family and social relations change and mobility can decrease. These variations require new settings, and of course special care. A nursing home is a place dedicated to help with this situation. Sometimes nursing homes can be perceived as mere institutions by society, and even by future residents. Inside, senior citizens are suppose to spend the rest of their lives doing the same activities day after day. How can we improve these days? Archite...

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  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. 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.

  5. 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

  6. Returning home

    DEFF Research Database (Denmark)

    Agergaard, Jytte; Brøgger, Ditte

    2016-01-01

    flows. By focusing on these educational migrants, this paper explores how they connect to their rural homes. Guided by a critical reading of the migration-development scholarship, the paper examines how migrants and their relatives make sense of educational migrants’ remitting and returning practices......, and by comparing three groups of educational migrants, the migrants’ reasons for staying connected and sending remittances are scrutinized. The paper finds that although educational migrants do not generate extensive economic remittances for local development in Nepal, they stay connected to their rural homes...

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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)

  15. 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

  16. 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.

  17. 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...

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Home Seismometer

    Science.gov (United States)

    Horiuchi, S.; Yamamoto, S.; Nakmaura, H.; Wu, C.; Rydelek, P.; Kachi, M.

    2007-12-01

    We have developed an automated system for analyzing Hi-net seismograms for earthquake early warning (EEW) in Japan. Because of limitations imposed by station spacing, our system generally cannot issue an EEW to areas within about 30 km distance of the earthquake's hypocenter. We estimate that about 10 times the number of stations would be needed to issue an EEW in these areas, but the overhead would be cost prohibitive for governmental agencies. The practical deployment of EEW in Japan has started in October, 2007 and millions of people are expected to purchase and install the receiving/alarm unit of EEW. Since most of these units are connected to internet and equipped with a CPU and memory, we realized that the addition of an inexpensive seismometer and digitizer would transform the receiver into a real-time seismic observatory, which we are calling a home seismometer; these modifications only cost about $20. The home seismometer can help to generate alerts at the time of the occurrence of a large local earthquake by using locally observed data. Also, home seismograms can be used to estimate the amplification factor in sedimentary layers, which will be used to determine the site correction for shaking intensity by comparing the waveform data from the home seismometer against those from nearby Hi-net or K-NET stations. This amplification factor, which is essentially the basis of a shake-map with very-high spatial resolution, will help to establish a safety index of houses/buildings for large earthquakes, since a structure located at a site with large seismic amplification can be damaged more seriously than those with small amplification factors. The installation of home seismometers will create an extremely dense seismic network that is without precedence. We are developing an automatic system that collects waveform data from all home seismometer installations, calculates earthquake parameters in real-time, and then sends back alarms signals based on computed

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

    Directory of Open Access Journals (Sweden)

    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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. The Medical Home

    Science.gov (United States)

    ... Videos for Educators Search English Español The Medical Home KidsHealth / For Parents / The Medical Home What's in ... for your child. What Does the Term "Medical Home" Mean? A medical home isn't a place ...

  10. Home education: globalization otherwise?:

    OpenAIRE

    Beck, Christian W.

    2006-01-01

    Home Education seems to be a successful way to educate. Academic results and socialization processes in home education are promising. Already home education is global, home educators everywhere educate their children themselves without schools. They develop new forms of local and international co-operation. Is home education an impulse to a renewing of modern education? Is home education globalization otherwise?

  11. 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

  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

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    Braun Werner

    2002-11-01

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

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

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    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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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...

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. Respiratory Home Health Care

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    ... Healthy Living > Living With Lung Disease > Respiratory Home Health Care Font: Aerosol Delivery Oxygen Resources Immunizations Pollution Nutrition ... Disease Articles written by Respiratory Experts Respiratory Home Health Care Respiratory care at home can contribute to improved ...

  9. Exercise at Home

    Science.gov (United States)

    ... Home Health Insights Exercise & Weight Exercise at Home Exercise at Home Make an Appointment Ask a Question ... with the movement and contact your provider. Posture Exercises Better posture means better breathing and movement. Axial ...

  10. Home Care Services

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    Home care is care that allows a person with special needs stay in their home. It might be for people who are getting ... are chronically ill, recovering from surgery, or disabled. Home care services include Personal care, such as help ...

  11. HOME Grantee Areas

    Data.gov (United States)

    Department of Housing and Urban Development — The HOME Investment Partnership Program (HOME) is authorized under Title II of the Cranston-Gonzalez National Affordable Housing Act. HOME provides formula grants to...

  12. 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

  13. 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

    -specific PCR. BLASTP program analysis revealed that GCRG213p peptide shared 83.0% alignment with the C-terminal region of L1 endonuclease (L1-EN). GCRG213p sequence possesses the important residues that compose the conserved features of L1-EN. GCRG213p could be a variant of L1-EN, a functional member of L1-EN family. Overexpression of GCRG213p is common in both primary gastric cancer and lymph node metastasis. These findings provide evidence of somatic L1 expression in gastric cancer, and its potential consequences in the form of tumor

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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)

  4. 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.

  5. Home safe home: Evaluation of a childhood home safety program.

    Science.gov (United States)

    Stewart, Tanya Charyk; Clark, Andrew; Gilliland, Jason; Miller, Michael R; Edwards, Jane; Haidar, Tania; Batey, Brandon; Vogt, Kelly N; Parry, Neil G; Fraser, Douglas D; Merritt, Neil

    2016-09-01

    The London Health Sciences Centre Home Safety Program (HSP) provides safety devices, education, a safety video, and home safety checklist to all first-time parents for the reduction of childhood home injuries. The objective of this study was to evaluate the HSP for the prevention of home injuries in children up to 2 years of age. A program evaluation was performed with follow-up survey, along with an interrupted time series analysis of emergency department (ED) visits for home injuries 5 years before (2007-2013) and 2 years after (2013-2015) implementation. Spatial analysis of ED visits was undertaken to assess differences in home injury rates by dissemination areas controlling differences in socioeconomic status (i.e., income, education, and lone-parent status) at the neighborhood level. A total of 3,458 first-time parents participated in the HSP (a 74% compliance rate). Of these, 20% (n = 696) of parents responded to our questionnaire, with 94% reporting the program to be useful (median, 6; interquartile range, 2 on a 7-point Likert scale) and 81% learning new strategies for preventing home injuries. The median age of the respondent's babies were 12 months (interquartile range, 1). The home safety check list was used by 87% of respondents to identify hazards in their home, with 95% taking action to minimize the risk. The time series analysis demonstrated a significant decline in ED visits for home injuries in toddlers younger than2 years of age after HSP implementation. The declines in ED visits for home injuries remained significant over and above each socioeconomic status covariate. Removing hazards, supervision, and installing safety devices are key facilitators in the reduction of home injuries. Parents found the HSP useful to identify hazards, learn new strategies, build confidence, and provide safety products. Initial finding suggests that the program is effective in reducing home injuries in children up to 2 years of age. Therapeutic/care management study

  6. 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.

  7. 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

  8. 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...

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Meals in nursing homes

    DEFF Research Database (Denmark)

    Kofod, Jens Erik; Birkemose, A.

    2004-01-01

    Undernutrition is present among 33% of nursing home residents in Denmark. Hence, it is relevant to examine the meal situation at nursing homes to single out factors that may increase or reduce the residents' food intake. in the ongoing Danish nursing home debate it is claimed that a new type...... of nursing home improves the residents' meal situation with a positive effect on nutrition. The aim of this work is to test the general hypothesis that (i) residents appreciate the meal situation in these nursing homes and (ii) nutritional status of the residents is improved in this type of nursing home....... This study was carried out in four Danish nursing homes at various locations in Denmark. The methods used are qualitative interviews and observations at four nursing homes in combination with measurement of body mass index (BMI) at two of the four nursing homes. Undernutrition is defined as a BMI below 20...

  5. Eldercare at Home: Choosing a Nursing Home

    Science.gov (United States)

    ... the way. Moving from one's home to a nursing home is a big life change. Adjusting to the move and becoming comfortable will ... residents. Ask them what they like and what changes they would suggest. What ... nursing assistants and observe them with residents. Nursing assistants ...

  6. HOME Rent Limits

    Data.gov (United States)

    Department of Housing and Urban Development — In accordance with 24 CFR Part 92.252, HUD provides maximum HOME rent limits. The maximum HOME rents are the lesser of: The fair market rent for existing housing for...

  7. Nursing Home Quality Initiative

    Data.gov (United States)

    U.S. Department of Health & Human Services — This Nursing Home Quality Initiative (NHQI) website provides consumer and provider information regarding the quality of care in nursing homes. NHQI discusses quality...

  8. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... UOAA). The skills kit contains: A booklet with information on the operation, home skills such as emptying and changing a pouch, problem solving, and home management. A DVD with demonstration of each skill Stoma ...

  9. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... kit contains: A booklet with information on the operation, home skills such as emptying and changing a pouch, problem solving, and home management. A DVD with demonstration of each skill Stoma ...

  10. Home Health Compare

    Data.gov (United States)

    U.S. Department of Health & Human Services — Home Health Compare has information about the quality of care provided by Medicare-certified home health agencies throughout the nation. Medicare-certified means the...

  11. Home Improvements Prevent Falls

    Science.gov (United States)

    ... turn JavaScript on. Feature: Falls and Older Adults Home Improvements Prevent Falls Past Issues / Winter 2014 Table ... and ensure your safety. "Safe-ty-fy" Your Home Some Questions for Your Provider Will my medicines ...

  12. Eye Injuries at Home

    Science.gov (United States)

    ... Steps to Safer Champagne Celebrations Eye Injuries at Home Leer en Español: Lesiones de los ojos en ... chore is being done. Preventing Eye Injuries at Home Wearing protective eyewear will prevent 90 percent of ...

  13. Home Canning and Botulism

    Science.gov (United States)

    ... this? Submit What's this? Submit Button Past Emails Home Canning and Botulism Language: English (US) Español (Spanish) ... myself and others safe when it comes to home-canned foods? Many cases of foodborne botulism have ...

  14. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Careers at ACS Careers at ACS About ACS Career Types Working at ACS ... American College of Surgeons Education Patients and Family Skills Programs Ostomy Home Skills Program Ostomy Home Skills ...

  15. TRAVEL AND HOME LEAVE

    CERN Multimedia

    Human Resources Division

    2002-01-01

    Administrative procedures for : Travel to the home station and home leave (hl) Additional travel to the home station (at) Travel to the home station and home leave for family reasons (hlf) As part of the process of simplifying administrative procedures, HR and AS Divisions have devised a new, virtually automatic procedure for payment of travel expenses to the home station. The changes are aimed at rationalising administrative procedures and not at reducing benefits. The conditions of eligibility are unchanged. The new procedure, which will be operational with effect from 1st June 2002, will greatly simplify the administrative processing of claims for travel expenses and the recording of home leaves. Currently, requests for payment are introduced manually into the Advances and Claims system (AVCL) by divisional secretariats. All travel to the home station starting prior to 1st June 2002 will be processed according to the existing system whereas that starting on 1st June and after will be processed accordi...

  16. Home Health Care

    Science.gov (United States)

    ... content Skip Navigation Department of Health and Human Services Your Browser does not support javascript, so the search function on this page is disabled 1-800-677-1116 Home > Resources > Factsheets > Home ...

  17. Genetics Home Reference: microphthalmia

    Science.gov (United States)

    ... navigation Home Page Search Home Health Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Share: Email ... COLOBOMA 9 MICROPHTHALMIA, ISOLATED, WITH CORECTOPIA OPTIC DISC ANOMALIES WITH RETINAL AND/OR MACULAR DYSTROPHY Sources for ...

  18. National Nursing Home Survey

    Science.gov (United States)

    The National Nursing Home Survey provides includes characteristics such as size of nursing home facilities, ownership, Medicare/Medicaid certification, occupancy rate, number of days of care provided, and expenses.

  19. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Overview The skills kit contains: A booklet with information on the operation, home skills such as emptying and changing a pouch, problem solving, and home management. A DVD with demonstration of each skill Stoma ...

  20. Community Nursing Home (CNH)

    Data.gov (United States)

    Department of Veterans Affairs — The Community Nursing Home (CNH) database contains a list of all Community Nursing Home facilities under local contract to Veterans Health Administration (VHA). CNH...

  1. School@Home.

    Science.gov (United States)

    Hammons, Christopher W.

    2001-01-01

    Describes home schooling movement and argues home schooling is viable alternative to public education system. Discusses increase in home-schooled students applying to college, taking and performing well on college entrance exams (ACT and SAT), engaging in extracurricular activities, and succeeding in college. Addresses and refutes criticisms of…

  2. Schooling at Home.

    Science.gov (United States)

    Long, Joyce Fleck

    2001-01-01

    Presents one family's experience with home schooling, explaining that no two home schools are alike, which is both a strength and a weakness of the movement. The paper discusses the parent's educational philosophy and the family's personal curriculum and pedagogical choices. It concludes by examining the growing trend in home schooling. (SM)

  3. What Is Home Schooling?

    Science.gov (United States)

    Ohio State Legislative Office of Education Oversight, Columbus.

    The Ohio Department of Education estimates that 15,000 children were being home-schooled in Ohio, based on a 1991 survey of school superintendents. This document presents an overview of home schooling and describes the nature and extent of home schooling in Ohio. Data are based on a review of literature, information received from national and…

  4. Home Schooling Goes Mainstream

    Science.gov (United States)

    Gaither, Milton

    2009-01-01

    This article reports that while home schooling may have particular appeal to celebrities, over the last decade families of all kinds have embraced the practice for widely varying reasons: no longer is home schooling exclusive to Christian fundamentalism and the countercultural Left. Along with growing acceptance of home schooling nationally has…

  5. Home in the Making

    DEFF Research Database (Denmark)

    Kreuzer, Maria; von Wallpach, Sylvia; Muehlbacher, Hans

    2016-01-01

    In a context of unprecedented migration home reaches high relevance. This study aims at understanding the (re-)construction of home by first generation consumer migrants. The findings provide insights into consumers’ (re-)construction of various dimensions of home and identify “inner home” as a new...

  6. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Ostomy Home Skills Program Ostomy Home Skills Program Adult Ostomy Pediatric Ostomy Programa de Destrezas para manejo Doméstico de Ostomía Ostomy Home Skills Program Adult Ostomy Pediatric Ostomy Programa de Destrezas para manejo ...

  7. Asthma Home Environment Checklist

    Science.gov (United States)

    This checklist guides home care visitors in identifying environmental asthma triggers most commonly found in homes. It includes sections on the building, home interior and room interior and provides low-cost action steps for remediation. EPA 402-F-03-030.

  8. Digital Living at Home

    DEFF Research Database (Denmark)

    Andersen, Pernille Viktoria Kathja; Christiansen, Ellen Tove

    2013-01-01

    of these user voices has directed us towards a ‘home-keeping’ design discourse, which opens new horizons for design of digital home control systems by allowing users to perform as self-determined controllers and groomers of their habitat. The paper concludes by outlining the implications of a ‘home......Does living with digital technology inevitably lead to digital living? Users talking about a digital home control system, they have had in their homes for eight years, indicate that there is more to living with digital technology than a functional-operational grip on regulation. Our analysis...

  9. Strategy Guideline. Demonstration Home

    Energy Technology Data Exchange (ETDEWEB)

    Hunt, A.; Savage, C.

    2012-12-01

    This guideline will provide a general overview of the different kinds of demonstration home projects, a basic understanding of the different roles and responsibilities involved in the successful completion of a demonstration home, and an introduction into some of the lessons learned from actual demonstration home projects. Also, this guideline will specifically look at the communication methods employed during demonstration home projects. And lastly, we will focus on how to best create a communication plan for including an energy efficient message in a demonstration home project and carry that message to successful completion.

  10. Strategy Guideline: Demonstration Home

    Energy Technology Data Exchange (ETDEWEB)

    Savage, C.; Hunt, A.

    2012-12-01

    This guideline will provide a general overview of the different kinds of demonstration home projects, a basic understanding of the different roles and responsibilities involved in the successful completion of a demonstration home, and an introduction into some of the lessons learned from actual demonstration home projects. Also, this guideline will specifically look at the communication methods employed during demonstration home projects. And lastly, we will focus on how to best create a communication plan for including an energy efficient message in a demonstration home project and carry that message to successful completion.

  11. LHC@home gets new home

    CERN Multimedia

    Oates, John

    2007-01-01

    "The distributed computing project LHC@home is moving to London from Cern in Switzerland. Researchers at Qeen Mary University have been trialling the system since June, but are now ready for the offical launch" (1 page)

  12. Home Within Me

    DEFF Research Database (Denmark)

    Kreuzer, Maria; Mühlbacher, Hans; von Wallpach, Sylvia

    2017-01-01

    In an increasingly globalized, digitalized and perceived unmanageable world, consumers strive for belongingness, identification and security and re-discover the importance of home. Home is central to peoples’ individual as well as collective identities and their self-development (McCracken, 1989......). Home, however, is a multi-dimensional concept and reaching a universal definition is nearly impossible (Moore, 2000). Therefore, this research project aims to answer the following research questions: 1) What is the meaning of home? 2) How do consumers experience home? And 3) What is the role...... of products and consumption rituals for experiencing home? To answer these questions, two qualitative studies covering 32 in-depth autobiographical interviews were conducted to elicit emotional, symbolic and cultural meanings and experiences related to home. Interviews took place in the same geographical area...

  13. 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.

  14. 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.

  15. 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 ...

  16. 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

  17. 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 ...

  18. 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.

  19. 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.

  20. 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

  1. Home range and travels

    Science.gov (United States)

    Stickel, L.F.; King, John A.

    1968-01-01

    The concept of home range was expressed by Seton (1909) in the term 'home region,' which Burr (1940, 1943) clarified with a definition of home range and exemplified in a definitive study of Peromyscus in the field. Burt pointed out the ever-changing characteristics of home-range area and the consequent absence of boundaries in the usual sense--a finding verified by investigators thereafter. In the studies summarized in this paper, sizes of home ranges of Peromyscus varied within two magnitudes, approximately from 0.1 acre to ten acres, in 34 studies conducted in a variety of habitats from the seaside dunes of Florida to the Alaskan forests. Variation in sizes of home ranges was correlated with both environmental and physiological factors; with habitat it was conspicuous, both in the same and different regions. Food supply also was related to size of home range, both seasonally and in relation to habitat. Home ranges generally were smallest in winter and largest in spring, at the onset of the breeding season. Activity and size also were affected by changes in weather. Activity was least when temperatures were low and nights were bright. Effects of rainfall were variable. Sizes varied according to sex and age; young mice remained in the parents' range until they approached maturity, when they began to travel more widely. Adult males commonly had larger home ranges than females, although there were a number of exceptions. An inverse relationship between population density and size of home range was shown in several studies and probably is the usual relationship. A basic need for activity and exploration also appeared to influence size of home range. Behavior within the home range was discussed in terms of travel patterns, travels in relation to home sites and refuges, territory, and stability of size of home range. Travels within the home range consisted of repeated use of well-worn trails to sites of food, shelter, and refuge, plus more random exploratory travels

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

    Science.gov (United States)

    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.

  5. 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.

  6. 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.

  7. 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

  8. [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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. 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

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. 6. Home deliveries

    African Journals Online (AJOL)

    Sitwala

    determine factors associated with home deliveries. Main outcome .... Multiple logistic regression analyses were used to assess and estimate the factors and magnitude of effect on home deliveries. The variables in the model were age or age group, marital .... This finding coupled with lack of transport, made it very difficult for ...

  3. Home Education in Russia

    Science.gov (United States)

    Staroverova, T. I.

    2011-01-01

    From the eighteenth through the early twentieth centuries, home education (home schooling) by tutors and governesses in Russia was a customary form of schooling for an overwhelming majority of members of the nobility. Social and political transformations of the twentieth century led to substantial changes as the state got actively involved with…

  4. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Adult Ostomy Pediatric Ostomy Programa de Destrezas para manejo Doméstico de Ostomía Ostomy Home Skills Program Adult Ostomy Pediatric Ostomy Programa de Destrezas para manejo Doméstico de Ostomía The Ostomy Home Skills Kit ...

  5. Technologies for Home Networking

    DEFF Research Database (Denmark)

    A broad overview of the home networking field, ranging from wireless technologies to practical applications. In the future, it is expected that private networks (e.g. home networks) will become part of the global network ecosystem, participating in sharing their own content, running IP...

  6. European Home Energy

    DEFF Research Database (Denmark)

    Tommerup, Henrik M.

    2009-01-01

    An important aim of the european energy performance of buildings directive is to improve the overall energy efficiency of new homes......An important aim of the european energy performance of buildings directive is to improve the overall energy efficiency of new homes...

  7. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... de Destrezas para manejo Doméstico de Ostomía Ostomy Home Skills Program Adult Ostomy Pediatric Ostomy Programa de Destrezas para manejo Doméstico de Ostomía The Ostomy Home Skills Kit supports patients with educational and simulation ...

  8. Home Teaching and Herbart.

    Science.gov (United States)

    Rust, Val D.; Reed, Frances

    1979-01-01

    Viewing the growing disenchantment with state-controlled schooling, the authors predict that home teaching will become an established educational alternative within a short time, and they reflect on the teachings and writings of Johann Friedrich Herbart, an eighteenth-century advocate of educating children at home. (Editor/SJL)

  9. HomePort

    DEFF Research Database (Denmark)

    Madsen, Per Printz

    2009-01-01

    In the last couple of year's computer based home control systems are getting more and more common in modern homes. For instance these systems take care of light control, heat control and security systems.  The latest trend is to use wireless communication like Z-Wave and ZigBee to interconnect di...

  10. Health Begins at Home

    Centers for Disease Control (CDC) Podcasts

    2009-03-30

    Clean and well-maintained homes can prevent many illnesses and injuries. This podcast discusses how good health begins at home.  Created: 3/30/2009 by Coordinating Center for Environmental Health and Injury Prevention (CCEHIP).   Date Released: 3/30/2009.

  11. Home-based care

    African Journals Online (AJOL)

    Mrs. Patience Edoho Samson-Akpan

    PLWHA. The recommendation was that home based care should be encouraged and given priority by stake holders in the management of PLWHA. KEY WORDS: home-based care, quality of life, basic nursing care, psychosocial care. INTRODUCTION. HIV/AIDS is a chronic progressive disease which threatens the quality ...

  12. Home Study Advertising Handbook.

    Science.gov (United States)

    Lambert, Michael P., Ed.; Welch, Sally R., Ed.

    This handbook contains a collections of nine articles on the subject of direct-response advertising. The handbook gives advice on how to create effective advertisements for home study courses. The nine articles are the following: "Overview of Home Study Advertising in the 1990s" (Michael P. Lambert); "Ad Features that Sell"…

  13. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Payment Models Surgeons as Institutional Employees Our ... Ostomy Associations of America (UOAA). The skills kit contains: A booklet with information on the operation, home skills such as emptying and changing a pouch, problem solving, and home management. A DVD with demonstration ...

  14. The "H" Word: Home Schooling.

    Science.gov (United States)

    Butler, Shery

    2000-01-01

    This article discusses home schooling gifted children, including reasons families choose to home school their children, laws regulating home schooling, the educational background of parents who home school, and curriculum options. Advantages and disadvantages of home schooling are explored, along with data indicating the higher achievement of home…

  15. Creating a new home

    DEFF Research Database (Denmark)

    Gram-Hanssen, Kirsten; Bech-Danielsen, Claus

    2012-01-01

    Housing research is increasingly focusing on how different groups of residents use their dwelling and transform it into a home. In this article, we look at the homes of immigrants in Danish social housing. The article is based on qualitative interviews with Somali, Iraqi and Turkish immigrants...... might find another meaning in the concept of 'home' than their Danish neighbours. Thus the main issue for our research is to ascertain the extent to which immigrants are able to identify with their dwelling and to establish 'home' in Danish social housing. Does the meaning of the dwelling amongst...... immigrants differ from the one we know from other residents? And to what degree does the physical framework of Danish social housing support or maybe conflict with regard to immigrants' expectations, traditions and routines. Our analysis suggests that the home is as important for immigrants...

  16. 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.

  17. 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.

  18. 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.

  19. 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

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

    Science.gov (United States)

    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.

  1. 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.

  2. 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.

  3. 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.

  4. Biometrics for home networks security

    KAUST Repository

    Ansari, Imran Shafique

    2009-01-01

    Hacking crimes committed to the home networks are increasing. Advanced network protection is not always possible for the home networks. In this paper we will study the ability of using biometric systems for authentication in home networks. ©2009 IEEE.

  5. Genetics Home Reference: Peters anomaly

    Science.gov (United States)

    ... navigation Home Page Search Home Health Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Share: Email Facebook Twitter Home Health Conditions Peters anomaly Peters anomaly Printable PDF Open All Close All ...

  6. [Home blood transfusion].

    Science.gov (United States)

    Gay, V; Prévôt, G; Amico, I; Bonnet, B; Mansard, M-O

    2010-12-01

    The development of alternatives to hospitalization including home medical care (HAD), an aging population and a more secure transfusion raises the question of the feasibility of home blood transfusion. The legislation allows the home blood transfusion under specified conditions, but when they are met, the texts on nursing care and the transfusion gesture may hamper this progress. We report our experience of 3 years: a protocol was established to do home blood transfusions by trained transfusion nurses from the HAD. Six patients were eligible for transfusion at home but only three of them could be treated at home. Moreover, since late 2009, the Nursing Department no longer allows this practice for legal reasons. At the same time, a questionnaire was sent to 224 HAD to find out about their practice on the subject. In the light of practices in different countries, earnings for the quality of life of the patient, lack of space in hospitals and the aging population, it seems essential to change the law to permit a rational transfusion, thoughtful, safe for the patient at home and for caregivers who are involved. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

  7. 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

  8. 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.

  9. Condensation in insulated homes

    Energy Technology Data Exchange (ETDEWEB)

    Wiley, R A

    1978-05-28

    A research proposal on condensation in insulated homes is presented. Information is provided on: justification for condensation control; previous work and present outlook (good vapor barrier, condensation and retrofit insulation, vapor barrier decreases condensation, brick-veneer walls, condensation in stress-skin panels, air-conditioned buildings, retrofitting for conservation, study on mobile homes, high indoor relative humidity, report on various homes); and procedure (after funding has been secured). Measures are briefly described on opening walls, testing measures, and retrofitting procedures. An extensive bibliography and additional informative citations are included. (MCW)

  10. 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.

  11. 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...

  12. Managing menopause at home

    Science.gov (United States)

    ... or supplements. Eat high calcium foods, such as cheese, leafy green vegetables, low-fat milk and other ... unable to manage your symptoms of menopause with home care only. Also call if you have any ...

  13. Genetics Home Reference: trichothiodystrophy

    Science.gov (United States)

    ... features of trichothiodystrophy can include dry, scaly skin (ichthyosis); abnormalities of the fingernails and toenails; clouding of ... 2 links) The Merck Manual for Healthcare Professionals: Ichthyosis The Merck Manual Home Edition for Patients and ...

  14. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Up to Date with ACS Association Management JACS Jobs Events Find a Surgeon Patients and Family Contact My Profile Shop ( 0 ) Cart Donate American College of Surgeons Education Patients and Family Skills Programs Ostomy Home Skills ...

  15. Home Health Quality Initiative

    Data.gov (United States)

    U.S. Department of Health & Human Services — The instrument-data collection tool used to collect and report performance data by home health agencies is called the Outcome and Assessment Information Set (OASIS)....

  16. Genetics Home Reference: vitiligo

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Vitiligo Vitiligo Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Vitiligo is a condition that causes patchy loss of ...

  17. Nursing Home Compare Data

    Data.gov (United States)

    U.S. Department of Health & Human Services — These are the official datasets used on the Medicare.gov Nursing Home Compare Website provided by the Centers for Medicare and Medicaid Services. These data allow...

  18. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... SESAP Sampler SRGS Resources in Surgical Education ACS Fundamentals of Surgery Curriculum Transition to Practice Program ACS/ ... login or create account first) Skills Kits Broadcast Rights for Hospitals Ostomy Home Skills Hospital Quality Improvement ...

  19. Work and Home

    Science.gov (United States)

    ... continue to grow and their sense of personal identity is strengthened. Whether those activities are at home, ... Financials News Press Room MS Prevalence Charitable Ratings Corporate Support Helpful Links Donate Society Store MSConnection Blog ...

  20. Nursing Home Data Compendium

    Data.gov (United States)

    U.S. Department of Health & Human Services — The compendium contains figures and tables presenting data on all Medicare- and Medicaid-certified nursing homes in the United States as well as the residents in...

  1. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Trauma and EMS Cancer and Research Health Information Technology Scope of Practice Pediatric Issues Other Federal Legislative ... The Ostomy Home Skills Kit supports patients with educational and simulation materials to learn and practice the ...

  2. Heart failure - home monitoring

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/patientinstructions/000113.htm Heart failure - home monitoring To use the sharing features on this page, please enable JavaScript. Heart failure is a condition in which the heart is ...

  3. Genetics Home Reference: shingles

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Shingles Shingles Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Shingles (also known as herpes zoster) results from infection ...

  4. Genetics Home Reference: neuroblastoma

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Neuroblastoma Neuroblastoma Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Neuroblastoma is a type of cancer that most often ...

  5. Genetics Home Reference: hemophilia

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Hemophilia Hemophilia Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Hemophilia is a bleeding disorder that slows the blood ...

  6. Home Oxygen Therapy

    Science.gov (United States)

    ... over 90%. This system has a number of advantages. Since a concentrator essentially makes its own oxygen, there is no need for resupplies by the home care company. However, you must have a small cylinder as ...

  7. Genetics Home Reference: schizophrenia

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Schizophrenia Schizophrenia Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Schizophrenia is a brain disorder classified as a psychosis, ...

  8. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Medical Student Core Curriculum ACS/ASE Medical Student Simulation-Based Surgical Skills Curriculum Cancer Education Cancer Education ... Home Skills Kit supports patients with educational and simulation materials to learn and practice the skills needed ...

  9. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... and changing a pouch, problem solving, and home management. A DVD with demonstration of each skill Stoma Practice Model Stoma supplies (measurement guide, marking pen, scissors, sample pouch) Ostomy ...

  10. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Centers National Cancer Database National Accreditation Program for Rectal Cancer Oncology Medical Home Accreditation Program Stereotactic Breast ... collaboration with the American Society of Colon and Rectal Surgeons (ASCRS), American Urological Association (AUA), Certified Enterostomal ...

  11. Home Health Care Agencies

    Data.gov (United States)

    U.S. Department of Health & Human Services — A list of all Home Health Agencies that have been registered with Medicare. The list includes addresses, phone numbers, and quality measure ratings for each agency.

  12. Home Health PPS

    Data.gov (United States)

    U.S. Department of Health & Human Services — Under prospective payment, Medicare pays home health agencies (HHAs) a predetermined base payment. The payment is adjusted for the health condition and care needs of...

  13. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... at ACS ACS and Veterans Diversity at ACS Benefits ... Profile Shop ( 0 ) Cart Donate American College of Surgeons Education Patients and Family Skills Programs Ostomy Home Skills ...

  14. Home Health Compare Data

    Data.gov (United States)

    U.S. Department of Health & Human Services — These are the official datasets used on the Medicare.gov Home Health Compare Website provided by the Centers for Medicare and Medicaid Services. These data allow you...

  15. Genetics Home Reference: achondroplasia

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Achondroplasia Achondroplasia Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Achondroplasia is a form of short-limbed dwarfism. The ...

  16. Genetics Home Reference: adermatoglyphia

    Science.gov (United States)

    ... individuals have had difficulty entering countries that require fingerprinting for identification. In some families, adermatoglyphia occurs without ... Genetics Home Reference Celebrates Its 15th Anniversary National DNA Day 2018 Newborn Screening Saves Lives Act Turns ...

  17. Genetics Home Reference: phenylketonuria

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Phenylketonuria Phenylketonuria Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Phenylketonuria (commonly known as PKU) is an inherited disorder ...

  18. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Specific Registry Surgeon Specific Registry News and Updates Account Setup Resources and FAQs Features of the SSR ... Today Ostomy Home Skills Kit (login or create account first) Skills Kits Broadcast Rights for Hospitals Ostomy ...

  19. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Stay Up to Date with ACS Association Management Jobs Events Find a Surgeon Patients and Family Contact My Profile Shop ( 0 ) Cart Donate American College of Surgeons Education Patients and Family Skills Programs Ostomy Home Skills ...

  20. Home garden plums

    Science.gov (United States)

    The paper is to provide extension information on plums for home owners in Georgia and other Southeastern states. It includes seven sections: introduction, varieties, planting, pruning, fertilization, pests/diseases, and long term care....

  1. Genetics Home Reference: retinoblastoma

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Retinoblastoma Retinoblastoma Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Retinoblastoma is a rare type of eye cancer that ...

  2. Genetics Home Reference: coloboma

    Science.gov (United States)

    ... 447-70. Review. Citation on PubMed Gongal PA, French CR, Waskiewicz AJ. Aberrant forebrain signaling during early ... 3):191-7. Citation on PubMed More from Genetics Home Reference Bulletins March is Trisomy Awareness Month ...

  3. Pervasive Home Environments

    Science.gov (United States)

    Bull, P.; Limb, R.; Payne, R.

    An increasing number of computers and other equipment, such as games consoles and multimedia appliances for the home, have networking capability. The rapid growth of broadband in the home is also fuelling the demand for people to network their homes. In the near future we will see a number of market sectors trying to 'own' the home by providing gateways either from the traditional ISP or from games and other service providers. The consumer is bombarded with attractive advertising to acquire the latest technological advances, but is left with a plethora of different appliances, which have a bewildering range of requirements and features in terms of networking, user interface, and higher-level communications protocols. In many cases, these are proprietary, preventing interworking. Such technical and usability anarchy confuses the consumer and could ultimately suppress market adoption.

  4. HOME Income Limits

    Data.gov (United States)

    Department of Housing and Urban Development — HOME Income Limits are calculated using the same methodology that HUD uses for calculating the income limits for the Section 8 program. These limits are based on HUD...

  5. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... measurement guide, marking pen, scissors, sample pouch) Ostomy self-care checklist Evaluation (Complete the Ostomy Patient Survey . We need your opinion!) Program outcomes The ACS Ostomy Home ...

  6. Home Health PPS - Reports

    Data.gov (United States)

    U.S. Department of Health & Human Services — Abt Associates July 21, 2010 Analysis of 2000-2008 Home Health Case-mix Change Report estimates the extent to which the observed increases in average case-mix were...

  7. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Federal Legislation Federal Legislation Health Care Reform Summary Letters on Health Care Reform Medicare Physician Payment Medical ... The Ostomy Home Skills Kit supports patients with educational and simulation materials to learn and practice the ...

  8. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Subscribe ACS Case Reviews Login CME Test Login Author Instructions Sample Article Chapter Competition Contact Resources in ... login or create account first) Skills Kits Broadcast Rights for Hospitals Ostomy Home Skills Hospital Quality Improvement ...

  9. Genetics Home Reference: depression

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Depression Depression Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Depression (also known as major depression or major depressive ...

  10. Nursing Home Compare

    Data.gov (United States)

    U.S. Department of Health & Human Services — The data that is used by the Nursing Home Compare tool can be downloaded for public use. This functionality is primarily used by health policy researchers and the...

  11. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... ACS ACS and Veterans Diversity at ACS Benefits ... Profile Shop ( 0 ) Cart Donate American College of Surgeons Education Patients and Family Skills Programs Ostomy Home Skills ...

  12. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... skills such as emptying and changing a pouch, problem solving, and home management. A DVD with demonstration ... Patients and Family Surgeons Residents and Associates Medical Students International Surgeons Media ACS Links About ACS ACS Foundation ...

  13. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Trauma and EMS Cancer and Research Health Information Technology Scope of Practice Pediatric Issues Other Federal Legislative ... create account first) Skills Kits Broadcast Rights for Hospitals Ostomy Home Skills Hospital Quality Improvement Package The ...

  14. High performance homes

    DEFF Research Database (Denmark)

    Beim, Anne; Vibæk, Kasper Sánchez

    2014-01-01

    Can prefabrication contribute to the development of high performance homes? To answer this question, this chapter defines high performance in more broadly inclusive terms, acknowledging the technical, architectural, social and economic conditions under which energy consumption and production occur....... Consideration of all these factors is a precondition for a truly integrated practice and as this chapter demonstrates, innovative project delivery methods founded on the manufacturing of prefabricated buildings contribute to the production of high performance homes that are cost effective to construct, energy...

  15. Australia's Bond Home Bias

    OpenAIRE

    Anil V. Mishra; Umaru B. Conteh

    2014-01-01

    This paper constructs the float adjusted measure of home bias and explores the determinants of bond home bias by employing the International Monetary Fund's high quality dataset (2001 to 2009) on cross-border bond investment. The paper finds that Australian investors' prefer investing in countries with higher economic development and more developed bond markets. Exchange rate volatility appears to be an impediment for cross-border bond investment. Investors prefer investing in countries with ...

  16. Review of home phototherapy.

    Science.gov (United States)

    Rajpara, Anand N; O'Neill, Jenna L; Nolan, Bridgit V; Yentzer, Brad A; Feldman, Steve R

    2010-12-15

    Outpatient phototherapy is a safe, effective, and low-cost treatment modality for moderate to severe psoriasis. Barriers to outpatient phototherapy including patient inconvenience, patient co-pays, decreased physician compensation, and insurance disincentive structures have led to decreased use and underutilization of phototherapy. Home phototherapy can potentially overcome many of the barriers associated with outpatient treatment but is not widely used because of concerns over safety and efficacy, lack of resident and physician education, and lack of insurance coverage. The purpose of this study is to review the use of phototherapy with emphasis on the safety, efficacy, and practical use of home phototherapy. A comprehensive Pubmed literature search was done using the keywords NB-UVB, narrowband UVB, BB-UVB, broadband UVB, PUVA, psoralen and UVA, UVA, history of phototherapy, mechanism of phototherapy, phototherapy in dermatology, home phototherapy, and phototherapy for psoriasis. All relevant articles were reviewed. Home NB-UVB phototherapy can be as safe, effective, and cost-effective as outpatient phototherapy. Further, home UVB is more convenient for patients, has higher patient satisfaction, and a lower treatment burden compared to outpatient phototherapy. Home NB-UVB should be considered as a treatment option for patients eligible for phototherapy.

  17. 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

  18. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  20. 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

  1. 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.

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

    Directory of Open Access Journals (Sweden)

    Rafael Nisa-Martínez

    Full Text Available Bacterial group II introns are self-splicing catalytic RNAs and mobile retroelements that have an open reading frame encoding an intron-encoded protein (IEP with reverse transcriptase (RT and RNA splicing or maturase activity. Some IEPs carry a DNA endonuclease (En domain, which is required to cleave the bottom strand downstream from the intron-insertion site for target DNA-primed reverse transcription (TPRT of the inserted intron RNA. Host factors complete the insertion of the intron. By contrast, the major retrohoming pathway of introns with IEPs naturally lacking endonuclease activity, like the Sinorhizobium meliloti intron RmInt1, is thought to involve insertion of the intron RNA into the template for lagging strand DNA synthesis ahead of the replication fork, with possible use of the nascent strand to prime reverse transcription of the intron RNA. The host factors influencing the retrohoming pathway of such introns have not yet been described. Here, we identify key candidates likely to be involved in early and late steps of RmInt1 retrohoming. Some of these host factors are common to En+ group II intron retrohoming, but some have different functions. Our results also suggest that the retrohoming process of RmInt1 may be less dependent on the intracellular free Mg2+ concentration than those of other group II introns.

  3. 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.

  4. 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.

  5. Smart assistants for smart homes

    OpenAIRE

    Rasch, Katharina

    2013-01-01

    The smarter homes of tomorrow promise to increase comfort, aid elderly and disabled people, and help inhabitants save energy. Unfortunately, smart homes today are far from this vision – people who already live in such a home struggle with complicated user interfaces, inflexible home configurations, and difficult installation procedures. Under these circumstances, smart homes are not ready for mass adoption. This dissertation addresses these issues by proposing two smart assistants for smart h...

  6. 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...

  7. Reflections: Volunteering at Home.

    Science.gov (United States)

    Hu, Amanda

    2016-08-01

    Many young people look forward to volunteering abroad and overlook the ample volunteer opportunities at home. There are several advantages to volunteering at home: you help people in your own community; you can make a long-term commitment; and you have continuity of care for your patients. There are >1200 free clinics in the United States whose main goal is to provide care to the indigent population. These free clinics are always looking for volunteers with specialized medical training. This article reviews the medically related and unrelated volunteer opportunities available in the United States. Volunteering at home is a worthwhile experience, and I encourage the otolaryngology community to explore these opportunities. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2016.

  8. At Home with Students

    DEFF Research Database (Denmark)

    Lyngsø, Anita

    2015-01-01

    This article reflects the methodological challenges presented in the research process, where the principle of 'following the field’ means that the researcher must also follow students engaged in online activities in their own homes. The ethnographic studies are a part of a PhD project on “NETeduc......This article reflects the methodological challenges presented in the research process, where the principle of 'following the field’ means that the researcher must also follow students engaged in online activities in their own homes. The ethnographic studies are a part of a PhD project...... on “NETeducation,” a full-scale development project in nursing education (Lyngsø, 2014). With a focus on online professional education as the starting point, the process of research will follow the shifting learning process, through phases in the virtual classroom and in the students’ own homes. Research in online...

  9. HomePort

    DEFF Research Database (Denmark)

    Le Guilly, Thibaut; Olsen, Petur; Ravn, Anders Peter

    2013-01-01

    Ambient Intelligence systems use many sensors and actuators, with a diversity of networks, protocols and technologies which makes it impossible to access the devices in a common manner. This paper presents the HomePort software, which provides an open source RESTful interface to heterogeneous...... sensor networks, allowing a simple unified access to virtually any kind of protocol using well known standards. HomePort includes means to provide event notification, as well as a tracing mechanism. The software is implemented and we report on initial experiments and provide an evaluation that shows...

  10. Future of home hemodialysis.

    Science.gov (United States)

    Friedman, E A

    1977-01-01

    An attempt to prophesy the future of home hemodialysis is based on anticipated near and long term technological improvements. Dialysis treatments will become shorter, due to pressure filtration, while extracting molecules in the 300 to 3000 dalton weight range through more permeable membranes. Portable and wearable dialysis systems will increase patient mobility. By the 21st century, implantable bionic kidneys and perfect host tolerance to renal allografts and xenografts will relegate home hemodialysis to history texts. A non-surgical definitive treatment for uremia is envisioned in the colonization of the human bowel with nitrogenn recycling bacteria.

  11. Windows Home Server users guide

    CERN Document Server

    Edney, Andrew

    2008-01-01

    Windows Home Server brings the idea of centralized storage, backup and computer management out of the enterprise and into the home. Windows Home Server is built for people with multiple computers at home and helps to synchronize them, keep them updated, stream media between them, and back them up centrally. Built on a similar foundation as the Microsoft server operating products, it's essentially Small Business Server for the home.This book details how to install, configure, and use Windows Home Server and explains how to connect to and manage different clients such as Windows XP, Windows Vist

  12. 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

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

    Directory of Open Access Journals (Sweden)

    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

  14. Home Start Evaluation Study.

    Science.gov (United States)

    High/Scope Educational Research Foundation, Ypsilanti, MI.

    Case studies of eight Home Start programs are given as the third section of an evaluation study. Communities involved are Binghamton, New York; Franklin, North Carolina; Cleveland, Ohio; Harrogate, Tennessee; Houston, Texas; Weslaco, Texas; Millville, Utah; Parkersburg, West Virginia. Although each study varies in format, each describes in detail…

  15. Home Network Security

    NARCIS (Netherlands)

    Scholten, Hans; van Dijk, Hylke

    2008-01-01

    Service discovery and secure and safe service usage are essential elements in the deployment of home and personal networks. Because no system administrator is present, setup and daily operation of such a network has to be automated as much as possible with a high degree of user friendliness. To

  16. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... manejo Doméstico de Ostomía The Ostomy Home Skills Kit supports patients with educational and simulation materials to ... the skills needed for optimal postoperative recovery. The kit supports the entire surgical team with quality, comprehensive ...

  17. Exploring Home Schooling.

    Science.gov (United States)

    Boyer, Wanda A. R.

    2002-01-01

    Examines concerns expressed by home-schooling parents in the context of guidelines of the World Organization for Early Childhood Education and the Association for Childhood Education International. Connects guidelines to recent literature to suggest effective strategies for meeting parental needs and responding to the diverse responsibilities of…

  18. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... and Safety Conference Registry Login SCR Training and Testing ... Programa de Destrezas para manejo Doméstico de Ostomía The Ostomy Home Skills Kit supports patients with educational and simulation materials to learn and practice the skills needed for ...

  19. At Home with Students

    DEFF Research Database (Denmark)

    Lyngsø, Anita

    2015-01-01

    This article reflects the methodological challenges presented in the research process, where the principle of 'following the field’ means that the researcher must also follow students engaged in online activities in their own homes. The ethnographic studies are a part of a PhD project on “NETeduc......This article reflects the methodological challenges presented in the research process, where the principle of 'following the field’ means that the researcher must also follow students engaged in online activities in their own homes. The ethnographic studies are a part of a PhD project...... on “NETeducation,” a full-scale development project in nursing education (Lyngsø, 2014). With a focus on online professional education as the starting point, the process of research will follow the shifting learning process, through phases in the virtual classroom and in the students’ own homes. Research in online...... to this division due to the “netstudents” activities in studying online at home. On the other hand, the dichotomy between online and offline contexts is found to be inadequate, during the observations conducted. In light of some preliminary findings, the challenges of observing online and offline activities almost...

  20. Future Home Network Requirements

    DEFF Research Database (Denmark)

    Charbonnier, Benoit; Wessing, Henrik; Lannoo, Bart

    This paper presents the requirements for future Home Area Networks (HAN). Firstly, we discuss the applications and services as well as their requirements. Then, usage scenarios are devised to establish a first specification for the HAN. The main requirements are an increased bandwidth (towards 1...

  1. Doing Home Works

    DEFF Research Database (Denmark)

    Nelund, Sidsel

    2013-01-01

    of aesthetic analysis of artworks and ethnographic fieldwork (Georgina Born); and 3) the use of generative ethnographic stories as a writing tool (Helen Verran). The latter two, especially, are then employed in analysing the Beirut-based extended exhibition, ‘Home Works: A Forum on Cultural Practices...

  2. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... emptying and changing a pouch, problem solving, and home management. A DVD with demonstration of each skill Stoma Practice Model Stoma supplies (measurement guide, marking pen, scissors, sample pouch) Ostomy self-care checklist Evaluation (Complete the Ostomy Patient Survey . We ...

  3. Home Ventilator Guide

    Science.gov (United States)

    HOME VENTILATOR GUIDE This project is made possible by a bequest from ventilator user Ira Holland. ©Copyright 2017 Post-Polio Health ... proper balance between the two. What is a ventilator? A ventilator, also known as a respirator, is ...

  4. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Contact My Profile Shop ( 0 ) Cart Donate American College of Surgeons Education Patients and Family Skills Programs Ostomy Home Skills ... facs.org Copyright © 1996-2018 by the American College of Surgeons, Chicago, IL 60611-3295 | Privacy Policy | Terms of Use

  5. Personalized Home-Networks

    DEFF Research Database (Denmark)

    Soler, José; Gandy, Michael

    2007-01-01

    The paper provides details of a home-networking architecture based on an enhanced residential gateway. Initially the need for mechanisms allowing user-dependent network behavior is described and afterwards details of an initial implementation are provided in terms of architectural description...

  6. Solar Electricity for Homes

    Science.gov (United States)

    Roman, Harry T.

    2012-01-01

    Every day, the sun showers the Earth with millions of times more energy than its people use. The only problem is that energy is spread out over the entire Earth's surface and must be harvested. Engineers are learning to capture and use some of this energy to make electricity for homes. Solar panels make up the heart of a solar system. They can be…

  7. Protokoller til Home Automation

    DEFF Research Database (Denmark)

    Kjær, Kristian Ellebæk

    2008-01-01

    computer, der kan skifte mellem foruddefinerede indstillinger. Nogle gange kan computeren fjernstyres over internettet, så man kan se hjemmets status fra en computer eller måske endda fra en mobiltelefon. Mens nævnte anvendelser er klassiske indenfor home automation, er yderligere funktionalitet dukket op...

  8. Home vision tests

    Science.gov (United States)

    ... missing areas. If any lines appear distorted or broken, note their location on the grid using a pen or pencil. DISTANCE VISION This is the standard eye chart doctors use, which has been adapted for home use. The chart is attached to a wall ...

  9. Home Energy Conservation Primer.

    Science.gov (United States)

    DeLuca, V. William; And Others

    This guide was prepared to support a program of training for community specialists in contemporary and practical techniques of home energy conservation. It is designed to assist professionals in efficient operation of energy conservation programs and to provide ideas for expanding education operations. Eight major sections are presented: (1)…

  10. Home Health Compare: Find a Home Health Agency

    Science.gov (United States)

    ... page could not be loaded. The Medicare.gov Home page currently does not fully support browsers with " ... widget - Select to show Back to top Footer Home A federal government website managed and paid for ...

  11. Factors predicting a home death among home palliative care recipients.

    Science.gov (United States)

    Ko, Ming-Chung; Huang, Sheng-Jean; Chen, Chu-Chieh; Chang, Yu-Ping; Lien, Hsin-Yi; Lin, Jia-Yi; Woung, Lin-Chung; Chan, Shang-Yih

    2017-10-01

    Awareness of factors affecting the place of death could improve communication between healthcare providers and patients and their families regarding patient preferences and the feasibility of dying in the preferred place.This study aimed to evaluate factors predicting home death among home palliative care recipients.This is a population-based study using a national representative sample retrieved from the National Health Insurance Research Database. Subjects receiving home palliative care, from 2010 to 2012, were analyzed to evaluate the association between a home death and various characteristics related to illness, individual, and health care utilization. A multiple-logistic regression model was used to assess the independent effect of various characteristics on the likelihood of a home death.The overall rate of a home death for home palliative care recipients was 43.6%. Age; gender; urbanization of the area where the patients lived; illness; the total number of home visits by all health care professionals; the number of home visits by nurses; utilization of nasogastric tube, endotracheal tube, or indwelling urinary catheter; the number of emergency department visits; and admission to intensive care unit in previous 1 year were not significantly associated with the risk of a home death. Physician home visits increased the likelihood of a home death. Compared with subjects without physician home visits (31.4%) those with 1 physician home visit (53.0%, adjusted odds ratio [AOR]: 3.23, 95% confidence interval [CI]: 1.93-5.42) and those with ≥2 physician home visits (43.9%, AOR: 2.23, 95% CI: 1.06-4.70) had higher likelihood of a home death. Compared with subjects with hospitalization 0 to 6 times in previous 1 year, those with hospitalization ≥7 times in previous 1 year (AOR: 0.57, 95% CI: 0.34-0.95) had lower likelihood of a home death.Among home palliative care recipients, physician home visits increased the likelihood of a home death. Hospitalizations ≥7

  12. Telemedicine in Neonatal Home Care

    DEFF Research Database (Denmark)

    Holm, Kristina Garne; Brødsgaard, Anne; Zachariassen, Gitte

    2016-01-01

    BACKGROUND: For the majority of preterm infants, the last weeks of hospital admission mainly concerns tube feeding and establishment of breastfeeding. Neonatal home care (NH) was developed to allow infants to remain at home for tube feeding and establishment of breastfeeding with regular home...... visits from neonatal nurses. For hospitals covering large regions, home visits may be challenging, time consuming, and expensive and alternative approaches must be explored. OBJECTIVE: To identify parental needs when wanting to provide neonatal home care supported by telemedicine. METHODS: The study used...... participatory design and qualitative methods. Data were collected from observational studies, individual interviews, and focus group interviews. Two neonatal units participated. One unit was experienced in providing neonatal home care with home visits, and the other planned to offer neonatal home care...

  13. Home-Use Tests - Cholesterol

    Science.gov (United States)

    ... Medical Procedures In Vitro Diagnostics Home Use Tests Cholesterol Share Tweet Linkedin Pin it More sharing options ... a home-use test kit to measure total cholesterol. What cholesterol is: Cholesterol is a fat (lipid) ...

  14. Home apnea monitor use - infants

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/patientinstructions/000755.htm Home apnea monitor use - infants To use the sharing ... portable. Why is an Apnea Monitor Used at Home? A monitor may be needed when: Your baby ...

  15. Blood pressure monitors for home

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/007482.htm Blood pressure monitors for home To use the sharing features ... ask you to keep track of your blood pressure at home. To do this, you will need ...

  16. Genetics Home Reference: Fraser syndrome

    Science.gov (United States)

    ... navigation Home Page Search Home Health Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Share: Email ... of the genitalia and the urinary tract (genitourinary anomalies). Other tissues and organs can also be affected. ...

  17. Genetics Home Reference: Greenberg dysplasia

    Science.gov (United States)

    ... navigation Home Page Search Home Health Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Share: Email ... K. Mutations causing Greenberg dysplasia but not Pelger anomaly uncouple enzymatic from structural functions of a nuclear ...

  18. Genetics Home Reference: RAPADILINO syndrome

    Science.gov (United States)

    ... navigation Home Page Search Home Health Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Share: Email ... Rapadilino syndrome Hospital for Sick Children: Radial Ray Anomaly MalaCards: rapadilino syndrome March of Dimes: Cleft Lip ...

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. 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.

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

    Science.gov (United States)

    Naganuma, Takahiro; Nakayama, Tomohiro; Sato, Naoyuki; Fu, Zhenyan; Soma, Masayoshi; Yamaguchi, Mai; Shimodaira, Masanori; Aoi, Noriko; Usami, Ron

    2010-02-01

    Oxidative DNA damage is involved in the pathophysiology of essential hypertension (EH), which is a multifactorial disorder. Apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/REF-1) is an essential endonuclease in the base excision repair pathway of oxidatively damaged DNA, in addition to having reducing properties that promote the binding of redox-sensitive transcription factors. Blood pressure in APE1/REF-1-knockout mice is reported to be significantly higher than in wild-type mice. The aim of this study was to investigate the relationship between EH and the human APE1/REF-1 gene through a haplotype-based case-control study using single-nucleotide polymorphisms (SNPs). We selected five SNPs in the human APE1/REF-1 gene (rs1760944, rs3136814, rs17111967, rs3136817, and rs1130409), and performed case-control studies in 265 EH patients and 266 age-matched normotensive (NT) subjects. rs17111967 was found to show nonheterogeneity among Japanese subjects. There were no significant differences in the overall distribution of genotypes or alleles for each SNP between EH and NT groups. In the overall distribution of the haplotype-based case-control study constructed based on rs1760944, rs3136817, and rs1130409, the frequency of the G-T-T haplotype was significantly higher in the EH group than in the NT group (2.1% vs. 0.0%, P = 0.001). Multiple logistic regression analysis also revealed significant differences for the G-T-T haplotype, even after adjustment for confounding factors (OR = 8.600, 95% CI: 1.073-68.951, P = 0.043). Based on the present results, the G-T-T haplotype appears to be a genetic marker of EH, and the APE1/REF-1 gene appears to be a susceptibility gene for EH.

  3. Home-based Healthcare Technology

    DEFF Research Database (Denmark)

    Verdezoto, Nervo

    of these systems target a specific treatment or condition and might not be sufficient to support the care management work at home. Based on a case study approach, my research investigates home-based healthcare practices and how they can inform future design of home-based healthcare technology that better account...

  4. Can Intimacy Justify Home Education?

    Science.gov (United States)

    Merry, Michael S.; Howell, Charles

    2009-01-01

    Many parents cite intimacy as one of their reasons for deciding to educate at home. It seems intuitively obvious that home education is conducive to intimacy because of the increased time families spend together. Yet what is not clear is whether intimacy can provide justification for one's decision to home educate. To see whether this is so, we…

  5. Home advantage in professional tennis

    NARCIS (Netherlands)

    Koning, Ruud H.

    2011-01-01

    Home advantage is a pervasive phenomenon in sport. It has been established in team sports such as basketball, baseball, American football, and European soccer. Attention to home advantage in individual sports has so far been limited. The aim of this study was to examine home advantage in

  6. When Globalization Hits Home

    DEFF Research Database (Denmark)

    Borrás, Susana; Haakonsson, Stine Jessen

    . The dynamics are generally differentiated according to the type of lead firm strategy, i.e. knowledge augmenting or knowledge exploiting. The qualitative and exploratory findings point towards some effects on national innovation networks. Hence, the paper concludes by hypothesizing that the globalization...... of lead firms’ innovation has a mobilization effect on preexisting national innovation networks. The hypothesis says that lead firms’ engagement in global innovation networks can mobilize the organizations that are part of the lead firms’ innovation networks at home. This mobilization effect differs......Lead firms are increasingly reorganizing their innovation activities into global innovation networks. Such reorganization has potentially major impact on their existing home-based innovation networks. Based on 31 interviews in four case studies of lead firms in the Danish food industry, the paper...

  7. The Home of Man

    DEFF Research Database (Denmark)

    Hauberg, Jørgen; Bjerrum, Peter

    2018-01-01

    Abstract: Accordingly, Le Corbusier in “Towards a New Architecture”, 1922, the question was: “Architecture or revolution” – also stated in “The Home of Man”, 1942: “… people live in poor conditions, this is the real, the most profound reason for the battles and upheavals of our time.” The ninetee......Abstract: Accordingly, Le Corbusier in “Towards a New Architecture”, 1922, the question was: “Architecture or revolution” – also stated in “The Home of Man”, 1942: “… people live in poor conditions, this is the real, the most profound reason for the battles and upheavals of our time...

  8. When Globalization Hits Home

    DEFF Research Database (Denmark)

    Borrás, Susana; Haakonsson, Stine Jessen

    Lead firms are increasingly reorganizing their innovation activities into global innovation networks. Such reorganization has potentially major impact on their existing home-based innovation networks. Based on 31 interviews in four case studies of lead firms in the Danish food industry, the paper...... analyzes the dynamics of five key features of home-based innovation networks: 1) size of the national networks, 2) type of organization, 3) content of collaboration within the network, 4) concurrent globalization of organizations in the network, and 5) degree of formalization of network interactions....... The dynamics are generally differentiated according to the type of lead firm strategy, i.e. knowledge augmenting or knowledge exploiting. The qualitative and exploratory findings point towards some effects on national innovation networks. Hence, the paper concludes by hypothesizing that the globalization...

  9. High performance homes

    DEFF Research Database (Denmark)

    Beim, Anne; Vibæk, Kasper Sánchez

    2014-01-01

    Can prefabrication contribute to the development of high performance homes? To answer this question, this chapter defines high performance in more broadly inclusive terms, acknowledging the technical, architectural, social and economic conditions under which energy consumption and production occur....... Consideration of all these factors is a precondition for a truly integrated practice and as this chapter demonstrates, innovative project delivery methods founded on the manufacturing of prefabricated buildings contribute to the production of high performance homes that are cost effective to construct, energy...... efficient to operate and valuable for building communities. Herein discussed are two successful examples of low energy prefabricated housing projects built in Copenhagen Denmark, which embraced both the constraints and possibilities offered by prefabrication....

  10. [Aromatherapy in nursing homes].

    Science.gov (United States)

    Barré, Lucile

    2015-01-01

    Pierre Delaroche de Clisson hospital uses essential oils as part of its daily organisation for the treatment of pain and the development of palliative care. The setting up of this project, in nursing homes and long-term care units, is the fruit of a complex mission carried out by a multidisciplinary team, which had to take into account the risks involved and overcome a certain amount of reluctance. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  11. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Workforce Trauma and EMS Cancer and Research Health Information Technology Scope of Practice Pediatric Issues Other Federal Legislative Issues Regulatory Issues Regulatory Issues Regulatory Issues Stop Overregulating My OR ... Rectal Surgeons (ASCRS), American Urological Association (AUA), Certified Enterostomal Therapy Nurses (CETN), and the United Ostomy Associations of America (UOAA). The skills kit contains: A booklet with information on the operation, home skills such as emptying ...

  12. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Workforce Trauma and EMS Cancer and Research Health Information Technology Scope of Practice Pediatric Issues Other Federal Legislative Issues Regulatory Issues Regulatory Issues Regulatory Issues Stop Overregulating My OR ... American Urological Association (AUA), Certified Enterostomal Therapy Nurses (CETN), and the United Ostomy Associations of America (UOAA). Program Overview The skills kit contains: A booklet with information on the operation, home skills such as emptying ...

  13. ArduSmartHome

    OpenAIRE

    Sánchez Muñoz, Miguel Ángel

    2017-01-01

    Diseño e implementación de red de sensores inalámbricos para el control domótico de una vivienda basado en Arduino. Design and implementation of a wireless sensor network for home automation control based on Arduino. Disseny i implementació de xarxa de sensors sense fils per al control domòtic d'un habitatge basat en Arduino.

  14. Home automation on the move:

    OpenAIRE

    Imperl, Bojan; Jeřabek, Boro; Šoštarič, Andrej

    2003-01-01

    In this article we would like to show how an arbitrary home and building electronic system based on the home automation standards such as Xl0 might be addressed and controlled by an appropriate mobile technology. Urge for mobility of users, which may be at the same time either the inhabitants of these homes or even administrators and supporters, is growing. The possibility to control and observe the status of home appliances while being on the move away from home using our mobile phones is be...

  15. Lighting Options for Homes.

    Energy Technology Data Exchange (ETDEWEB)

    Baker, W.S.

    1991-04-01

    This report covers many aspects of various lighting options for homes. Types of light sources described include natural light, artificial light, incandescent lamps, fluorescent lamps, and high intensity discharge lamps. A light source selection guide gives the physical characteristics of these, design considerations, and common applications. Color, strategies for efficient lighting, and types of lighting are discussed. There is one section giving tips for various situations in specific rooms. Rooms and types of fixtures are shown on a matrix with watts saved by using the recommended type lighting for that room and room location. A major emphasis of this report is saving energy by utilizing the most suitable, recommended lighting option. (BN)

  16. The @Home project

    DEFF Research Database (Denmark)

    Coxon, Ian Robert

    2014-01-01

    of being overshadowed by what is increasingly seen as new (and substantial) pharmacological and health equipment business market opportunities in what is now viewed as a health 'industry'. The Centre for Innovation at Mayo Clinic (CFI) in Rochester USA, has conducted many projects which border on and have...... investigated various aspects of care provision and patient experience within a patient's home environment. Considerable work by other researchers inside and outside the health field has also contributed insights and platforms for moving healthcare in this direction. In most areas of the western world...

  17. Convents as homes.

    Science.gov (United States)

    Arias, Enrique Alberto

    2005-01-01

    The present article discusses convents as homes. Resulting from the study of a Gregorian source presently housed at DePaul University's Richardson library, this article probes the complexities and restrictions of convent life in 17th century Spain. The Sanctoral de Visperas (1653) functions as a backdrop for a consideration of how singing chant and attendant rituals enriched the lives of nuns. Also included are references to nuns from this period who were outstanding musicians and poets and whose works have recently received enthusiastic attention.

  18. Second-home electricity consumption

    International Nuclear Information System (INIS)

    Andersen, Frits M.; Christensen, Morten S.; Jensen, Ole Michael; Kofoed, Niels-Ulrik; Morthorst, Poul Erik

    2008-01-01

    In Denmark, electricity consumption in first and second homes has developed quite differently. Since 1990, electricity consumption in ordinary residences has grown moderately, while consumption in weekend and second homes has increased considerably. In turn, this development has been blamed on a growing number of luxury cottages, new legislation permitting senior citizens to have their permanent address in their second home and a growing number of electric appliances. In order to examine the growing electricity consumption in second homes and to estimate future demand, a multidisciplinary study combining top-down and bottom-up analyses was conducted, i.e., combining models using aggregated economic parameters and feasibility studies using technical parameters, respectively. The top-down estimation showed that changes in electricity consumption in second homes correlate to changes in income. The bottom-up estimation showed that consumption was mainly affected by the frequency with which second homes were used in the winter time. This indicates that additional second homes, increased full-time use and intensified use of electric appliances are the main reasons for the observed increases in electricity consumption. Luxury tourism use and senior citizens' that use a few per cent of the second homes as their home contribute to a minor degree to the overall increase of electricity consumption. Scenarios show that this development may accelerate with increased leisure time, increased use and more permanent settlement in second homes

  19. Second-home electricity consumption

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Frits M.; Morthorst, Poul Erik [Risoe, Systems Analysis Department, Technical University of Denmark, P.O. Box 49, DK-4000 Roskilde (Denmark); Christensen, Morten S.; Kofoed, Niels-Ulrik [Esbensen Consulting Engineers, Carl Jacobsens Vej 25D, DK-2500 Valby (Denmark); Jensen, Ole Michael [Danish Building Research Institute, Department of Energy and Environment, University of Aalborg, P.O. Box 119, DK-2970 Horsholm (Denmark)

    2008-01-15

    In Denmark, electricity consumption in first and second homes has developed quite differently. Since 1990, electricity consumption in ordinary residences has grown moderately, while consumption in weekend and second homes has increased considerably. In turn, this development has been blamed on a growing number of luxury cottages, new legislation permitting senior citizens to have their permanent address in their second home and a growing number of electric appliances. In order to examine the growing electricity consumption in second homes and to estimate future demand, a multidisciplinary study combining top-down and bottom-up analyses was conducted, i.e., combining models using aggregated economic parameters and feasibility studies using technical parameters, respectively. The top-down estimation showed that changes in electricity consumption in second homes correlate to changes in income. The bottom-up estimation showed that consumption was mainly affected by the frequency with which second homes were used in the winter time. This indicates that additional second homes, increased full-time use and intensified use of electric appliances are the main reasons for the observed increases in electricity consumption. Luxury tourism use and senior citizens' that use a few per cent of the second homes as their home contribute to a minor degree to the overall increase of electricity consumption. Scenarios show that this development may accelerate with increased leisure time, increased use and more permanent settlement in second homes. (author)

  20. Alert management for home healthcare based on home automation analysis.

    Science.gov (United States)

    Truong, T T; de Lamotte, F; Diguet, J-Ph; Said-Hocine, F

    2010-01-01

    Rising healthcare for elder and disabled people can be controlled by offering people autonomy at home by means of information technology. In this paper, we present an original and sensorless alert management solution which performs multimedia and home automation service discrimination and extracts highly regular home activities as sensors for alert management. The results of simulation data, based on real context, allow us to evaluate our approach before application to real data.

  1. Home Rx: The Health Benefits of Home Performance

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Jonathan [National Center for Healthy Housing (NCHH), Columbia, MD (United States); Jacobs, David [National Center for Healthy Housing (NCHH), Columbia, MD (United States); Reddy, Amanda [National Center for Healthy Housing (NCHH), Columbia, MD (United States); Tohn, Ellen [Tohn Environmental Strategies, Wayland, MA (United States); Cohen, Jonathan [Dept. of Energy (DOE), Washington DC (United States); Jacobsohn, Ely [Dept. of Energy (DOE), Washington DC (United States)

    2016-12-01

    Evidence in a new, groundbreaking U.S. Department of Energy report, Home Rx: The Health Benefits of Home Performance, shows that home performance upgrades can improve the quality of a home’s indoor environment by reducing the prevalence of harmful indoor air pollutants and contaminants. Until recently, no systematic review of this evidence had been conducted, limiting full understanding of the link between home performance and health. This new report summarizes current knowledge and identifies research gaps. The design characteristics and results of each of the 40 studies considered in the report are summarized in a searchable matrix.

  2. The Nest Home

    Energy Technology Data Exchange (ETDEWEB)

    Pickerill, Heath [Missouri Univ. of Science and Technology, Rolla, MO (United States)

    2016-07-11

    The purpose of the project was to build a competitive solar-powered house for the U.S. Department of Energy Solar Decathlon 2015 held in Irvine, California. The house, named the Nest Home, was an innovative design that works with the environment to meet the needs of the occupants, identified as a growing family. Reused materials were instrumental in the design. Three refurbished shipping containers composed the primary structure of the house, creating an open floor plan that defies common architecture for container homes. The exterior siding was made of deconstructed shipping pallets collected locally. Other recycled products included carpet composed of discarded fishing nets, denim batting made of recycled blue jeans that outperform traditional fiberglass insulation in sound proofing and thermal resistance, and kitchen cabinets that were purchased used and refinished. Collectively these elements formed a well-balanced blend of modern design, comfort, and sustainability. The house was Missouri University of Science and Technology’s sixth entry in the DOE Solar Decathlon. Missouri S&T has been invited to compete in six of the seven decathlons held, more than any other university worldwide. The house was brought back to Rolla after the Decathlon in California where it has been placed in its permanent location on the S&T campus.

  3. Telemedicine: extension to home care.

    Science.gov (United States)

    Kaye, L W

    1997-01-01

    Although there are multiple challenges facing the use of telemedicine in home health care, it seems likely that they will be resolved. This analysis projects significant increases in productivity and savings to be realized by implementing a telemedicine system (such as the Tevital Home Care System) compared with traditional home care services. Additional savings are expected as a result of the reduction in the utilization of medical services other than home health care. At the same time, telemedicine offers conceivable benefits such as improved access for consumers, extended coverage capability by home health care agencies, decreased inefficiencies attributable to the complications associated with agency personnel travel, improved agency capacity for preventive care, and, ultimately, heightened consumer satisfaction and both physical and psychological well-being. Taken together, these benefits make adoption of telemedicine technology by home care providers highly desirable.

  4. The Science of Home Automation

    Science.gov (United States)

    Thomas, Brian Louis

    Smart home technologies and the concept of home automation have become more popular in recent years. This popularity has been accompanied by social acceptance of passive sensors installed throughout the home. The subsequent increase in smart homes facilitates the creation of home automation strategies. We believe that home automation strategies can be generated intelligently by utilizing smart home sensors and activity learning. In this dissertation, we hypothesize that home automation can benefit from activity awareness. To test this, we develop our activity-aware smart automation system, CARL (CASAS Activity-aware Resource Learning). CARL learns the associations between activities and device usage from historical data and utilizes the activity-aware capabilities to control the devices. To help validate CARL we deploy and test three different versions of the automation system in a real-world smart environment. To provide a foundation of activity learning, we integrate existing activity recognition and activity forecasting into CARL home automation. We also explore two alternatives to using human-labeled data to train the activity learning models. The first unsupervised method is Activity Detection, and the second is a modified DBSCAN algorithm that utilizes Dynamic Time Warping (DTW) as a distance metric. We compare the performance of activity learning with human-defined labels and with automatically-discovered activity categories. To provide evidence in support of our hypothesis, we evaluate CARL automation in a smart home testbed. Our results indicate that home automation can be boosted through activity awareness. We also find that the resulting automation has a high degree of usability and comfort for the smart home resident.

  5. Home advantage in Greek football.

    Science.gov (United States)

    Armatas, Vasilis; Pollard, Richard

    2014-01-01

    Home advantage as it relates to team performance at football was examined in Superleague Greece using nine seasons of game-by-game performance data, a total of 2160 matches. After adjusting for team ability and annual fluctuations in home advantage, there were significant differences between teams. Previous findings regarding the role of territorial protection were strengthened by the fact that home advantage was above average for the team from Xanthi (P =0.015), while lower for teams from the capital city Athens (P =0.008). There were differences between home and away teams in the incidence of most of the 13 within-game match variables, but associated effect sizes were only moderate. In contrast, outcome ratios derived from these variables, and measuring shot success, had negligible effect sizes. This supported a previous finding that home and away teams differed in the incidence of on-the-ball behaviours, but not in their outcomes. By far the most important predictor of home advantage, as measured by goal difference, was the difference between home and away teams in terms of kicked shots from inside the penalty area. Other types of shots had little effect on the final score. The absence of a running track between spectators and the playing field was also a significant predictor of goal difference, worth an average of 0.102 goals per game to the home team. Travel distance did not affect home advantage.

  6. Home environment and childhood asthma.

    Science.gov (United States)

    Leen, M G; O'Connor, T; Kelleher, C; Mitchell, E B; Loftus, B G

    1994-01-01

    The prevalence of childhood asthma has increased dramatically in the past 20 years. The reasons for the increase are unclear but many authors suggest that changes in the home environment favourable to proliferation of the house dust mite are to blame. Our study aimed to compare home environment of children with asthma and controls. A questionnaire on home environment was administered to the parents of 134 children with asthma and 118 controls. Detailed studies of home temperature, humidity and dust mite allergen (DERp1) levels were performed in 20 homes (10 from each group). The questionnaire response rate was 86%. There were no significant differences between asthma and control homes with respect to social class, type of housing, smoking habits, pets, insulation, home heating, bedding, carpeting and domestic cleaning habits. A first degree family history of atopy was obtained in 42% of asthmatic families and in 16% of controls. Temperature, humidity and dust mite allergen levels were similar in both groups. The majority gave readings exceeding recommended norms. Values for DERp1 were above thresholds by a factor of 5 in 48%. Home environment does not significantly differ in children with or without asthma. The home environment is now generally mite friendly, and large segments of the childhood population are now exposed to high levels of DERp1. This may account for the increasing prevalence as more and more children with an atopic background develop overt symptoms in response to high levels of allergen load.

  7. 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

  8. Research on the model of home networking

    Science.gov (United States)

    Yun, Xiang; Feng, Xiancheng

    2007-11-01

    It is the research hotspot of current broadband network to combine voice service, data service and broadband audio-video service by IP protocol to transport various real time and mutual services to terminal users (home). Home Networking is a new kind of network and application technology which can provide various services. Home networking is called as Digital Home Network. It means that PC, home entertainment equipment, home appliances, Home wirings, security, illumination system were communicated with each other by some composing network technology, constitute a networking internal home, and connect with WAN by home gateway. It is a new network technology and application technology, and can provide many kinds of services inside home or between homes. Currently, home networking can be divided into three kinds: Information equipment, Home appliances, Communication equipment. Equipment inside home networking can exchange information with outer networking by home gateway, this information communication is bidirectional, user can get information and service which provided by public networking by using home networking internal equipment through home gateway connecting public network, meantime, also can get information and resource to control the internal equipment which provided by home networking internal equipment. Based on the general network model of home networking, there are four functional entities inside home networking: HA, HB, HC, and HD. (1) HA (Home Access) - home networking connects function entity; (2) HB (Home Bridge) Home networking bridge connects function entity; (3) HC (Home Client) - Home networking client function entity; (4) HD (Home Device) - decoder function entity. There are many physical ways to implement four function entities. Based on theses four functional entities, there are reference model of physical layer, reference model of link layer, reference model of IP layer and application reference model of high layer. In the future home network

  9. Associations Between Home Death and the Use and Type of Care at Home.

    Science.gov (United States)

    McEwen, Rebecca; Asada, Yukiko; Burge, Frederick; Lawson, Beverley

    2018-01-01

    Despite wishes for and benefits of home deaths, a discrepancy between preferred and actual location of death persists. Provision of home care may be an effective policy response to support home deaths. Using the population-based mortality follow-back study conducted in Nova Scotia, we investigated the associations between home death and formal care at home and between home death and the type of formal care at home. We found (1) the use of formal care at home at the end of life was associated with home death and (2) the use of formal home support services at home was associated with home death among those whose symptoms were well managed.

  10. 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.

  11. 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.

  12. Nursing home and nursing home physician: the Dutch experience.

    NARCIS (Netherlands)

    Schols, J.M.G.A.; Crebolder, H.F.J.M.; Weel, C. van

    2004-01-01

    Dutch nursing home care today includes a broad range of institutional and outreaching care functions. Medical care is an essential part of this care. Nursing home medicine in The Netherlands has developed as an officially acknowledged medical specialty. This is unique because The Netherlands is the

  13. Family and home characteristics correlate with mold in homes

    Science.gov (United States)

    Previously, we demonstrated that infants exposed to higher Environmental Relative Moldiness Index (ERMI) value homes were more likely to develop asthma by age seven. The purpose of this analysis was to determine what family and home characteristics were associated with higher ER...

  14. Home geriatric physiological measurements

    International Nuclear Information System (INIS)

    Tamura, Toshiyo

    2012-01-01

    In an ageing society, the elderly can be monitored with numerous physiological, physical and passive devices. Sensors can be installed in the home for continuous mobility assistance and unobtrusive disease prevention. This review presents several modern sensors, which improve the quality of life and assist the elderly, disabled people and their caregivers. The main concept of geriatric sensors is that they are capable of providing assistance without limiting or disturbing the subject's daily routine, giving him or her greater comfort, pleasure and well-being. Furthermore, this review includes associated technologies of wearable/implantable monitoring systems and the ‘smart-house’ project. This review concludes by discussing future challenges of the future aged society. (topical review)

  15. Avatars@Home

    Science.gov (United States)

    Morandell, Martin M.; Hochgatterer, Andreas; Wöckl, Bernhard; Dittenberger, Sandra; Fagel, Sascha

    Avatars are a common field of research for interfacing smart homes, especially for elderly people. The present study focuses on the usage of photo-realistic faces with different levels of movements (video, avatar and photo) as components of the graphical user interface (GUI) for Ambient Assisted Living (AAL) environments. Within a usability test, using the "Wizard of Oz" technique, these presentation modes were compared with a text and a voice only interface with users of the target groups: elderly people with (nMCI=12) and without (nMCI=12) Mild Cognitive Impairment (MCI). Results show that faces on the GUI were liked by both, elderly with and without cognitive restrictions. However, users' performance on executing tasks did not differ much between the different presentation modes.

  16. Nursing Home Response to Nursing Home Compare: The Provider Perspective.

    Science.gov (United States)

    Perraillon, Marcelo Coca; Brauner, Daniel J; Konetzka, R Tamara

    2017-08-01

    Nursing Home Compare (NHC) publishes composite quality ratings of nursing homes based on a five-star rating system, a system that has been subject to controversy about its validity. Using in-depth interviews, we assess the views of nursing home administrators and staff on NHC and unearth strategies used to improve ratings. Respondents revealed conflicting goals and strategies. Although nursing home managers monitor the ratings and expend effort to improve scores, competing goals of revenue maximization and avoidance of litigation often overshadow desire to score well on NHC. Some of the improvement strategies simply involve coding changes that have no effect on resident outcomes. Many respondents doubted the validity of the self-reported staffing data and stated that lack of risk adjustment biases ratings. Policy makers should consider nursing home incentives when refining the system, aiming to improve the validity of the self-reported domains to provide incentives for broader quality improvement.

  17. Home Haemodialysis in Ireland.

    Science.gov (United States)

    Kennedy, C; Connaughton, D; Murray, S; Ormond, J; Butler, A; Phelan, E; Young, J; Durack, L; Flavin, J; O'Grady, M; O'Kelly, P; Lavin, P; Leavey, S; Lappin, D; Giblin, L; Casserly, L; Plant, W D; Conlon, P

    2017-12-20

    Home haemodialysis (HHD) has the potential to impact positively on patient outcomes and health resource management. There has been rejuvenated international interest in HHD in recent years. We aimed to review the activity and outcomes of the Irish HHD Programme since inception (2009-2016). Patient data were collected using the national electronic Renal Patient database (eMEDRenal version 3.2.1) and individual centre records. All data were recorded in a coded fashion on a Microsoft Excel Spreadsheet and analysed with Stata SE software. 101 patients completed training and commenced HHD; a further 45 patients were assessed for HHD suitability but did not ultimately dialyse at home. Twenty patients switched to nocturnal HHD when this resource became available. The switch from conventional in-centre dialysis to HHD led to an increase in the mean weekly hours on HD and a reduction in medication burden for the majority of patients. The overall rate of arteriovenous fistula (AVF) as primary vascular access was 62%. Most HHD complications were related to access function or access-related infection. Over the seven-years, 29 HHD patients were transplanted and nine patients died. No deaths resulted directly from a HHD complication or technical issue. Patient and technique survival rates compared favourably to published international reports. However, we identified several aspects that require attention. A small number of patients were receiving inadequate dialysis and require targeted education. Ongoing efforts to increase AVF and self-needling rates in HD units must continue. Psychosocial support is critical during the transition between dialysis modalities. © The Author 2017. Published by Oxford University Press on behalf of the Association of Physicians. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  18. Firewise Landscaping for Woodland Homes

    OpenAIRE

    Close, David

    2015-01-01

    A home in a woodland setting is surrounded by flammable vegetation. Firewise landscaping can help you create a defensible space or buffer zone around your home. This publication details landscaping zones which should be used when planning for fire protections and rates common landscaping plants by flammability.

  19. Home education: The social motivation

    Directory of Open Access Journals (Sweden)

    Christian W. Beck

    2010-10-01

    Full Text Available Data from a Norwegian survey show correlation between a student’s socially related problems at school and the parent’s social motivation for home education. I argue that more time spent at school by a student could result in more socially related problems at school, which can explain an increase in social motivation for home education.

  20. Home Schooling in Rural Nebraska.

    Science.gov (United States)

    Morgan, Robert L.; Cruzeiro, Patricia; Holz, Jan

    1999-01-01

    A 1996-97 survey of 40 home schooling families in rural Nebraska examined family characteristics, parents' social and political attitudes, the rationale for home schooling, curriculum and supplementary materials, children's opportunities for social experiences, rural characteristics, parents' educational attitudes, and support from extended…