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Sample records for dna cleavage activities

  1. Distinct mechanisms for DNA cleavage by myoglobin with a designed heme active center.

    PubMed

    Zhao, Yuan; Du, Ke-Jie; Gao, Shu-Qin; He, Bo; Wen, Ge-Bo; Tan, Xiangshi; Lin, Ying-Wu

    2016-03-01

    Heme proteins perform diverse biological functions, of which myoglobin (Mb) is a representative protein. In this study, the O2 carrier Mb was shown to cleave double stranded DNA upon aerobic dithiothreitol-induced reduction, which is fine-tuned by an additional distal histidine, His29 or His43, engineered in the heme active center. Spectroscopic (UV-vis and EPR) and inhibition studies suggested that free radicals including singlet oxygen and hydroxyl radical are responsible for efficient DNA cleavage via an oxidative cleavage mechanism. On the other hand, L29E Mb, with a distinct heme active center involving three water molecules in the met form, was found to exhibit an excellent DNA cleavage activity that was not depending on O2. Inhibition and ligation studies demonstrated for the first time that L29E Mb cleaves double stranded DNA into both the nicked circular and linear forms via a hydrolytic cleavage mechanism, which resembles native endonucleases. This study provides valuable insights into the distinct mechanisms for DNA cleavage by heme proteins, and lays down a base for creating artificial DNA endonucleases by rational design of heme proteins. Moreover, this study suggests that the diverse functions of heme proteins can be fine-tuned by rational design of the heme active center with a hydrogen-bonding network. PMID:26775281

  2. Specific detection of the cleavage activity of mycobacterial enzymes using a quantum dot based DNA nanosensor

    NASA Astrophysics Data System (ADS)

    Jepsen, Morten Leth; Harmsen, Charlotte; Godbole, Adwait Anand; Nagaraja, Valakunja; Knudsen, Birgitta R.; Ho, Yi-Ping

    2015-12-01

    We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes.We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes. Electronic supplementary information (ESI) available: Characterization of the QD-based DNA Nanosensor. See DOI: 10.1039/c5nr06326d

  3. Synthesis of isatin thiosemicarbazones derivatives: In vitro anti-cancer, DNA binding and cleavage activities

    NASA Astrophysics Data System (ADS)

    Ali, Amna Qasem; Teoh, Siang Guan; Salhin, Abdussalam; Eltayeb, Naser Eltaher; Khadeer Ahamed, Mohamed B.; Majid, A. M. S. Abdul

    New derivatives of thiosemicarbazone Schiff base with isatin moiety were synthesized L1-L6. The structures of these compounds were characterized based on the spectroscopic techniques. Compound L6 was further characterized by XRD single crystal. The interaction of these compounds with calf thymus (CT-DNA) exhibited high intrinsic binding constant (kb = 5.03-33.00 × 105 M-1) for L1-L3 and L5 and (6.14-9.47 × 104 M-1) for L4 and L6 which reflect intercalative activity of these compounds toward CT-DNA. This result was also confirmed by the viscosity data. The electrophoresis studies reveal the higher cleavage activity of L1-L3 than L4-L6. The in vitro anti-proliferative activity of these compounds against human colon cancer cell line (HCT 116) revealed that the synthesized compounds (L3, L6 and L2) exhibited good anticancer potency.

  4. Metal-based netropsin mimics showing AT-selective DNA binding and DNA cleavage activity at red light.

    PubMed

    Patra, Ashis K; Bhowmick, Tuhin; Ramakumar, Suryanarayanarao; Chakravarty, Akhil R

    2007-10-29

    Copper(II) bis-arginate [Cu(l-arg)2](NO3)2 (1) and [Cu(l-arg)(phen)Cl]Cl (2) as mimics of the minor-groove-binding natural antibiotic netropsin show preferential binding to the AT-rich region of double-stranded DNA. The complexes with a d-d band near 600 nm display oxidative DNA cleavage activity on photoirradiation at UV-A light of 365 nm and at red light of 647.1 nm (Ar-Kr laser) in a metal-assisted photoexcitation process forming singlet oxygen (1O2) species in a type-2 pathway. PMID:17880211

  5. A zinc site in the C-terminal domain of RAG1 is essential for DNA cleavage activity

    PubMed Central

    Gwyn, Lori M.; Peak, Mandy M.; De, Pallabi; Rahman, Negar S.; Rodgers, Karla K.

    2009-01-01

    The recombination activating protein, RAG1, a key component of the V(D)J recombinase, binds multiple Zn2+ ions in its catalytically-required core region. However, the role of zinc in the DNA cleavage activity of RAG1 is not well-resolved. To address this issue, we determined the stoichiometry of Zn2+ ions bound to the catalytically active core region of RAG1 under various conditions. Using metal quantitation methods, we determined that core RAG1 can bind up to four Zn2+ ions. Stripping the full complement of bound Zn2+ ions to produce apo-protein abrogated DNA cleavage activity. Moreover, even partial removal of zinc-binding equivalents resulted in a significant diminishment of DNA cleavage activity, as compared to holo-Zn2+ core RAG1. Mutants of the intact core RAG1 and the isolated core RAG1 domains were studied to identify the location of zinc-binding sites. Significantly, the C-terminal domain in core RAG1 binds at least two Zn2+ ions, with one zinc-binding site containing C902 and C907 as ligands (termed the CC zinc site) and H937 and H942 coordinating a Zn2+ ion in a separate site (HH zinc site). The latter zinc-binding site is essential for DNA cleavage activity, given that the H937A and H942A mutants were defective in both in vitro DNA cleavage assays and cellular recombination assays. Furthermore, as mutation of the active site residue E962 reduces Zn2+ coordination, we propose that the HH zinc site is located in close proximity to the DDE active site. Overall, these results demonstrate that Zn2+ serves an important auxiliary role for RAG1 DNA cleavage activity. Furthermore, we propose that one of the zinc-binding sites is linked to the active site of core RAG1 directly or indirectly by E962. PMID:19500590

  6. Photocytotoxic oxovanadium(IV) complexes showing light-induced DNA and protein cleavage activity.

    PubMed

    Sasmal, Pijus K; Saha, Sounik; Majumdar, Ritankar; Dighe, Rajan R; Chakravarty, Akhil R

    2010-02-01

    Oxovanadium(IV) complexes [VO(L)(B)]Cl(2) (1-3), where L is bis(2-benzimidazolylmethyl)amine and B is 1,10-phenanthroline (phen), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq) or dipyrido[3,2-a:2',3'-c]phenazine (dppz), have been prepared, characterized, and their photo-induced DNA and protein cleavage activity studied. The photocytotoxicity of complex 3 has been studied using adenocarcinoma A549 cells. The phen complex 1, structurally characterized by single-crystal X-ray crystallography, shows the presence of a vanadyl group in six-coordinate VON(5) coordination geometry. The ligands L and phen display tridentate and bidentate N-donor chelating binding modes, respectively. The complexes exhibit a d-d band near 740 nm in 15% DMF-Tris-HCl buffer (pH 7.2). The phen and dpq complexes display an irreversible cathodic cyclic voltammetric response near -0.8 V in 20% DMF-Tris-HCl buffer having 0.1 M KCl as supporting electrolyte. The dppz complex 3 exhibits a quasi-reversible voltammogram near -0.6 V (vs SCE) that is assignable to the V(IV)-V(III) couple. The complexes bind to calf thymus DNA giving binding constant values in the range of 6.6 x 10(4)-2.9 x 10(5) M(-1). The binding site size, thermal melting and viscosity binding data suggest DNA surface and/or groove binding nature of the complexes. The complexes show poor "chemical nuclease" activity in dark in the presence of 3-mercaptopropionic acid or hydrogen peroxide. The dpq and dppz complexes are efficient photocleavers of plasmid DNA in UV-A light of 365 nm via a mechanistic pathway that involves formation of both singlet oxygen and hydroxyl radicals. The complexes show significant photocleavage of DNA in near-IR light (>750 nm) via hydroxyl radical pathway. Among the three complexes, the dppz complex 3 shows significant BSA and lysozyme protein cleavage activity in UV-A light of 365 nm via hydroxyl radical pathway. The dppz complex 3 also exhibits photocytotoxicity in non-small cell lung carcinoma/human lung

  7. Tunable DNA cleavage activity promoted by copper(ii) ternary complexes with N-donor heterocyclic ligands.

    PubMed

    Bortolotto, T; Silva-Caldeira, P P; Pich, C T; Pereira-Maia, E C; Terenzi, H

    2016-06-01

    Several small molecules have the capacity to cleave DNA promptly at high yields, even under mild conditions. Usually, this activity has no constraints, occurring without external or user control. Here, we demonstrate that UV-light exposure can greatly enhance the DNA cleavage activity promoted by four ternary copper(ii) complexes. A remarkable photocontrolled activity was achieved, which may be interesting for chemical and biochemical applications. PMID:27168172

  8. Substitution of conserved residues within the active site alters the cleavage religation equilibrium of DNA topoisomerase I.

    PubMed

    Colley, William C; van der Merwe, Marie; Vance, John R; Burgin, Alex B; Bjornsti, Mary-Ann

    2004-12-24

    Eukaryotic DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of camptothecin (CPT). Mutation of conserved residues in close proximity to the active site tyrosine (Tyr(727) of yeast Top1p) alters the DNA cleavage religation equilibrium, inducing drug-independent cell lethality. Previous studies indicates that yeast Top1T722Ap and Top1N726Hp cytotoxicity results from elevated levels of covalent enzyme-DNA intermediates. Here we show that Top1T722Ap acts as a CPT mimetic by exhibiting reduced rates of DNA religation, whereas increased Top1N726Hp.DNA complexes result from elevated DNA binding and cleavage. We also report that the combination of the T722A and N726H mutations in a single protein potentiates the cytotoxic action of the enzyme beyond that induced by co-expression of the single mutants. Moreover, the addition of CPT to cells expressing the double top1T722A/N726H mutant did not enhance cell lethality. Thus, independent alterations in DNA cleavage and religation contribute to the lethal phenotype. The formation of distinct cytotoxic lesions was also evidenced by the different responses induced by low levels of these self-poisoning enzymes in isogenic strains defective for the Rad9 DNA damage checkpoint, processive DNA replication, or ubiquitin-mediated proteolysis. Substitution of Asn(726) with Phe or Tyr also produces self-poisoning enzymes, implicating stacking interactions in the increased kinetics of DNA cleavage by Top1N726Hp and Top1N726Fp. In contrast, replacing the amide side chain of Asn(726) with Gln renders Top1N726Qp resistant to CPT, suggesting that the orientation of the amide within the active site is critical for effective CPT binding. PMID:15489506

  9. DNA Cleavage and Condensation Activities of Mono- and Binuclear Hybrid Complexes and Regulation by Graphene Oxide.

    PubMed

    Li, Shuo; Dai, Mingxing; Zhang, Chunping; Jiang, Bingying; Xu, Junqiang; Zhou, Dewen; Gu, Zhongwei

    2016-01-01

    Hybrid complexes with N,N'-bis(2-benzimidazolylmethyl)amine and cyclen moieties are novel enzyme mimics and controlled DNA release materials, which could interact with DNA through three models under different conditions. In this paper, the interactions between plasmid DNA and seven different complexes were investigated, and the methods to change the interaction patterns by graphene oxide (GO) or concentrations were also investigated. The cleavage of pUC19 DNA promoted by target complexes were via hydrolytic or oxidative mechanisms at low concentrations ranging from 3.13 × 10(-7) to 6.25 × 10(-5) mol/L. Dinuclear complexes 2a and 2b can promote the cleavage of plasmid pUC19 DNA to a linear form at pH values below 7.0. Furthermore, binuclear hybrid complexes could condense DNA as nanoparticles above 3.13 × 10(-5) mol/L and partly release DNA by graphene oxide with π-π stacking. Meanwhile, the results also reflected that graphene oxide could prevent DNA from breaking down. Cell viability assays showed dinuclear complexes were safe to normal human hepatic cells at relative high concentrations. The present work might help to develop novel strategies for the design and synthesis of DNA controllable releasing agents, which may be applied to gene delivery and also to exploit the new application for GO. PMID:27428945

  10. Activation of an Mg2+-dependent DNA endonuclease of avian myeloblastosis virus alpha beta DNA polymerase by in vitro proteolytic cleavage.

    PubMed Central

    Grandgenett, D P; Golomb, M; Vora, A C

    1980-01-01

    Partial chymotryptic digestion of purified avian myeloblastosis virus alpha beta DNA polymerase resulted in the activation of a Mg2+-dependent DNA endonuclease activity. Incubation of the polymerase-protease mixture in the presence of super-coiled DNA and Mg2+ permitted detection of the cleaved polymerase fragment possessing DNA nicking activity. Protease digestion conditions were established permitting selective cleavage of beta to alpha, which contained DNA polymerase and RNase H activity and to a family of polypeptides ranging in size from 30,000 to 34,000 daltons. These latter beta-unique fragments were purified by polyuridylate-Sepharose 4B chromatography and were shown to contain both DNA binding and DNA endonuclease activities. We have demonstrated that this group of polymerase fragments derived by chymotryptic digestion of alpha beta DNA polymerase is similar to the in vivo-isolated avian myeloblastosis virus p32pol in size, sequence, and DNA endonuclease activity. Images PMID:6154149

  11. Efficient DNA cleavage mediated by mononuclear mixed ligand copper(II) phenolate complexes: the role of co-ligand planarity on DNA binding and cleavage and anticancer activity.

    PubMed

    Jaividhya, Paramasivam; Dhivya, Rajkumar; Akbarsha, Mohamad Abdulkadhar; Palaniandavar, Mallayan

    2012-09-01

    The new mononuclear copper(II) complexes [Cu(L)(H(2)O)(2)](+)1 and [Cu(L)(diimine)](+)2-6, where LH=2-[(2-dimethylaminoethylimino)methyl]phenol and diimine=2,2'-bipyridine (bpy) (2), or 1,10-phenanthroline (phen) (3), or dipyrido[3,2-f:2',3'-h]quinoxaline (dpq) (4) or dipyrido[3,2-a:2',3'-c]phenazine (dppz) (5) or 11,12-dimethyldipyrido[3,2-a:2',3'-c]phenazine (dmdppz) (6), have been isolated and characterized. The X-ray crystal structures of 2 contains the monomeric complex molecule with a trigonal bipyramidal distorted square pyramidal (TBPDSP) coordination geometry, while 4 and 6 with square pyramidal distorted trigonal bipyramidal (SPDTBP) coordination geometry. The amine nitrogen of -NMe(2) group of the tridentate primary ligand is located at one of the corners of the square plane in 2 and 6 but in the axial position in 4. The interaction of the complexes with calf thymus DNA has been investigated using UV-visible and fluorescence spectroscopy, and viscosity measurements to understand the effect of diimine co-ligands on the mode and extent of DNA binding. The complexes 4 and 5 interact with calf thymus DNA more strongly than the other complexes through partial intercalation of the extended planar ring of the dpq (4) and dppz (5) co-ligands with the DNA base stack. All the complexes, except 1, effect the double strand DNA cleavage of plasmid DNA and 5 cleaves plasmid DNA in the absence of a reductant at a concentration (40 μM) lower than 4. It is remarkable that all the complexes display cytotoxicity against human breast cancer cell lines (MCF-7) and human cervical epidermoid carcinoma cell lines (ME 180) with potency higher than the currently used chemotherapeutic agent cisplatin and that 5 exhibits cytotoxicity higher than the other complexes. PMID:22841366

  12. The activation of DNA damage detection and repair responses in cleavage-stage rat embryos by a damaged paternal genome.

    PubMed

    Grenier, Lisanne; Robaire, Bernard; Hales, Barbara F

    2012-06-01

    Male germ cell DNA damage, after exposure to radiation, exogenous chemicals, or chemotherapeutic agents, is a major cause of male infertility. DNA-damaged spermatozoa can fertilize oocytes; this is of concern because there is limited information on the capacity of early embryos to repair a damaged male genome or on the fate of these embryos if repair is inadequate. We hypothesized that the early activation of DNA damage response in the early embryo is a critical determinant of its fate. The objective of this study was to assess the DNA damage response and mitochondrial function as a measure of the energy supply for DNA repair and general health in cleavage-stage embryos sired by males chronically exposed to an anticancer alkylating agent, cyclophosphamide. Male rats were treated with saline or cyclophosphamide (6 mg/kg/day) for 4 weeks and mated to naturally cycling females. Pronuclear two- and eight-cell embryos were collected for immunofluorescence analysis of mitochondrial function and biomarkers of the DNA damage response: γH2AX foci, 53BP1 reactivity, and poly(ADP-ribose) polymer formation. Mitochondrial activities did not differ between embryos sired by control- and cyclophosphamide-exposed males. At the two-cell stage, there was no treatment-related increase in DNA double-strand breaks; by the eight-cell stage, a significant increase was noted, as indicated by increased medium and large γH2AX foci. This was accompanied by a dampened DNA repair response, detected as a decrease in the nuclear intensity of poly(ADP-ribose) polymers. The micronuclei formed in cyclophosphamide-sired embryos contained large γH2AX foci and enhanced poly(ADP-ribose) polymer and 53BP1 reactivity compared with their nuclear counterparts. Thus, paternal cyclophosphamide exposure activated a DNA damage response in cleavage-stage embryos. Furthermore, this damage response may be useful in assessing embryo quality and developmental competence. PMID:22454429

  13. Synthesis, crystal structure, DNA binding and photo-induced DNA cleavage activity of (S-methyl-L-cysteine)copper(II) complexes of heterocyclic bases.

    PubMed

    Patra, Ashis K; Nethaji, Munirathinam; Chakravarty, Akhil R

    2007-02-01

    Ternary S-methyl-L-cysteine (SMe-l-cys) copper(II) complexes [Cu(SMe-L-cys)(B)(H(2)O)](X) (1-4), where the heterocyclic base B is 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), dipyridoquinoxaline (dpq, 3) and dipyridophenazine (dppz, 4), and X is ClO(4)(-) (1-3) or NO(3)(-) (4), are prepared and their DNA binding and cleavage properties studied. Complexes 2 and 4 are structurally characterized by X-ray crystallography. Both the crystal structures show distorted square-pyramidal (4+1) CuN(3)O(2) coordination geometry of the complexes in which the N,O-donor S-methyl-L-cysteine and N,N-donor heterocyclic base bind at the basal plane with a water molecule as the axial ligand. In addition, the dppz structure shows the presence of a 1D-chain formed due to covalent linkage of the carboxylate oxygen atom belonging to another molecule at the elongated axial site. The crystal structures show chemically significant non-covalent interactions like hydrogen bonding involving the axial aqua ligand and pi-pi interactions between dppz ligands. The complexes display a d-d band in the range of 605-654 nm in aqueous dimethylformamide (DMF) solution (9:1 v/v). The redox active complexes show quasireversible cyclic voltammetric response near 0.1 V in DMF assignable to the Cu(II)/Cu(I) couple. The complexes show good binding affinity to calf thymus (CT) DNA giving the order: 4 (dppz)>3 (dpq)>2 (phen)>1 (bpy). The intrinsic binding constants, obtained from UV-visible spectroscopic studies, are 1.3x10(4) and 2.15 x 10(4) M(-1) for 3 and 4, respectively. Control DNA cleavage experiments using pUC19 supercoiled (SC) DNA and minor groove binder distamycin suggest major groove binding propensity for the dppz complex, while the phen and dpq complexes bind at the minor groove of DNA. Complexes 2-4 show DNA cleavage activity in dark in the presence of a reducing agent 3-mercaptopropionic acid (MPA) via a mechanistic pathway involving formation of hydroxyl radical as the reactive

  14. RNA-activated DNA cleavage by the Type III-B CRISPR-Cas effector complex.

    PubMed

    Estrella, Michael A; Kuo, Fang-Ting; Bailey, Scott

    2016-02-15

    The CRISPR (clustered regularly interspaced short palindromic repeat) system is an RNA-guided immune system that protects prokaryotes from invading genetic elements. This system represents an inheritable and adaptable immune system that is mediated by multisubunit effector complexes. In the Type III-B system, the Cmr effector complex has been found to cleave ssRNA in vitro. However, in vivo, it has been implicated in transcription-dependent DNA targeting. We show here that the Cmr complex from Thermotoga maritima can cleave an ssRNA target that is complementary to the CRISPR RNA. We also show that binding of a complementary ssRNA target activates an ssDNA-specific nuclease activity in the histidine-aspartate (HD) domain of the Cmr2 subunit of the complex. These data suggest a mechanism for transcription-coupled DNA targeting by the Cmr complex and provide a unifying mechanism for all Type III systems. PMID:26848046

  15. The cleavage of nuclear DNA into high molecular weight DNA fragments occurs not only during apoptosis but also accompanies changes in functional activity of the nonapoptotic cells.

    PubMed

    Solov'yan, V T; Andreev, I O; Kolotova, T Y; Pogribniy, P V; Tarnavsky, D T; Kunakh, V A

    1997-08-25

    In this paper we demonstrate that apoptosis in primary culture of murine thymocytes and in continuously growing human cells is associated with the progressive disintegration of nuclear DNA into high molecular weight (HMW)-DNA fragments of about 50-150 kb. We also show that the formation of similarly sized HMW-DNA fragments takes place in the same cells in the absence of apoptotic inducers. Unlike an apoptotic fragmentation of nuclear DNA, the formation of HMW-DNA fragments in nonapoptotic cells is rapidly induced, has no correlation with the cell death, and is not associated with the development of oligonucleosomal "ladder" or apoptotic changes in nuclear morphology. The disintegration of DNA into HMW-fragments is also observed in nuclei isolated from healthy, nonapoptosizing tissues of various eukaryotes. We show that the formation of HMW-DNA fragments in the absence of apoptotic inducers is strongly dependent on the ionic detergents, is responsive to the topoisomerase II-specific poison, teniposide, and is completely reversible under conditions that favor topoisomerase II-dependent rejoining reaction. Also, we demonstrate that the formation of HMW-DNA fragments in continuously growing cell lines caused either by serum deprivation or monolayer establishment is of a transient nature and rapidly reverses to the control level following serum addition or dilution of monolayer. The results suggest that the cleavage of nuclear DNA into HMW-DNA fragments is associated not only with apoptosis but also accompanies changes in functional activity of nonapoptotic cells. PMID:9281361

  16. Enhancing Cell Nucleus Accumulation and DNA Cleavage Activity of Anti-Cancer Drug via Graphene Quantum Dots

    NASA Astrophysics Data System (ADS)

    Wang, Chong; Wu, Congyu; Zhou, Xuejiao; Han, Ting; Xin, Xiaozhen; Wu, Jiaying; Zhang, Jingyan; Guo, Shouwu

    2013-10-01

    Graphene quantum dots (GQDs) maintain the intrinsic layered structural motif of graphene but with smaller lateral size and abundant periphery carboxylic groups, and are more compatible with biological system, thus are promising nanomaterials for therapeutic applications. Here we show that GQDs have a superb ability in drug delivery and anti-cancer activity boost without any pre-modification due to their unique structural properties. They could efficiently deliver doxorubicin (DOX) to the nucleus through DOX/GQD conjugates, because the conjugates assume different cellular and nuclear internalization pathways comparing to free DOX. Also, the conjugates could enhance DNA cleavage activity of DOX markedly. This enhancement combining with efficient nuclear delivery improved cytotoxicity of DOX dramatically. Furthermore, the DOX/GQD conjugates could also increase the nuclear uptake and cytotoxicity of DOX to drug-resistant cancer cells indicating that the conjugates may be capable to increase chemotherapy efficacy of anti-cancer drugs that are suboptimal due to the drug resistance.

  17. DNA Cleavage, Cytotoxic Activities, and Antimicrobial Studies of Ternary Copper(II) Complexes of Isoxazole Schiff Base and Heterocyclic Compounds.

    PubMed

    Chityala, Vijay Kumar; Sathish Kumar, K; Macha, Ramesh; Tigulla, Parthasarathy; Shivaraj

    2014-01-01

    Novel mixed ligand bivalent copper complexes [Cu. L. A. ClO 4 ] and [Cu. L. A] where "L" is Schiff bases, namely 2-((3,4-dimethylisoxazol-5-ylimino)methyl)-4-bromophenol (DMIIMBP)/2-((3,4-dimethylisoxazol-5-ylimino)methyl)-4-chlorophenol (DMIIMCP), and "A" is heterocyclic compound, such as 1,10-phenanthroline (phen)/2,2(1)-bipyridyl (bipy)/8-hydroxyquinoline (oxine)/5-chloro-8-hydroxyquinoline (5-Cl-oxine), have been synthesized. These complexes have been characterized by IR, UV-Vis, ESR, elemental analysis, magnetic moments, TG, and DTA. On the basis of spectral studies and analytical data, five-coordinated square pyramidal/four-coordinated square planar geometry is assigned to all complexes. The ligands and their ternary complexes with Cu(II) have been screened for antimicrobial activity against bacteria and fungi by paper disc method. The antimicrobial studies of Schiff bases and their metal complexes showed significant activity and further it is observed that the metal complexes showed more activity than corresponding Schiff bases. In vitro antitumor activity of Cu(II) complexes was assayed against human cervical carcinoma (HeLa) cancer cells and it was observed that few complexes exhibit good antitumor activity on HeLa cell lines. The DNA cleavage studies have also been carried out on pBR 322 and it is observed that these Cu(II) complexes are capable of cleaving supercoiled plasmid DNA in the presence of H2O2 and UV light. PMID:24895493

  18. Mixed ligand ruthenium(III) complexes of benzaldehyde 4-methyl-3-thiosemicarbazones with triphenylphosphine/triphenylarsine co-ligands: Synthesis, DNA binding, DNA cleavage, antioxidative and cytotoxic activity

    NASA Astrophysics Data System (ADS)

    Sampath, K.; Sathiyaraj, S.; Raja, G.; Jayabalakrishnan, C.

    2013-08-01

    The new ruthenium(III) complexes with 4-methyl-3-thiosemicarbazone ligands, (E)-2-(2-chlorobenzylidene)-N-methylhydrazinecarbothioamide (HL1) and (E)-2-(2-nitrobenzylidene)-N-methylhydrazinecarbothioamide (HL2), were prepared and characterized by various physico-chemical and spectroscopic methods. The title compounds act as bidentate, monobasic chelating ligands with S and N as the donor sites and are preferably found in the thiol form in all the complexes studied. The molecular structure of HL1 and HL2 were determined by single crystal X-ray diffraction method. DNA binding of the ligands and complexes were investigated by absorption spectroscopy and IR spectroscopy. It reveals that the compounds bind to nitrogenous bases of DNA via intercalation. The oxidative cleavage of the complexes with CT-DNA inferred that the effects of cleavage are dose dependent. Antioxidant study of the ligands and complexes showed the significant antioxidant activity against DPPH radical. In addition, the in vitro cytotoxicity of the ligands and complexes against MCF-7 cell line was assayed which showed higher cytotoxic activity with the lower IC50 values indicating their efficiency in killing the cancer cells even at low concentrations.

  19. Evaluation of DNA-binding, DNA cleavage, antioxidant and cytotoxic activity of mononuclear ruthenium(II) carbonyl complexes of benzaldehyde 4-phenyl-3-thiosemicarbazones

    NASA Astrophysics Data System (ADS)

    Sampath, Krishnan; Sathiyaraj, Subbaiyan; Jayabalakrishnan, Chinnasamy

    2013-11-01

    Two 4-phenyl-3-thiosemicarbazone ligands, (E)-2-(2-chlorobenzylidene)-N-phenylhydrazinecarbothioamide (HL1) and (E)-2-(2-nitrobenzylidene)-N-phenylhydrazinecarbothioamide (HL2), and its ruthenium(II) complexes were synthesized and characterized by physico-chemical and spectroscopic methods. The Schiff bases act as bidentate, monobasic chelating ligands with S and N as the donor sites and are preferably found in the thiol form in all the complexes studied. The molecular structure of HL1 and HL2 were determined by single crystal X-ray diffraction method. DNA binding of the compounds was investigated by absorption spectroscopy which indicated that the compounds bind to DNA via intercalation. The oxidative cleavage of the complexes with CT-DNA inferred that the effects of cleavage are dose dependent. Antioxidant study of the ligands and complexes showed significant antioxidant activity against DPPH radical. In addition, the in vitro cytotoxicity of the ligands and complexes assayed against HeLa and MCF-7 cell lines showed higher cytotoxic activity with the lower IC50 values indicating their efficiency in killing the cancer cells even at low concentrations.

  20. Evaluation of DNA-binding, DNA cleavage, antioxidant and cytotoxic activity of mononuclear ruthenium(II) carbonyl complexes of benzaldehyde 4-phenyl-3-thiosemicarbazones.

    PubMed

    Sampath, Krishnan; Sathiyaraj, Subbaiyan; Jayabalakrishnan, Chinnasamy

    2013-11-01

    Two 4-phenyl-3-thiosemicarbazone ligands, (E)-2-(2-chlorobenzylidene)-N-phenylhydrazinecarbothioamide (HL(1)) and (E)-2-(2-nitrobenzylidene)-N-phenylhydrazinecarbothioamide (HL(2)), and its ruthenium(II) complexes were synthesized and characterized by physico-chemical and spectroscopic methods. The Schiff bases act as bidentate, monobasic chelating ligands with S and N as the donor sites and are preferably found in the thiol form in all the complexes studied. The molecular structure of HL(1) and HL(2) were determined by single crystal X-ray diffraction method. DNA binding of the compounds was investigated by absorption spectroscopy which indicated that the compounds bind to DNA via intercalation. The oxidative cleavage of the complexes with CT-DNA inferred that the effects of cleavage are dose dependent. Antioxidant study of the ligands and complexes showed significant antioxidant activity against DPPH radical. In addition, the in vitro cytotoxicity of the ligands and complexes assayed against HeLa and MCF-7 cell lines showed higher cytotoxic activity with the lower IC50 values indicating their efficiency in killing the cancer cells even at low concentrations. PMID:23845986

  1. Synthesis, characterization, DNA interactions, DNA cleavage, radical scavenging activity, antibacterial, anti-proliferative and docking studies of new transition metal complexes.

    PubMed

    Chennam, Kishan Prasad; Ravi, Mudavath; Ushaiah, B; Srinu, V; Eslavath, Ravi Kumar; Devi, Ch Sarala

    2016-01-01

    The compound N-(2-hydroxybenzylidene)-1-ethyl-1, 4-dihydro-7-methyl-4-oxo-1, 8 naphthyridine-3-carbohydrazide (LH) and its Cu (II), Co (II) and Zn (II) complexes were synthesized and characterized. The absorption spectral titrations and competitive DNA binding studies depicted those complexes of title compound bind to CT-DNA through intercalation. Interestingly [Cu (II)-(L2)] showed relatively high binding constant value (6.61 x 10(5) M(-1)) compared to [Co (II)-(L2)] (4.378× 10(5) M(-1)) and [Zn (II)-(L2)] (3.1x10(5) M(-1)). Ligand and its complexes were also examined for DNA nuclease activity against pBR-322 plasmid DNA, which showed that [Cu (II)-(L2)] had the best hydrolytic cleavage property displaying prominent double-strand DNA cleavage. In addition, antioxidant activities of the ligand and its metal complexes investigated through scavenging effects for DPPH radical in- vitro, indicated their potentiality as good antioxidants. The in vitro anti-bacterial study inferred the better anti-bacterial activity of [Cu (II)-(L2)] and this was also correlated theoretically by employing docking studies wherein [Cu (II)-(L2)] displayed good Gold score and Chem score. Finally the in vitro anti- proliferative activity of studied compounds was tested against HeLa and MCF-7 cell lines. Interestingly [Cu (II)-(L2)] displayed lower IC50 value and lower percentage of viability in both HeLa and MCF-7 cell lines. PMID:26545354

  2. Synthesis, structure, DNA binding and DNA cleavage activity of oxovanadium(IV) N-salicylidene-S-methyldithiocarbazate complexes of phenanthroline bases.

    PubMed

    Sasmal, Pijus K; Patra, Ashis K; Chakravarty, Akhil R

    2008-07-01

    Ternary oxovanadium(IV) complexes [VO(salmdtc)(B)] (1-3), where salmdtc is dianionic N-salicylidene-S-methyldithiocarbazate and B is N,N-donor phenanthroline bases like 1,10-phenanthroline (phen, 1), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 2) and dipyrido[3,2-a:2',3'-c]phenazine (dppz, 3), are prepared, characterized and their DNA binding and DNA cleavage activity studied. Complex 3 is structurally characterized by single-crystal X-ray crystallography. The molecular structure shows the presence of a vanadyl group in six-coordinate VN(3)O(2)S coordination geometry. The S-methyldithiocarbazate Schiff base acts as a tridentate NSO-donor ligand in a meridional binding mode. The N,N-donor heterocyclic base displays a chelating mode of binding with an N-donor site trans to the vanadyl oxo-group. The complexes show a d-d band in the range of 675-707 nm in DMF. They exhibit an irreversible oxidative cyclic voltammetric response near 0.9 V due to the V(V)/V(IV) couple and a quasi-reversible reductive V(IV)/V(III) redox couple near -1.0 V vs. SCE in DMF-0.1M TBAP. The complexes show good binding propensity to calf thymus DNA giving binding constant values in the range of 7.4 x 10(4)-2.3 x 10(5)M(-1). The thermal denaturation and viscosity binding data suggest DNA surface and/or groove binding nature of the complexes. The complexes show poor chemical nuclease activity in dark in the presence of 3-mercaptopropionic acid (MPA) or hydrogen peroxide. The dpq and dppz complexes show efficient DNA cleavage activity in UV-A light of 365 nm via a type-II mechanistic pathway involving formation of singlet oxygen ((1)O(2)) as the reactive species. PMID:18279964

  3. Copper complexes based on chiral Schiff-base ligands: DNA/BSA binding ability, DNA cleavage activity, cytotoxicity and mechanism of apoptosis.

    PubMed

    Zhou, Xue-Quan; Li, Yang; Zhang, Dong-Yan; Nie, Yan; Li, Zong-Jin; Gu, Wen; Liu, Xin; Tian, Jin-Lei; Yan, Shi-Ping

    2016-05-23

    Four copper(II) complexes with chiral Schiff-base ligands, [Cu(R-L(1))2]·EtOAc (1) and [Cu(S-L(1))2]·EtOAc (2), [Cu(R-L(2))2]·EtOAc (3) and [Cu(S-L(2))2]·EtOAc (4), (R/S-HL(1) = (R/S)-(1-naththyl)-salicylaldimine, R/S-HL(2) = (R/S)-(1-naththyl)-3-methoxysalicylaldimine, EtOAc = ethyl acetate) were synthesized to serve as artificial nucleases and anticancer drugs. All complexes and R/S-HL(1) ligands were structurally characterized by X-ray crystallography. The interaction of these complexes with CT-DNA was researched via several spectroscopy methods, which indicates that complexes bind to CT-DNA by moderate intercalation binding mode. Moreover, DNA cleavage experiments revealed that the complexes exhibited remarkable DNA cleavage activities in the presence of H2O2via the generation of hydroxyl radical. Particularly, complex 4 also could nick DNA with the production of (1)O2. And all complexes exhibited excellent cytotoxicity to MDA-MB-231, A549 and Hela human cancer cells in micromole magnitude. Furthermore, complex 4 exhibited comparable cytotoxic effect to cisplatin against the proliferation of MDA-MB-231 and A549 cancer cells, as well as showed better anticancer ability to the three cancer cells than the other complexes. The results of cell cycle analysis indicated that complexes 3-4 could induce G2/M phase cell cycle arrest. Furthermore, MDA-MB-231 cells treated with 3 and 4 were subjected to apoptosis and death by generation of ROS and the activation of caspase-3. Interestingly, the chiral complexes 3 and 4 may induce cell apoptosis through extrinsic and mitochondrial intrinsic pathway, respectively. PMID:26994692

  4. Evaluation of DNA binding, DNA cleavage, protein binding, radical scavenging and in vitro cytotoxic activities of ruthenium(II) complexes containing 2,4-dihydroxy benzylidene ligands.

    PubMed

    Mohanraj, Maruthachalam; Ayyannan, Ganesan; Raja, Gunasekaran; Jayabalakrishnan, Chinnasamy

    2016-12-01

    The new ruthenium(II) complexes with hydrazone ligands, 4-Methyl-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL(1)), 4-Methoxy-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL(2)), 4-Bromo-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL(3)), were synthesized and characterized by various spectro analytical techniques. The molecular structures of the ligands were confirmed by single crystal X-ray diffraction technique. The DNA binding studies of the ligands and complexes were examined by absorption, fluorescence, viscosity and cyclic voltammetry methods. The results indicated that the ligands and complexes could interact with calf thymus DNA (CT-DNA) through intercalation. The DNA cleavage activity of the complexes was evaluated by gel electrophoresis assay, which revealed that the complexes are good DNA cleaving agents. The binding interaction of the ligands and complexes with bovine serum albumin (BSA) was investigated using fluorescence spectroscopic method. Antioxidant studies showed that the complexes have a strong radical scavenging properties. Further, the cytotoxic effect of the complexes examined on cancerous cell lines showed that the complexes exhibit significant anticancer activity. PMID:27612830

  5. DNA Cleavage, Cytotoxic Activities, and Antimicrobial Studies of Ternary Copper(II) Complexes of Isoxazole Schiff Base and Heterocyclic Compounds

    PubMed Central

    Chityala, Vijay Kumar; Sathish Kumar, K.; Macha, Ramesh; Tigulla, Parthasarathy; Shivaraj

    2014-01-01

    Novel mixed ligand bivalent copper complexes [Cu. L. A. ClO4] and [Cu. L. A] where “L” is Schiff bases, namely 2-((3,4-dimethylisoxazol-5-ylimino)methyl)-4-bromophenol (DMIIMBP)/2-((3,4-dimethylisoxazol-5-ylimino)methyl)-4-chlorophenol (DMIIMCP), and “A” is heterocyclic compound, such as 1,10-phenanthroline (phen)/2,21-bipyridyl (bipy)/8-hydroxyquinoline (oxine)/5-chloro-8-hydroxyquinoline (5-Cl-oxine), have been synthesized. These complexes have been characterized by IR, UV-Vis, ESR, elemental analysis, magnetic moments, TG, and DTA. On the basis of spectral studies and analytical data, five-coordinated square pyramidal/four-coordinated square planar geometry is assigned to all complexes. The ligands and their ternary complexes with Cu(II) have been screened for antimicrobial activity against bacteria and fungi by paper disc method. The antimicrobial studies of Schiff bases and their metal complexes showed significant activity and further it is observed that the metal complexes showed more activity than corresponding Schiff bases. In vitro antitumor activity of Cu(II) complexes was assayed against human cervical carcinoma (HeLa) cancer cells and it was observed that few complexes exhibit good antitumor activity on HeLa cell lines. The DNA cleavage studies have also been carried out on pBR 322 and it is observed that these Cu(II) complexes are capable of cleaving supercoiled plasmid DNA in the presence of H2O2 and UV light. PMID:24895493

  6. Quantification of DNA cleavage specificity in Hi-C experiments

    PubMed Central

    Meluzzi, Dario; Arya, Gaurav

    2016-01-01

    Hi-C experiments produce large numbers of DNA sequence read pairs that are typically analyzed to deduce genomewide interactions between arbitrary loci. A key step in these experiments is the cleavage of cross-linked chromatin with a restriction endonuclease. Although this cleavage should happen specifically at the enzyme's recognition sequence, an unknown proportion of cleavage events may involve other sequences, owing to the enzyme's star activity or to random DNA breakage. A quantitative estimation of these non-specific cleavages may enable simulating realistic Hi-C read pairs for validation of downstream analyses, monitoring the reproducibility of experimental conditions and investigating biophysical properties that correlate with DNA cleavage patterns. Here we describe a computational method for analyzing Hi-C read pairs to estimate the fractions of cleavages at different possible targets. The method relies on expressing an observed local target distribution downstream of aligned reads as a linear combination of known conditional local target distributions. We validated this method using Hi-C read pairs obtained by computer simulation. Application of the method to experimental Hi-C datasets from murine cells revealed interesting similarities and differences in patterns of cleavage across the various experiments considered. PMID:26264668

  7. In vitro cytotoxicity, DNA cleavage and SOD-mimic activity of copper(II) mixed-ligand quinolinonato complexes.

    PubMed

    Buchtík, Roman; Trávníček, Zdeněk; Vančo, Ján

    2012-11-01

    Six mixed-ligand copper(II) complexes with the composition [Cu(qui)(L)]BF(4)·xH(2)O (1-6), where Hqui=2-phenyl-3-hydroxy-4(1H)-quinolinone, L=2,2'-bipyridine (bpy) (1), 1,10-phenanthroline (phen) (2), bis(2-pyridyl)amine (ambpy) (3), 5-methyl-1,10-phenanthroline (mphen) (4), 5-nitro-1,10-phenanthroline (nphen) (5) and bathophenanthroline (bphen) (6), were prepared, fully characterized and studied for their in vitro cytotoxicity on human osteosarcoma (HOS) and human breast adenocarcinoma (MCF7) cancer cell lines. The overall promising results of the cytotoxicity were found for all the complexes, while the best results were achieved for complex 6, with IC(50)=2.6 ± 0.8 μM (HOS), and 1.3 ± 0.5 μM (MCF7). The interactions of the Cu(II) complexes 1-6 with calf thymus DNA were investigated by the UV-visible spectral titration. An agarose-gel electrophoretic method of oxidative damage determination to circular plasmid pUC19 was used to assess the ability of the complexes to act as chemical nucleases. A high effectiveness of DNA cleavage was observed for 2, 4 and 5. In vitro antioxidative activity of the complexes was studied by the superoxide dismutase-mimic (SOD-mimic) method. The best result was afforded by complex 1 with IC(50)=4.7 ± 1.0 μM, which corresponds to 10.2% of the native Cu,Zn-SOD enzyme activity. The ability of the tested complexes to interact with sulfur-containing biomolecules (cysteine and reduced glutathione) at physiological levels was proved by electrospray-ionization mass spectrometry (ESI-MS). PMID:23022693

  8. Sequence specificity of DNA cleavage by Micrococcus luteus. gamma. endonuclease

    SciTech Connect

    Hentosh, P.; Henner, W.D.; Reynolds, R.J.

    1985-04-01

    DNA fragments of defined sequence have been used to determine the sites of cleavage by ..gamma..-endonuclease activity in extracts prepared from Micrococcus luteus. End-labeled DNA restriction fragments of pBR322 DNA that had been irradiated under nitrogen in the presence of potassium iodide or t-butanol were treated with M. luteus ..gamma.. endonuclease and analyzed on irradiated DNA preferentially at the positions of cytosines and thymines. DNA cleavage occurred immediately to the 3' side of pyrimidines in irradiated DNA and resulted in fragments that terminate in a 5'-phosphoryl group. These studies indicate that both altered cytosines and thymines may be important DNA lesions requiring repair after exposure to ..gamma.. radiation.

  9. Synthesis, Characterization, and Biological Activities of Pendant Arm-Pyridyltetrazole Copper(II) Complexes: DNA Binding/Cleavage Activity and Cytotoxic Studies.

    PubMed

    Mustafa, Shaik; Rao, Bommuluri Umamaheswara; Surendrababu, Manubolu Surya; Raju, Kalidindi Krishnam; Rao, Gollapalli Nageswara

    2015-10-01

    2-(1H-Tetrazol-5-yl)pyridine (L) has been reacted separately with Me2NCH2CH2Cl⋅HCl and ClCH2CH2OH to yield two regioisomers in each case, N,N-dimethyl-2-[5-(pyridin-2-yl)-1H-tetrazol-1-yl]ethanamine (L1)/N,N-dimethyl-2-[5-(pyridin-2-yl)-2H-tetrazol-2-yl]ethanamine (L2) and 2-[5-(pyridin-2-yl)-1H-tetrazol-1-yl]ethanol (L3)/2-[5-(pyridin-2-yl)-2H-tetrazol-2-yl]ethanol (L4), respectively. These ligands, L1-L4, have been coordinated with CuCl2 ⋅H2O in 1 : 1 composition to furnish the corresponding complexes 1-4. EPR Spectra of Cu complexes 1 and 3 were characteristic of square planar geometry, with nuclear hyperfine spin 3/2. Single X-ray crystallographic studies of 3 revealed that the Cu center has a square planar structure. DNA binding studies were carried out by UV/VIS absorption; viscosity and thermal denaturation studies revealed that each of these complexes are avid binders of calf thymus DNA. Investigation of nucleolytic cleavage activities of the complexes was carried out on double-stranded pBR322 circular plasmid DNA by using a gel electrophoresis experiment under various conditions, where cleavage of DNA takes place by oxidative free-radical mechanism (OH(⋅)). In vitro anticancer activities of the complexes against MCF-7 (human breast adenocarcinoma) cells revealed that the complexes inhibit the growth of cancer cells. The IC50 values of the complexes showed that Cu complexes exhibit comparable cytotoxic activities compared to the standard drug cisplatin. PMID:26460557

  10. Synthesis and characterization, antimicrobial activity, DNA binding and DNA cleavage studies of new 5-chloro-2-[4-phenylthiazol-2-yl-iminomethyl]phenol metal complexes

    NASA Astrophysics Data System (ADS)

    Alaghaz, Abdel-Nasser M. A.; Zayed, Mohamed E.; Alharbi, Suliman A.

    2015-02-01

    New Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II) and Cd(II) complexes derived from bidentate Schiff base ligand, 5-chloro-2-[4-phenylthiazol-2-yl-iminomethyl]phenol (HL) have been synthesized. The molar ratio for all synthesized complexes is M: L = 1:2 which was established from the results of chemical analysis. The complexes have been characterized by elemental analysis, spectral (IR, UV-Vis, (1H and 13C) NMR, mass, ESR, XRD, CV, fluorescence, and magnetic as well as thermal analysis measurements. The IR spectra of the prepared complexes were suggested that the Schiff base ligand behaves as a bi-dentate ligand through the azomethine nitrogen atom and phenolic oxygen atom. The crystal field splitting, Racah repulsion and nepheloauxetic parameters and determined from the electronic spectra of the complexes. The presence of co-ordinated water molecules were confirmed by thermal studies. The spectroscopic studies suggest the octahedral geometry. From the modeling studies, the bond length, bond angle, core-core interaction, heat of formation, electronic energy, binding energy, HOMO, LUMO and dipole moment had been calculated to confirm the geometry of the ligand and their investigated complexes. Also, the thermal behavior and the kinetic parameters of degradation were determined using Coats-Redfern, Horowitz-Metzger and Piloyan-Novikova methods. Moreover, the in vitro antibacterial studies of all compounds screened against pathogenic bacteria (two Gram +ve and three Gram -ve) and three antifungal to assess their inhibiting potential. The assay indicated that the inhibition potential is metal ion dependent. The interaction of the complexes with calf thymus DNA (CT-DNA) has been investigated by UV absorption method, and the mode of CT-DNA binding to the complexes has been explored. Furthermore, the DNA cleavage activity by the complexes was performed.

  11. Synthesis, DNA binding and cleavage activities of copper (II) thiocyanate complex with 4-( N, N-dimethylamino)pyridine and N, N-dimethylformamide

    NASA Astrophysics Data System (ADS)

    Chen, Feng-juan; Xu, Min; Xi, Pin-xian; Liu, Hong-yang; Zeng, Zheng-zhi

    2011-10-01

    Two novel copper(II) thiocyanate complexes with 4-( N, N-dimethylamino) pyridine and N, N-dimethylformamide( 1) and with4-( N, N-dimethylamino) pyridine ( 2) have been synthesized and characterized. The crystal and molecular structures of complexes 1 and 2 were determined by single-crystal X-ray diffraction. Antioxidative activity tests in vitro showed that complex 1 has significant antioxidative activity against hydroxyl free radicals from the Fenton reaction and also oxygen free radicals, which is better than standard antioxidants like vitamin C and mannitol. The interaction of complex 1 with calf thymus DNA was investigated by spectroscopic, cyclic voltammetry, and viscosity measurements. Results suggest that complex 1 can bind to DNA via partial intercalation mode. Moreover, complex 1 has been found to cleavage of plasmid DNA pBR322.

  12. Synthesis, DNA binding and cleavage activities of copper (II) thiocyanate complex with 4-(N,N-dimethylamino)pyridine and N,N-dimethylformamide.

    PubMed

    Chen, Feng-juan; Xu, Min; Xi, Pin-xian; Liu, Hong-yang; Zeng, Zheng-zhi

    2011-10-15

    Two novel copper(II) thiocyanate complexes with 4-(N,N-dimethylamino) pyridine and N,N-dimethylformamide (1) and with 4-(N,N-dimethylamino) pyridine (2) have been synthesized and characterized. The crystal and molecular structures of complexes 1 and 2 were determined by single-crystal X-ray diffraction. Antioxidative activity tests in vitro showed that complex 1 has significant antioxidative activity against hydroxyl free radicals from the Fenton reaction and also oxygen free radicals, which is better than standard antioxidants like vitamin C and mannitol. The interaction of complex 1 with calf thymus DNA was investigated by spectroscopic, cyclic voltammetry, and viscosity measurements. Results suggest that complex 1 can bind to DNA via partial intercalation mode. Moreover, complex 1 has been found to cleavage of plasmid DNA pBR322. PMID:21723777

  13. A mechanistic approach for the DNA binding of chiral enantiomeric L- and D-tryptophan-derived metal complexes of 1,2-DACH: cleavage and antitumor activity.

    PubMed

    Arjmand, Farukh; Muddassir, Mohd

    2011-03-01

    A new chiral series of potential antitumor metal-based complexes 1-3(a and b) of L- and D-tryptophan have been synthesized and thoroughly characterized. Both enantiomers of 1-3 bind DNA noncovalently via phosphate interaction with slight preference of metal center for covalent coordination to nucleobases. The K(b) values of L-enantiomer, however, possess higher propensity for DNA binding in comparison with the D-enantiomeric analogs. The relative trend in K(b) values is as follows: 2(a) > 2(b) > 3(a) > 1(a) > 3(b) > 1(b). These observations together with the findings of circular dichoric and fluorescence studies reveal maximal potential of L-enantiomeric form of copper complex to bind DNA, thereby exerting its therapeutic effect. The complex 2a exhibits a remarkable DNA cleavage activity with pBR322DNA in the presence of different activators such as H(2) O(2) , ascorbic acid, 3-mercaptopropionic acid, and glutathione, suggesting the involvement of active oxygen species for the DNA scission. In vitro anticancer activity of complexes 1-3(a) were screened against 14 different human carcinoma cell lines of different histological origin, and the results reveal that 2a shows significant antitumor activity in comparison with both 1a and 3a and is particularly selective for MIAPACA2 (pancreatic cancer cell line). PMID:20928895

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  16. Highly sensitive fluorescence assay of DNA methyltransferase activity via methylation-sensitive cleavage coupled with nicking enzyme-assisted signal amplification.

    PubMed

    Zhao, Yongxi; Chen, Feng; Wu, Yayan; Dong, Yanhua; Fan, Chunhai

    2013-04-15

    Herein, using DNA adenine methylation (Dam) methyltransferase (MTase) as a model analyte, a simple, rapid, and highly sensitive fluorescence sensing platform for monitoring the activity and inhibition of DNA MTase was developed on the basis of methylation-sensitive cleavage and nicking enzyme-assisted signal amplification. In the presence of Dam MTase, an elaborately designed hairpin probe was methylated. With the help of methylation-sensitive restriction endonuclease DpnI, the methylated hairpin probe could be cleaved to release a single-stranded DNA (ssDNA). Subsequently, this released ssDNA would hybridize with the molecular beacon (MB) to open its hairpin structure, resulting in the restoration of fluorescence signal as well as formation of the double-stranded recognition site for nicking enzyme Nt.BbvCI. Eventually, an amplified fluorescence signal was observed through the enzymatic recycling cleavage of MBs. Based on this unique strategy, a very low detection limit down to 0.06 U/mL was achieved within a short assay time (60 min) in one step, which is superior to those of most existing approaches. Owing to the specific site recognition of MTase toward its substrate, the proposed sensing system was able to readily discriminate Dam MTase from other MTase such as M.SssI and even detect the target in complex biological matrix. Furthermore, the application of the proposed sensing strategy for screening Dam MTase inhibitors was also demonstrated with satisfactory results. This novel method not only provides a promising platform for monitoring activity and inhibition of DNA MTases, but also shows great potentials in biological process researches, drugs discovery and clinical diagnostics. PMID:23202331

  17. Dual role of glutathione in modulating camptothecin activity: depletion potentiates activity, but conjugation enhances the stability of the topoisomerase I-DNA cleavage complex.

    PubMed

    Gamcsik, M P; Kasibhatla, M S; Adams, D J; Flowers, J L; Colvin, O M; Manikumar, G; Wani, M; Wall, M E; Kohlhagen, G; Pommier, Y

    2001-11-01

    Depletion of glutathione (GSH) in MCF-7 and MDA-MB-231 cell lines by pretreatment with the GSH synthesis inhibitor buthionine sulfoximine potentiated the activity of 10,11-methylenedioxy-20(S)-camptothecin, SN-38 [7-ethyl-10-hydroxy-20(S)-camptothecin], topotecan, and 7-chloromethyl-10,11-methylenedioxy-20(S)-camptothecin (CMMDC). The greatest potentiation was observed with the alkylating camptothecin CMMDC. Buthionine sulfoximine pretreatment also increased the number of camptothecin-induced DNA-protein crosslinks, indicating that GSH affects the mechanism of action of camptothecin. We also report that GSH interacts with CMMDC to form a stable conjugate, 7-(glutathionylmethyl)-10,11-methylenedioxy-20(S)-camptothecin (GSMMDC), which is formed spontaneously in buffered solutions and in MCF-7 cells treated with CMMDC. GSMMDC was synthesized and found to be nearly as active as 10,11-methylenedioxy-20(S)-camptothecin in a topoisomerase (topo) I-mediated DNA nicking assay. The resulting topo I cleavage complexes were remarkably stable. In cell culture, GSMMDC displayed potent growth-inhibitory activity against U937 and P388 leukemia cell lines. GSMMDC was not active against a topo I-deficient P388 cell line, indicating that topo I is its cellular target. Peptide-truncated analogues of GSMMDC were prepared and evaluated. All three derivatives [7-(gamma-glutamylcysteinylmethyl)-10,11-methylenedioxy-20(S)-camptothecin, 7-(cysteinylglycylmethyl)-10,11-methylenedioxy-20(S)-camptothecin, and 7-(cysteinylmethyl)-10,11-methylenedioxy-20(S)-camptothecin] displayed topo I and cell growth-inhibitory activity. These results suggest that 7-peptidyl derivatives represent a new class of camptothecin analogues. PMID:12467234

  18. Activities of Human Immunodeficiency Virus (HIV) Integration Protein In vitro: Specific Cleavage and Integration of HIV DNA

    NASA Astrophysics Data System (ADS)

    Bushman, Frederic D.; Craigie, Robert

    1991-02-01

    Growth of human immunodeficiency virus (HIV) after infection requires the integration of a DNA copy of the viral RNA genome into a chromosome of the host. Here we present a simple in vitro system that carries out the integration reaction and the use of this system to probe the mechanism of integration. The only HIV protein necessary is the integration (IN) protein, which has been overexpressed in insect cells and then partially purified. DNA substrates are supplied as oligonucleotides that match the termini of the linear DNA product of reverse transcription. In the presence of HIV IN protein, oligonucleotide substrates are cleaved to generate the recessed 3' ends that are the precursor for integration, and the cleaved molecules are efficiently inserted into a DNA target. Analysis of reaction products reveals that HIV IN protein joins 3' ends of the viral DNA to 5' ends of cuts made by IN protein in the DNA target. We have also used this assay to characterize the sequences at the ends of the viral DNA involved in integration. The assay provides a simple screen for testing candidate inhibitors of HIV IN protein; some such inhibitors might have useful antiviral activity.

  19. Induction of cell death by ternary copper(II) complexes of L-tyrosine and diimines: role of coligands on DNA binding and cleavage and anticancer activity.

    PubMed

    Ramakrishnan, Sethu; Rajendiran, Venugopal; Palaniandavar, Mallayan; Periasamy, Vaiyapuri Subbarayan; Srinag, Bangalore Suresh; Krishnamurthy, Hanumanthappa; Akbarsha, Mohammad Abdulkader

    2009-02-16

    viscosity of DNA bound to 1 decreases, indicating the shortening of the DNA chain length by means of the formation of kinks or bends. All complexes exhibit effective DNA (pUC19 DNA) cleavage at 100 microM complex concentrations, and the order of DNA cleavage ability varies as 3 > 2 > 4 > 1. Interestingly, 3 exhibits a DNA cleavage rate constant that is higher than that of the other complexes only at 100 microM concentration, whereas 4 exhibits the highest cleavage rate constant at 80 microM complex concentration. The oxidative DNA cleavage follows the order 4 > 3 > 2 > 1. Mechanistic studies reveal that the DNA cleavage pathway involves hydroxyl radicals. Interestingly, only 4 displays efficient photonuclease activity upon irradiation with 365 nm light, which occurs through double-strand DNA breaks involving hydroxyl radicals. Furthermore, cytotoxicity studies on the nonsmall lung cancer (H-460) cell line show that the IC(50) values of 2-4 are more or less equal to cisplatin for the same cell line, indicating that they have the potential to act as very effective anticancer drugs in a time-dependent manner. The study of cytological changes reveals the higher induction of apoptosis and mitotic catastrophe for 4 and 3, respectively. The alkaline single-cell gel electrophoresis (comet assay), DNA laddering, and AO/EB and Hoechst 33258 staining assays have also been employed in finding the extent of DNA damage. Flow cytometry analysis shows an increase in the percentage of cells with apoptotic morphological features in the sub-G(0)/G(1) phase for 4, whereas it shows mitotic catastrophe for 3. PMID:19140687

  20. Synthesis of mononuclear copper(II) complexes of acyclic Schiff's base ligands: Spectral, structural, electrochemical, antibacterial, DNA binding and cleavage activity

    NASA Astrophysics Data System (ADS)

    Jayamani, Arumugam; Thamilarasan, Vijayan; Sengottuvelan, Nallathambi; Manisankar, Paramasivam; Kang, Sung Kwon; Kim, Young-Inn; Ganesan, Vengatesan

    2014-03-01

    The mononuclear copper(II) complexes (1&2) of ligands L1 [N,N";-bis(2-hydroxy-5-methylbenzyl)-1,4-bis(3-iminopropyl)piperazine] or L2 [N,N";-bis(2-hydroxy-5-bromobenzyl)-1,4-bis(3-iminopropyl) piperazine] have been synthesized and characterised. The single crystal X-ray study had shown that ligands L1 and L2 crystallize in a monoclinic crystal system with P21/c space group. The mononuclear copper(II) complexes show one quasireversible cyclic voltammetric response near cathodic region (-0.77 to -0.85 V) in DMF assignable to the Cu(II)/Cu(I) couple. Binding interaction of the complexes with calf thymus DNA (CT DNA) investigated by absorption studies and fluorescence spectral studies show good binding affinity to CT DNA, which imply both the copper(II) complexes can strongly interact with DNA efficiently. The copper(II) complexes showed efficient oxidative cleavage of plasmid pBR322 DNA in the presence of 3-mercaptopropionic acid as reducing agent through a mechanistic pathway involving formation of singlet oxygen as the reactive species. The Schiff bases and their Cu(II) complexes have been screened for antibacterial activities which indicates that the complexes exhibited higher antimicrobial activity than the free ligands.

  1. Reversed DNA strand cleavage specificity in initiation of Cre-LoxP recombination induced by the His289Ala active-site substitution.

    PubMed

    Gelato, Kathy A; Martin, Shelley S; Baldwin, Enoch P

    2005-11-25

    During the first steps of site-specific recombination, Cre protein cleaves and religates a specific homologous pair of LoxP strands to form a Holliday junction (HJ) intermediate. The HJ is resolved into recombination products through exchange of the second homologous strand pair. CreH289A, containing a His to Ala substitution in the conserved R-H-R catalytic motif, has a 150-fold reduced recombination rate and accumulates HJs. However, to produce these HJs, CreH289A exchanges the opposite set of strands compared to wild-type Cre (CreWT). To investigate how CreH289A and CreWT impose strand exchange order, we characterized their reactivities and strand cleavage preferences toward LoxP duplex and HJ substrates containing 8bp spacer substitutions. Remarkably, CreH289A had different and often opposite strand exchange preferences compared to CreWT with nearly all substrates. CreH289N was much less perturbed, implying that overall recombination rate and strand exchange depend more on His289 hydrogen bonding capability than on its acid/base properties. LoxP substitutions immediately 5' (S1 nucleotide) or 3' (S1' nucleotide) of the scissile phosphate had large effects on substrate utilization and strand exchange order. S1' substitutions, designed to alter base-unstacking events concomitant with Cre-induced LoxP bending, caused HJ accumulation and dramatically inverted the cleavage preferences. That pre-formed HJs were resolved via either strand in vitro suggests that inhibition of the "conformational switch" isomerization required to trigger the second strand exchange accounts for the observed HJ accumulation. Rather than reflecting CreWT behavior, CreH289A accumulates HJs of opposite polarity through a combination of its unique cleavage specificity and an HJ isomerization defect. The overall implication is that cleavage specificity is mediated by sequence-dependent DNA deformations that influence the scissile phosphate positioning and reactivity. A role of His289 may be to

  2. DNA Binding, Cleavage and Antibacterial Activity of Mononuclear Cu(II), Ni(II) and Co(II) Complexes Derived from Novel Benzothiazole Schiff Bases.

    PubMed

    Vamsikrishna, Narendrula; Kumar, Marri Pradeep; Tejaswi, Somapangu; Rambabu, Aveli; Shivaraj

    2016-07-01

    A series of novel bivalent metal complexes M(L1)2 and M(L2)2 where M = Cu(II), Ni(II), Co(II) and L1 = 2-((benzo [d] thiazol-6-ylimino)methyl)-4-bromophenol [BTEMBP], L2 = 1-((benzo [d] thiazol-6-ylimino)methyl) naphthalen-2-ol [BTEMNAPP] were synthesized. All the compounds have been characterized by elemental analysis, SEM, Mass, (1)H NMR, (13)C NMR, UV-Vis, IR, ESR, spectral data and magnetic susceptibility measurements. Based on the analytical and spectral data four-coordinated square planar geometry is assigned to all the complexes. DNA binding properties of these complexes have been investigated by electronic absorption spectroscopy, fluorescence and viscosity measurements. It is observed that these binary complexes strongly bind to calf thymus DNA by an intercalation mode. DNA cleavage efficacy of these complexes was tested in presence of H2O2 and UV light by gel electrophoresis and found that all the complexes showed better nuclease activity. Finally the compounds were screened for antibacterial activity against few pathogens and found that the complexes have potent biocidal activity than their free ligands. PMID:27165038

  3. Comparative studies of UV-induced DNA cleavage by structural isomers of an iodinated DNA ligand

    SciTech Connect

    Martin, R.F.; Green, A.; Denison, L.; Pardee, M.; Kelly, D.P.; Roberts, M.; Rose, M.; Reum, M.

    1994-06-15

    The purpose was to evaluate the importance of the position of the halogen atom in iodinated DNA-binding bibenzimidazoles, with respect to sensitization of UV-A-induced DNA breakage. Three analogues of iodoHoechst 33258, denoted ortho-, meta- and paraiodoHoechst, according to the site of iodine substitution, were synthesized. Plasmid DNA (pBR322) was used to assay UV-A-induced DNA single-strand breaks (ssbs). The location of the sites of strand breakage was determined by DNA sequencing gel analysis, using a [sup 32]P-endlabelled oligoDNA with a single binding site for the ligands. A clear trend in decreasing activity of sensitization of UV-induced DNA ssbs was established: Ortho- > meta-, para- > iodoHoechst 33258. The sequencing gel studies showed that orthoiodoHoechst was distinct from the other three compounds, with respect to the sites of DNA strand breakage and the chemistry of the cleavage reaction. The position of iodine substitution in iodinated bibenzimidazoles determines the location of the carbon-centered radical on the ligand in the minor groove of DNA. DNA strand cleavage is mediated by abstraction of a nearby deoxyribosyl H-atom. Hence, the position of the radical species determines: which deoxyribosyl group is attacked (i.e., site of cleavage relative to the ligand binding site); which H-atom is abstracted, more specifically which of the five deoxyribosyl carbons is involved (i.e., the chemistry of the cleavage reaction), and the stereochemistry of the transition state for the H-atom abstraction (and hence the efficiency or extent of strand breakage). The ortho-compound represents the best example to date of iodinated DNA ligands designed as potential radiation sensitizers, as an extension of the well-established sensitization by halogenated DNA precursors. 30 refs., 3 figs.

  4. Synthesis and structure of dicopper(II) complexes bridged by N-(5-chloro-2-hydroxyphenyl)-N'-[3-(methy lamino)propyl]oxamide: evaluation of DNA/protein binding, DNA cleavage, and in vitro anticancer activity.

    PubMed

    Xu, Xiao-Wen; Li, Xue-Jie; Zhu, Ling; Li, Yan-Tuan; Wu, Zhi-Yong; Yan, Cui-Wei

    2015-06-01

    Three new dicopper(II) complexes bridged by N-(5-chloro-2-hydroxyphenyl)-N'-[3-(methylamino)-propyl]oxamide (H3chmpoxd) and end-capped with 1,10-phenanthroline (phen); 2,2'-diamino-4,4'-bithiazole (dabt); and 2,2'-bipyridine (bpy), namely [Cu2(chmpoxd)(H2O)(phen)](ClO4)⋅CH3CN (1), [Cu2(chmpoxd)(dabt)(C2H5OH)](NO3) (2) and [Cu2(chmpoxd)(H2O)(bpy)](NO3)⋅CH3CN (3), were synthesized and structurally characterized. The single-crystal X-ray diffraction analysis revealed that both the copper(II) ions bridged by the cis-chmpoxd(3-) ligands in the three complexes are in square-planar and square-pyramidal environments, respectively. The reactivity towards herring sperm DNA (HS-DNA) and protein bovine serum albumin (BSA) indicated that these copper(II) complexes can interact with the DNA in the mode of intercalation, and bind to BSA responsible for quenching of tryptophan fluorescence by the static quenching mechanism. The cytotoxicity and DNA cleavage suggested that all the dicopper(II) complexes are active against the selected tumor cell lines, and the complex 1 exhibits the cleavage capacity for plasmid DNA. PMID:25837411

  5. Synthesis, characterization, optical band gap, in vitro antimicrobial activity and DNA cleavage studies of some metal complexes of pyridyl thiosemicarbazone

    NASA Astrophysics Data System (ADS)

    Yousef, T. A.; Abu El-Reash, G. M.; El-Gammal, O. A.; Bedier, R. A.

    2013-03-01

    A new series of Cr(III), Mn(II), Ni(II), Zn(II) and Hg(II) complexes of Schiff-bases derived from the condensation of 4-(2-pyridyl)-3-thiosemicarbazide and pyruvic acid (H2PTP) have been synthesized and characterized by spectroscopic studies. Schiff-base exhibit thiol-thione tautomerism wherein sulfur plays an important role in the coordination. The coordination possibility of the Schiff-bases towards metal ions have been proposed in the light of elemental analysis, spectral (IR, UV-vis, 1H NMR and 13C NMR), magnetic and thermal studies. IR spectra show that H2PTP is coordinated to the metal ions in a mononegative tridentate manner except in Cr(III) complex in which the ligand exhibits mononegative bidentate manner. The parameters total energy, binding energy, isolated atomic energy, electronic energy, heat of formation, dipole moment, HOMO and LUMO were calculated for the ligand and its complexes. Furthermore, the kinetic and thermodynamic parameters for the different decomposition steps were calculated using the Coats-Redfern and Horowitz-Metzger methods. Also, the optical band gap (Eg) of the metal complexes has been calculated. The optical transition energy (Eg) is direct and equals 3.20, 3.27 and 3.26 eV for Cr, Mn and Ni complexes, respectively. The synthesized ligand, in comparison to its metal complexes is screened for its antibacterial activity against the bacterial species, Bacillus thuringiensis, Staphylococcus aureus, Pseudomonas aeuroginosa and Escherichia coli. The results show that the metal complexes be more potent in activity antibacterial than the parent Shciff base ligand towards one or more bacterial species. Finally, the biochemical studies showed that, Mn complex have powerful and complete degradation effect on DNA.

  6. Irreversible and reversible topoisomerase II DNA cleavage stimulated by clerocidin: sequence specificity and structural drug determinants.

    PubMed

    Binaschi, M; Zagotto, G; Palumbo, M; Zunino, F; Farinosi, R; Capranico, G

    1997-05-01

    In contrast to other topoisomerase II poisons, the microbial terpenoid clerocidin was shown to stimulate irreversible topoisomerase II-mediated DNA cleavage. To establish the structural determinants for drug activity, in this study we have investigated intensity patterns and sequence specificity of clerocidin-stimulated DNA cleavage using 5'-end 32P-labeled DNA fragments. At a majority of the sites, clerocidin-stimulated cleavage did not revert upon NaCl addition; nevertheless, at some sites, cleavage completely reverted. Statistical analyses showed that drug-preferred bases were different in the two cases: guanine and cytosine were highly preferred at position -1 at irreversible and reversible sites, respectively. These results demonstrated that cleavage irreversibility was site selective and required a guanine at the 3' end of the cut. Further experiments revealed that some irreversible sites showed an abnormal electrophoretic mobility in sequencing gels with respect to cleaved bands generated by 4-(9-acridinylamino)methanesulfon-m-anisidide, suggesting a chemical alteration of the DNA strand. Interestingly, the ability to stimulate irreversible cleavage progressively decreased over time when clerocidin was stored in ethanol. Under these conditions, nuclear magnetic resonance measurements demonstrated that the drug underwent structural modifications that involved the C-12-C-15 side chain. Thus, the results indicate that a specific moiety of clerocidin may react with the DNA (guanine at -1) in the ternary complex, resulting in cleavage irreversibility and in altered DNA mobility in sequencing gels. PMID:9135013

  7. Synthesis, spectroscopic characterisation, DNA cleavage, superoxidase dismutase activity and antibacterial properties of some transition metal complexes of a novel bidentate Schiff base derived from isatin and 2-aminopyrimidine.

    PubMed

    Nitha, L P; Aswathy, R; Mathews, Niecy Elsa; Kumari, B Sindhu; Mohanan, K

    2014-01-24

    Complexes of manganese(II), cobalt(II), nickel(II), copper(II) and zinc(II) with a Schiff base, formed by the condensation of isatin with 2-aminopyrimidine have been synthesised and characterised through elemental analysis, molar conductance measurements, magnetic susceptibility, IR, UV-Vis, (1)HNMR, FAB mass and EPR spectral studies. The spectral data revealed that the ligand acts as neutral bidentate, coordinating to the metal ion through the carbonyl oxygen and azomethine nitrogen. Molar conductance values adequately support the electrolytic nature of the complexes. On the basis of the above observations the complexes have been formulated as [M(ISAP)2]X2, where M=Mn(II), Co(II), Ni(II), Cu(II) and Zn(II); X=Cl, OAc; ISAP=2-[N-indole-2-one]aminopyrimidine. The ligand and copper(II) complex were subjected to X-ray diffraction studies. The DNA cleavage study was monitored by gel electrophoresis method. The superoxide dismutase (SOD) mimetic activities of the ligand and the metal complexes were checked using NBT assay. The in vitro antibacterial activity of the synthesized compounds has been tested against gram negative and gram positive bacteria. PMID:24051284

  8. Synthesis, spectroscopic characterisation, DNA cleavage, superoxidase dismutase activity and antibacterial properties of some transition metal complexes of a novel bidentate Schiff base derived from isatin and 2-aminopyrimidine

    NASA Astrophysics Data System (ADS)

    Nitha, L. P.; Aswathy, R.; Mathews, Niecy Elsa; Sindhu kumari, B.; Mohanan, K.

    2014-01-01

    Complexes of manganese(II), cobalt(II), nickel(II), copper(II) and zinc(II) with a Schiff base, formed by the condensation of isatin with 2-aminopyrimidine have been synthesised and characterised through elemental analysis, molar conductance measurements, magnetic susceptibility, IR, UV-Vis, 1HNMR, FAB mass and EPR spectral studies. The spectral data revealed that the ligand acts as neutral bidentate, coordinating to the metal ion through the carbonyl oxygen and azomethine nitrogen. Molar conductance values adequately support the electrolytic nature of the complexes. On the basis of the above observations the complexes have been formulated as [M(ISAP)2]X2, where M = Mn(II), Co(II), Ni(II), Cu(II) and Zn(II); X = Cl, OAc; ISAP = 2-[N-indole-2-one]aminopyrimidine. The ligand and copper(II) complex were subjected to X-ray diffraction studies. The DNA cleavage study was monitored by gel electrophoresis method. The superoxide dismutase (SOD) mimetic activities of the ligand and the metal complexes were checked using NBT assay. The in vitro antibacterial activity of the synthesized compounds has been tested against gram negative and gram positive bacteria.

  9. DNA binding, photo-induced DNA cleavage and cytotoxicity studies of lomefloxacin and its transition metal complexes

    NASA Astrophysics Data System (ADS)

    Ragheb, Mohamed A.; Eldesouki, Mohamed A.; Mohamed, Mervat S.

    2015-03-01

    This work was focused on a study of the DNA binding and cleavage properties of lomefloxacin (LMF) and its ternary transition metal complexes with glycine. The nature of the binding interactions between compounds and calf thymus DNA (CT-DNA) was studied by electronic absorption spectra, fluorescence spectra and thermal denaturation experiments. The obtained results revealed that LMF and its complexes could interact with CT-DNA via partial/moderate intercalative mode. Furthermore, the DNA cleavage activities of the compounds were investigated by gel electrophoresis. Mechanistic studies of DNA cleavage suggest that singlet oxygen (1O2) is likely to be the cleaving agent via an oxidative pathway, except for Cu(II) complex which proceeds via both oxidative and hydrolytic pathways. Antimicrobial and antitumor activities of the compounds were also studied against some kinds of bacteria, fungi and human cell lines.

  10. Comparison of the specificities and catalytic activities of hammerhead ribozymes and DNA enzymes with respect to the cleavage of BCR-ABL chimeric L6 (b2a2) mRNA.

    PubMed

    Kuwabara, T; Warashina, M; Tanabe, T; Tani, K; Asano, S; Taira, K

    1997-08-01

    With the eventual goal of developing a treatment for chronic myelogenous leukemia (CML), attempts have been made to design hammerhead ribozymes that can specifically cleave BCR-ABL fusion mRNA. In the case of L6 BCR-ABL fusion mRNA (b2a2 type; BCR exon 2 is fused to ABL exon 2), which has no effective cleavage sites for conventional hammerhead ribozymes near the BCR-ABL junction, it has proved very difficult to cleave the chimeric mRNA specifically. Several hammerhead ribozymes with relatively long junction-recognition sequences have poor substrate-specificity. Therefore, we explored the possibility of using newly selected DNA enzymes that can cleave RNA molecules with high activity to cleave L6 BCR-ABL fusion (b2a2) mRNA. In contrast to the results with the conventional ribozymes, the newly designed DNA enzymes, having higher flexibility for selection of cleavage sites, were able to cleave this chimeric RNA molecule specifically at sites close to the junction. Cleavage occurred only within the abnormal BCR-ABL mRNA, without any cleavage of the normal ABL or BCR mRNA. Thus, these chemically synthesized DNA enzymes seem to be potentially useful for application in vivo , especially for the treatment of CML, if we can develop exogenous delivery strategies. PMID:9224607

  11. Activation of α-secretase cleavage.

    PubMed

    Postina, Rolf

    2012-01-01

    Alpha-secretase-mediated cleavage of the amyloid precursor protein (APP) releases the neuroprotective APP fragment sαAPP and prevents amyloid β peptide (Aβ) generation. Moreover, α-secretase-like cleavage of the Aβ transporter 'receptor for advanced glycation end products' counteracts the import of blood Aβ into the brain. Assuming that Aβ is responsible for the development of Alzheimer's disease (AD), activation of α-secretase should be preventive. α-Secretase-mediated APP cleavage can be activated via several G protein-coupled receptors and receptor tyrosine kinases. Protein kinase C, mitogen-activated protein kinases, phosphatidylinositol 3-kinase, cAMP and calcium are activators of receptor-induced α-secretase cleavage. Selective targeting of receptor subtypes expressed in brain regions affected by AD appears reasonable. Therefore, the PACAP receptor PAC1 and possibly the serotonin 5-HT(6) receptor subtype are promising targets. Activation of APP α-secretase cleavage also occurs upon blockade of cholesterol synthesis by statins or zaragozic acid A. Under physiological statin concentrations, the brain cholesterol content is not influenced. Statins likely inhibit Aβ production in the blood by α-secretase activation which is possibly sufficient to inhibit AD development. A disintegrin and metalloproteinase 10 (ADAM10) acts as α-secretase on APP. By targeting the nuclear retinoic acid receptor β, the expression of ADAM10 and non-amyloidogenic APP processing can be enhanced. Excessive activation of ADAM10 should be avoided because ADAM10 and also ADAM17 are not APP-specific. Both ADAM proteins cleave various substrates, and therefore have been associated with tumorigenesis and tumor progression. PMID:21883223

  12. Conversion of Topoisomerase I Cleavage Complexes on the Leading Strand of Ribosomal DNA into 5′-Phosphorylated DNA Double-Strand Breaks by Replication Runoff

    PubMed Central

    Strumberg, Dirk; Pilon, André A.; Smith, Melanie; Hickey, Robert; Malkas, Linda; Pommier, Yves

    2000-01-01

    Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3′ DNA ends are extended by DNA polymerase in vivo closely to the 5′ ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5′ ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5′ kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA. PMID:10805740

  13. Reversed DNA Strand Cleavage Specificity in Initiation of Cre–LoxP Recombination Induced by the His289Ala Active-site Substitution

    PubMed Central

    Gelato, Kathy A.; Martin, Shelley S.; Baldwin, Enoch P.

    2010-01-01

    During the first steps of site-specific recombination, Cre protein cleaves and religates a specific homologous pair of LoxP strands to form a Holliday junction (HJ) intermediate. The HJ is resolved into recombination products through exchange of the second homologous strand pair. CreH289A, containing a His to Ala substitution in the conserved R-H-R catalytic motif, has a 150-fold reduced recombination rate and accumulates HJs. However, to produce these HJs, CreH289A exchanges the opposite set of strands compared to wild-type Cre (CreWT). To investigate how CreH289A and CreWT impose strand exchange order, we characterized their reactivities and strand cleavage preferences toward LoxP duplex and HJ substrates containing 8 bp spacer substitutions. Remarkably, CreH289A had different and often opposite strand exchange preferences compared to CreWT with nearly all substrates. CreH289N was much less perturbed, implying that overall recombination rate and strand exchange depend more on His289 hydrogen bonding capability than on its acid/base properties. LoxP substitutions immediately 5′(S1 nucleotide) or 3′(S1′nucleotide) of the scissile phosphate had large effects on substrate utilization and strand exchange order. S1′substitutions, designed to alter base-unstacking events concomitant with Cre-induced LoxP bending, caused HJ accumulation and dramatically inverted the cleavage preferences. That pre-formed HJs were resolved via either strand in vitro suggests that inhibition of the “conformational switch” isomerization required to trigger the second strand exchange accounts for the observed HJ accumulation. Rather than reflecting CreWT behavior, CreH289A accumulates HJs of opposite polarity through a combination of its unique cleavage specificity and an HJ isomerization defect. The overall implication is that cleavage specificity is mediated by sequence-dependent DNA deformations that influence the scissile phosphate positioning and reactivity. A role of His289

  14. The pattern of DNA cleavage intensity around indels.

    PubMed

    Chen, Wei; Zhang, Liqing

    2015-01-01

    Indels (insertions and deletions) are the second most common form of genetic variations in the eukaryotic genomes and are responsible for a multitude of genetic diseases. Despite its significance, detailed molecular mechanisms for indel generation are still unclear. Here we examined 2,656,597 small human and mouse germline indels, 16,742 human somatic indels, 10,599 large human insertions, and 5,822 large chimpanzee insertions and systematically analyzed the patterns of DNA cleavage intensities in the 200 base pair regions surrounding these indels. Our results show that DNA cleavage intensities close to the start and end points of indels are significantly lower than other regions, for both small human germline and somatic indels and also for mouse small indels. Compared to small indels, the patterns of DNA cleavage intensity around large indels are more complex, and there are two low intensity regions near each end of the indels that are approximately 13 bp apart from each other. Detailed analyses of a subset of indels show that there is slight difference in cleavage intensity distribution between insertion indels and deletion indels that could be contributed by their respective enrichment of different repetitive elements. These results will provide new insight into indel generation mechanisms. PMID:25660536

  15. Cleavage of supercoiled plasmid DNA by autoantibody Fab fragment: application of the flow linear dichroism technique.

    PubMed Central

    Gololobov, G V; Chernova, E A; Schourov, D V; Smirnov, I V; Kudelina, I A; Gabibov, A G

    1995-01-01

    A highly effective method consisting of two affinity chromatography steps and ion-exchange and gel-filtration chromatography steps was developed for purification of autoantibodies from human sera with DNA-hydrolyzing activity. Antibody Fab fragment, which had been purified 130-fold, was shown to catalyze plasmid DNA cleavage. The flow linear dichroism technique was used for quantitative and qualitative studying of supercoiled plasmid DNA cleavage by these autoantibodies in comparison with DNase I and EcoRI restriction endonuclease. The DNA autoantibody Fab fragment was shown to hydrolyze plasmid DNA by Mg(2+)-dependent single-strand multiple nicking of the substrate. Kinetic properties of the DNA autoantibody Fab fragment were evaluated from the flow linear dichroism and agarose gel electrophoresis data and revealed a high affinity (Kobsm = 43 nM) and considerable catalytic efficiency (kappcat/Kobsm = 0.32 min-1.nM-1) of the reaction. Images Fig. 2 PMID:7816827

  16. Mixed ligand complexation of some transition metal ions in solution and solid state: Spectral characterization, antimicrobial, antioxidant, DNA cleavage activities and molecular modeling

    NASA Astrophysics Data System (ADS)

    Shobana, Sutha; Dharmaraja, Jeyaprakash; Selvaraj, Shanmugaperumal

    2013-04-01

    Equilibrium studies of Ni(II), Cu(II) and Zn(II) mixed ligand complexes involving a primary ligand 5-fluorouracil (5-FU; A) and imidazoles viz., imidazole (him), benzimidazole (bim), histamine (hist) and L-histidine (his) as co-ligands(B) were carried out pH-metrically in aqueous medium at 310 ± 0.1 K with I = 0.15 M (NaClO4). In solution state, the stoichiometry of MABH, MAB and MAB2 species have been detected. The primary ligand(A) binds the central M(II) ions in a monodentate manner whereas him, bim, hist and his co-ligands(B) bind in mono, mono, bi and tridentate modes respectively. The calculated Δ log K, log X and log X' values indicate higher stability of the mixed ligand complexes in comparison to binary species. Stability of the mixed ligand complex equilibria follows the Irving-Williams order of stability. In vitro biological evaluations of the free ligand(A) and their metal complexes by well diffusion technique show moderate activities against common bacterial and fungal strains. Oxidative cleavage interaction of ligand(A) and their copper complexes with CT DNA is also studied by gel electrophoresis method in the presence of oxidant. In vitro antioxidant evaluations of the primary ligand(A), CuA and CuAB complexes by DPPH free radical scavenging model were carried out. In solid, the MAB type of M(II)sbnd 5-FU(A)sbnd his(B) complexes were isolated and characterized by various physico-chemical and spectral techniques. Both the magnetic susceptibility and electronic spectral analysis suggest distorted octahedral geometry. Thermal studies on the synthesized mixed ligand complexes show loss of coordinated water molecule in the first step followed by decomposition of the organic residues subsequently. XRD and SEM analysis suggest that the microcrystalline nature and homogeneous morphology of MAB complexes. Further, the 3D molecular modeling and analysis for the mixed ligand MAB complexes have also been carried out.

  17. Thiosemicarbazone Cu(II) and Zn(II) complexes as potential anticancer agents: syntheses, crystal structure, DNA cleavage, cytotoxicity and apoptosis induction activity.

    PubMed

    Shao, Jia; Ma, Zhong-Ying; Li, Ang; Liu, Ya-Hong; Xie, Cheng-Zhi; Qiang, Zhao-Yan; Xu, Jing-Yuan

    2014-07-01

    Four novel thiosemicarbazone metal complexes, [Cu(Am4M)(OAc)]·H2O (1), [Zn(HAm4M)Cl2] (2), [Zn2(Am4M)2Br2] (3) and [Zn2(Am4M)2(OAc)2]·2MeOH (4) [HAm4M=(Z)-2-(amino(pyridin-2-yl)methylene)-N-methylhydrazinecarbothioamide], have been synthesized and characterized by X-ray crystallography, elemental analysis, ESI-MS and IR. X-ray analysis revealed that complexes 1 and 2 are mononuclear, which possess residual coordination sites for Cu(II) ion in 1 and good leaving groups (Cl(-)) for Zn(II) ion in 2. Both 3 and 4 displayed dinuclear units, in which the metal atoms are doubly bridged by S atoms of two Am4M(-) ligands in 3 and by two acetate ions in bi- and mono-dentate forms, respectively, in 4. Their antiproliferative activities on human epithelial cervical cancer cell line (HeLa), human liver hepatocellular carcinoma cell line (HepG-2) and human gastric cancer cell line (SGC-7901) were screened. Inspiringly, IC50 value (11.2±0.9 μM) of complex 1 against HepG-2 cells was nearly 0.5 fold of that against human hepatic cell lines LO2, showing a lower toxicity to human liver cells. Additionally, it displayed a stronger inhibition on the viability of HepG-2 cells than cisplatin (IC50=25±3.1 μM), suggesting complex 1 might be a potential high efficient antitumor agent. Furthermore, fluorescence microscopic observation and flow cytometric analysis revealed that complex 1 could significantly suppress HepG-2 cell viability and induce apoptosis. Several indexes, such as DNA cleavage, reactive oxygen species (ROS) generation, comet assay and cell cycle analysis indicated that the antitumor mechanism of complex 1 on HepG-2 cells might be via ROS-triggered apoptosis pathway. PMID:24690556

  18. Drosophila topoisomerase II double-strand DNA cleavage: analysis of DNA sequence homology at the cleavage site.

    PubMed Central

    Sander, M; Hsieh, T S

    1985-01-01

    In order to study the sequence specificity of double-strand DNA cleavage by Drosophila topoisomerase II, we have mapped and sequenced 16 strong and 47 weak cleavage sites in the recombinant plasmid p pi 25.1. Analysis of the nucleotide and dinucleotide frequencies in the region near the site of phosphodiester bond breakage revealed a nonrandom distribution. The nucleotide frequencies observed would occur by chance with a probability less than 0.05. The consensus sequence we derived is 5'GT.A/TAY decrease ATT.AT..G 3', where a dot means no preferred nucleotide, Y is for pyrimidine, and the arrow shows the point of bond cleavage. On average, strong sites match the consensus better than weak sites. Images PMID:2987816

  19. DNA binding, photoactivated DNA cleavage and cytotoxic activity of Cu(II) and Co(II) based Schiff-base azo photosensitizers

    NASA Astrophysics Data System (ADS)

    Pradeepa, S. M.; Bhojya Naik, H. S.; Vinay Kumar, B.; Indira Priyadarsini, K.; Barik, Atanu; Prabhakara, M. C.

    2015-04-01

    A new class of Cu(II) and Co(II) complexes of azo-containing Schiff base of the type [Cu(L1)2] and [Co(L1)2], where L1 = 4-[(E)-{2-hydroxy-3-[(E)-(4-bromophenyl)diazenyl]benzylidene}amino]benzoic acid have been synthesized and characterized. Extension of conjugation and the presence of free carboxylic acid group of the ligand L1 increased the wavelength of the complexes from visible region to the near IR region (620-850 nm). The Cu(II) and Co(II) complexes interacted with CT-DNA via intercalative mode with the respective Kb value of 3.2 × 104 M-1 and 2.9 × 104 M-1 and acted as proficient photocleavers of SC pUC19 DNA in UV-A light, forming 1O2 as the reactive oxygen species with the quantum yield of 0.38 and 0.36, respectively. Furthermore, the Cu(II) and Co(II) complexes showed photocytotoxicity toward two selected tumor cell lines MCF-7 and A549 by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) method, and the Cu(II) complex exhibits higher photocytotoxicity than Co(II) complex against each of the selected cell lines, this result is identical with their DNA binding ability order.

  20. Synthesis, characterization, antimicrobial, DNA-cleavage and antioxidant activities of 3-((5-chloro-2-phenyl-1H-indol-3-ylimino)methyl)quinoline-2(1H)-thione and its metal complexes

    NASA Astrophysics Data System (ADS)

    Vivekanand, B.; Mahendra Raj, K.; Mruthyunjayaswamy, B. H. M.

    2015-01-01

    Schiff base 3-((5-chloro-2-phenyl-1H-indol-3-ylimino)methyl)quinoline-2(1H)-thione and its Cu(II), Co(II), Ni(II), Zn(II) and Fe(III), complexes have been synthesized and characterized by elemental analysis, UV-Visible, IR, 1H NMR, 13C NMR and mass spectra, molar conductance, magnetic susceptibility, ESR and TGA data. The ligand and its metal complexes have been screened for their antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa, antifungal activity against Aspergillus niger and Aspergillus flavus in minimum inhibition concentration (MIC) by cup plate method respectively, antioxidant activity using 1,1-diphenyl-2-picryl hydrazyl (DPPH), which was compared with that of standard drugs vitamin-C and vitamin-E and DNA cleavage activity using calf-thymus DNA.

  1. Increasing cleavage specificity and activity of restriction endonuclease KpnI

    PubMed Central

    Vasu, Kommireddy; Nagamalleswari, Easa; Zahran, Mai; Imhof, Petra; Xu, Shuang-yong; Zhu, Zhenyu; Chan, Siu-Hong; Nagaraja, Valakunja

    2013-01-01

    Restriction enzyme KpnI is a HNH superfamily endonuclease requiring divalent metal ions for DNA cleavage but not for binding. The active site of KpnI can accommodate metal ions of different atomic radii for DNA cleavage. Although Mg2+ ion higher than 500 μM mediates promiscuous activity, Ca2+ suppresses the promiscuity and induces high cleavage fidelity. Here, we report that a conservative mutation of the metal-coordinating residue D148 to Glu results in the elimination of the Ca2+-mediated cleavage but imparting high cleavage fidelity with Mg2+. High cleavage fidelity of the mutant D148E is achieved through better discrimination of the target site at the binding and cleavage steps. Biochemical experiments and molecular dynamics simulations suggest that the mutation inhibits Ca2+-mediated cleavage activity by altering the geometry of the Ca2+-bound HNH active site. Although the D148E mutant reduces the specific activity of the enzyme, we identified a suppressor mutation that increases the turnover rate to restore the specific activity of the high fidelity mutant to the wild-type level. Our results show that active site plasticity in coordinating different metal ions is related to KpnI promiscuous activity, and tinkering the metal ion coordination is a plausible way to reduce promiscuous activity of metalloenzymes. PMID:23963701

  2. FOB1 affects DNA topoisomerase I in vivo cleavages in the enhancer region of the Saccharomyces cerevisiae ribosomal DNA locus

    PubMed Central

    Di Felice, Francesca; Cioci, Francesco; Camilloni, Giorgio

    2005-01-01

    In Saccharomyces cerevisiae the FOB1 gene affects replication fork blocking activity at the replication fork block (RFB) sequences and promotes recombination events within the rDNA cluster. Using in vivo footprinting assays we mapped two in vivo Fob1p-binding sites, RFB1 and RFB3, located in the rDNA enhancer region and coincident with those previously reported to be in vitro binding sites. We previously provided evidences that DNA topoisomerase I is able to cleave two sites within this region. The results reported in this paper, indicate that the DNA topoisomerase I cleavage specific activity at the enhancer region is affected by the presence of Fob1p and independent of replication and transcription activities. We thus hypothesize that the binding to DNA of Fob1p itself may be the cause of the DNA topoisomerase I activity in the rDNA enhancer. PMID:16269824

  3. Structural basis for DNA cleavage by the potent antiproliferative agent (-)-lomaiviticin A.

    PubMed

    Woo, Christina M; Li, Zhenwu; Paulson, Eric K; Herzon, Seth B

    2016-03-15

    (-)-Lomaiviticin A (1) is a complex antiproliferative metabolite that inhibits the growth of many cultured cancer cell lines at low nanomolar-picomolar concentrations. (-)-Lomaiviticin A (1) possesses a C2-symmetric structure that contains two unusual diazotetrahydrobenzo[b]fluorene (diazofluorene) functional groups. Nucleophilic activation of each diazofluorene within 1 produces vinyl radical intermediates that affect hydrogen atom abstraction from DNA, leading to the formation of DNA double-strand breaks (DSBs). Certain DNA DSB repair-deficient cell lines are sensitized toward 1, and 1 is under evaluation in preclinical models of these tumor types. However, the mode of binding of 1 to DNA had not been determined. Here we elucidate the structure of a 1:1 complex between 1 and the duplex d(GCTATAGC)2 by NMR spectroscopy and computational modeling. Unexpectedly, we show that both diazofluorene residues of 1 penetrate the duplex. This binding disrupts base pairing leading to ejection of the central AT bases, while placing the proreactive centers of 1 in close proximity to each strand. DNA binding may also enhance the reactivity of 1 toward nucleophilic activation through steric compression and conformational restriction (an example of shape-dependent catalysis). This study provides a structural basis for the DNA cleavage activity of 1, will guide the design of synthetic DNA-activated DNA cleavage agents, and underscores the utility of natural products to reveal novel modes of small molecule-DNA association. PMID:26929332

  4. Predictors of Hepatitis B Cure Using Gene Therapy to Deliver DNA Cleavage Enzymes: A Mathematical Modeling Approach

    PubMed Central

    Schiffer, Joshua T.; Swan, Dave A.; Stone, Daniel; Jerome, Keith R.

    2013-01-01

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

  5. Synthesis, crystal structures and characterization of late first row transition metal complexes derived from benzothiazole core: anti-tuberculosis activity and special emphasis on DNA binding and cleavage property.

    PubMed

    Netalkar, Priya P; Netalkar, Sandeep P; Budagumpi, Srinivasa; Revankar, Vidyanand K

    2014-05-22

    Air and moisture stable coordination compounds of late first row transition metals, viz. Co(II), Ni(II), Cu(II) and Zn(II), with a newly designed ligand, 2-(2-benzo[d]thiazol-2-yl)hydrazono)propan-1-ol (LH), were prepared and successfully characterized using various spectro-analytical techniques. The molecular structures of the ligand and nickel complex were unambiguously determined by single-crystal X-ray diffraction method. The [Ni(LH)2]Cl2.3H2O complex is stabilized by intermolecular CH⋯π stacking interactions between the methyl hydrogen and the C18 atom of the phenyl ring (C11-H11B⋯C18) forming 1D zig-zag chain structure. Both, the ligand and its copper complex, were electrochemically active in the working potential range, showing quasi-reversible redox system. The interactions of all the compounds with calf thymus DNA have been comprehensively investigated using electronic absorption spectroscopy, viscosity, electrochemistry and thermal denaturation studies. The cleavage reaction on pBR322 DNA has been monitored by agarose gel electrophoresis. The results showed that the ligand can bind to CT-DNA through partial intercalation, whereas the complexes bind electrostatically. Further, [Ni(LH)2]Cl2.3H2O and [CuLCl(H2O)2] complexes in the series have high binding and cleavage affinity towards pBR322 DNA. Additionally, all the compounds were screened for anti-tuberculosis activity. All the complexes revealed an MIC value of 0.8 μg/mL, which is almost 8 times active than standard used (Streptomycin, 6.25 μg/mL). PMID:24721314

  6. Mitoxantrone resistance in HL-60 leukemia cells: Reduced nuclear topoisomerase II catalytic activity and drug-induced DNA cleavage in association with reduced expression of the topoisomerase II. beta. isoform

    SciTech Connect

    Harker, W.G.; Slade, D.L.; Parr, R.L. ); Drake, F.H. )

    1991-10-15

    Mitoxantrone-resistant variants of the human HL-60 leukemia cell line are cross-resistant to several natural product and synthetic antineoplastic agents. The resistant cells (HL-60/MX2) retain sensitivity to the Vinca alkaloids vincristine and vinblastine, drugs that are typically associated with the classical multidrug resistance phenotype. Mitoxantrone accumulation and retention are equivalent in the sensitive and resistant cell types, suggesting that mitoxantrone resistance inn HL-60/MX2 cells might be associated with an alteration in the type II DNA topoisomerases. The authors discovered that topoisomerase II catalytic activity in 1.0 M NaCl nuclear extracts from the HL-60/MX2 variant was reduced 4- to 5-fold compared to that in the parental HL-60 cells. Studies were designed to minimize the proteolytic degradation of the topoisomerase II enzymes by extraction of whole cells with hot SDS. When nuclear extracts from the two cell types were normalized for equivalent catalytic activity, mitoxantrone inhibited the decatenation of kDNA by these extracts to an equal extent but levels of mitoxantrone-induced cleavage of {sup 32}P-labeled pBR322 DNA by nuclear extracts from HL-60/MX2 cells were 3- to 4-fold lower than in comparable HL-60 extracts. Resistance to the topoisomerase II inhibitor mitoxantrone in HL-60/MX2 is associated with reduced nuclear and whole cell topoisomerase II catalytic activity, immunologically undetectable levels of the 180-kDa topoisomerase II isozyme, and reduced mitoxantrone-induced cleavage of radiolabeled DNA by topoisomerase II in nuclear extracts from these cells.

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

    PubMed Central

    2016-01-01

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

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

    PubMed

    Vann, Kendra R; Sedgeman, Carl A; Gopas, Jacob; Golan-Goldhirsh, Avi; Osheroff, Neil

    2015-07-28

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

  9. Intronic cleavage and polyadenylation regulates gene expression during DNA damage response through U1 snRNA

    PubMed Central

    Devany, Emral; Park, Ji Yeon; Murphy, Michael R; Zakusilo, George; Baquero, Jorge; Zhang, Xiaokan; Hoque, Mainul; Tian, Bin; Kleiman, Frida E

    2016-01-01

    The DNA damage response involves coordinated control of gene expression and DNA repair. Using deep sequencing, we found widespread changes of alternative cleavage and polyadenylation site usage on ultraviolet-treatment in mammalian cells. Alternative cleavage and polyadenylation regulation in the 3ʹ untranslated region is substantial, leading to both shortening and lengthening of 3ʹ untranslated regions of genes. Interestingly, a strong activation of intronic alternative cleavage and polyadenylation sites is detected, resulting in widespread expression of truncated transcripts. Intronic alternative cleavage and polyadenylation events are biased to the 5ʹ end of genes and affect gene groups with important functions in DNA damage response and cancer. Moreover, intronic alternative cleavage and polyadenylation site activation during DNA damage response correlates with a decrease in U1 snRNA levels, and is reversible by U1 snRNA overexpression. Importantly, U1 snRNA overexpression mitigates ultraviolet-induced apoptosis. Together, these data reveal a significant gene regulatory scheme in DNA damage response where U1 snRNA impacts gene expression via the U1-alternative cleavage and polyadenylation axis. PMID:27462460

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

    PubMed Central

    Csefalvay, Eva; Lapkouski, Mikalai; Guzanova, Alena; Csefalvay, Ladislav; Baikova, Tatsiana; Bialevich, Vitali; Shamayeva, Katsiaryna; Janscak, Pavel; Kuta Smatanova, Ivana; Panjikar, Santosh; Carey, Jannette; Weiserova, Marie; Ettrich, Rüdiger

    2015-01-01

    Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling. PMID:26039067

  11. Alkyl and aryl sulfonyl p-pyridine ethanone oximes are efficient DNA photo-cleavage agents.

    PubMed

    Andreou, Nicolaos-Panagiotis; Dafnopoulos, Konstantinos; Tortopidis, Christos; Koumbis, Alexandros E; Koffa, Maria; Psomas, George; Fylaktakidou, Konstantina C

    2016-05-01

    Sulfonyloxyl radicals, readily generated upon UV irradiation of p-pyridine sulfonyl ethanone oxime derivatives, effectively cleave DNA, in a pH independent manner, and under either aerobic or anaerobic conditions. p-Pyridine sulfonyl ethanone oxime derivatives were synthesized from the reaction of p-pyridine ethanone oxime with the corresponding sulfonyl chlorides in good to excellent yields. All compounds, at a concentration of 100μM, were irradiated at 312nm for 15min, after incubation with supercoiled circular pBluescript KS II DNA and resulted in extended single- and double- strand cleavages. The cleavage ability was found to be concentration dependent, with some derivatives exhibiting activity even at nanomolar levels. Besides that, p-pyridine sulfonyl ethanone oxime derivatives showed good affinity to DNA, as it was observed with UV interaction and viscosity experiments with CT DNA and competitive studies with ethidium bromide. The compounds interact to CT DNA probably by non-classical intercalation (i.e. groove-binding) and at a second step they may intercalate within the DNA base pairs. The fluorescence emission spectra of pre-treated EB-DNA exhibited a significant or moderate quenching. Comparing with the known aryl carbonyloxyl radicals the sulfonyloxyl ones are more powerful, with both aryl and alkyl sulfonyl substituted derivatives to exhibit DNA photo-cleaving ability, in significantly lower concentrations. These properties may serve in the discovery of new leads for "on demand" biotechnological and medical applications. PMID:26945644

  12. Studies of viomycin, an anti-tuberculosis antibiotic: copper(ii) coordination, DNA degradation and the impact on delta ribozyme cleavage activity.

    PubMed

    Stokowa-Sołtys, K; Barbosa, N A; Kasprowicz, A; Wieczorek, R; Gaggelli, N; Gaggelli, E; Valensin, G; Wrzesiński, J; Ciesiołka, J; Kuliński, T; Szczepanik, W; Jeżowska-Bojczuk, M

    2016-05-17

    Viomycin is a basic peptide antibiotic, which is among the most effective agents against multidrug-resistant tuberculosis. In this paper we provide the characteristics of its acid base properties, coordination preferences towards the Cu(ii) ions, as well as the reactivity of the resulting complexes against plasmid DNA and HDV ribozyme. Careful coordination studies throughout the wide pH range allow for the characterisation of all the Cu(ii)-viomycin complex species. The assignment of proton chemical shifts was achieved by NMR experiments, while the DTF level of theory was applied to support molecular structures of the studied complexes. The experiments with the plasmid DNA reveal that at the physiological levels of hydrogen peroxide the Cu(ii)-viomycin complex is more aggressive against DNA than uncomplexed metal ions. Moreover, the degradation of DNA by viomycin can be carried out without the presence of transition metal ions. In the studies of antigenomic delta ribozyme catalytic activity, viomycin and its complex are shown to modulate the ribozyme functioning. The molecular modelling approach allows the indication of two different locations of viomycin binding sites to the ribozyme. PMID:27143296

  13. Cleavage of supercoiled circular double-stranded DNA induced by a eukaryotic cambialistic superoxide dismutase from Cinnamomum camphora.

    PubMed

    Wang, Bao-Zhong; Wei, Xu-Bin; Liu, Wang-Yi

    2004-09-01

    A eukaryotic cambialistic superoxide dismutase (SOD) has been purified to homogeneity from mature seeds of the disease- and insect-resistant camphor tree (Cinnamomum camphora). Besides the known role of this SOD in protecting cells against oxidative stress, it can induce the cleavage of supercoiled double-stranded DNA into nicked and linear DNA. It can not cleave linear DNA or RNA, demonstrating there is no DNase or RNase in the purified cambialistic SOD. Furthermore, the SOD can linearize circular pGEM-4Z DNA that is relaxed by topoisomerase I. This result indicates that the DNA-cleaving activity requires substrates being topologically constrained. The supercoiled DNA-cleaving activity of the cambialistic SOD can be inhibited by either SOD inhibitor (azide) or catalase and hydroxyl radical scavengers (ethanol and mannitol). The chelator of iron, diethylenetriaminepentaacetic acid (DTPA), also inhibits the supercoiled DNA-cleaving activity. These results show that the dismutation activity is crucial for the supercoiled DNA cleavage. The modification of tryptophan residue of the cambialistic SOD with N-bromosuccinimide (NBS) shows that these two activities are structurally correlative. The reaction mechanism is proposed that the hydroxyl radical formed in a transition-metal-catalyzing Fenton-type reaction contributes to the DNA-cleaving activity. In addition, the cleavage sites in supercoiled pGEM-4Z DNA are random. PMID:15346198

  14. Recognition and cleavage of 5-methylcytosine DNA by bacterial SRA-HNH proteins

    PubMed Central

    Han, Tiesheng; Yamada-Mabuchi, Megumu; Zhao, Gong; Li, Li; liu, Guang; Ou, Hong-Yu; Deng, Zixin; Zheng, Yu; He, Xinyi

    2015-01-01

    SET and RING-finger-associated (SRA) domain is involved in establishment and maintenance of DNA methylation in eukaryotes. Proteins containing SRA domains exist in mammals, plants, even microorganisms. It has been established that mammalian SRA domain recognizes 5-methylcytosine (5mC) through a base-flipping mechanism. Here, we identified and characterized two SRA domain-containing proteins with the common domain architecture of N-terminal SRA domain and C-terminal HNH nuclease domain, Sco5333 from Streptomyces coelicolor and Tbis1 from Thermobispora bispora. Both sco5333 and tbis1 cannot establish in methylated Escherichia coli hosts (dcm+), and this in vivo toxicity requires both SRA and HNH domain. Purified Sco5333 and Tbis1 displayed weak DNA cleavage activity in the presence of Mg2+, Mn2+ and Co2+ and the cleavage activity was suppressed by Zn2+. Both Sco5333 and Tbis1 bind to 5mC-containing DNA in all sequence contexts and have at least a preference of 100 folds in binding affinity for methylated DNA over non-methylated one. We suggest that linkage of methyl-specific SRA domain and weakly active HNH domain may represent a universal mechanism in competing alien methylated DNA but to maximum extent minimizing damage to its own chromosome. PMID:25564526

  15. Photocytotoxicity and DNA cleavage activity of L-arg and L-lys Schiff base oxovanadium(IV) complexes having phenanthroline bases.

    PubMed

    Sasmal, Pijus K; Majumdar, Ritankar; Dighe, Rajan R; Chakravarty, Akhil R

    2010-08-14

    Oxovanadium(IV) complexes [VO(sal-argH)(B)]Cl (1-3) and [VO(sal-lysH)(B)]Cl (4-6), where sal-argH2 and sal-lysH2 are N-salicylidene-L-arginine and N-salicylidene-L-lysine Schiff bases and B is a phenanthroline base, viz. 1,10-phenanthroline (phen in 1 and 4); dipyrido[3,2-d:2',3'-f]quinoxaline (dpq in 2 and 5) and dipyrido[3,2-a:2',3'-c]phenazine (dppz in 3 and 6), have been prepared, characterized and their DNA photocleavage activity studied. Complex 1, characterized by X-ray crystallography, shows the presence of a vanadyl group in V(IV)O3N3 coordination geometry with a tridentate Schiff base having a pendant guanidinium moiety and bidentate phen ligand. The complexes exhibit a d-d band at 715 nm in 20% DMF-Tris-HCl buffer. The complexes are redox active showing cathodic and anodic responses near -1.0 V and 0.85 V (vs. SCE) for the V(IV)-V(III) and V(V)-V(IV) couples, respectively, in DMF-Tris-HCl buffer. The complexes bind to calf thymus DNA giving Kb values in the range of 3.8 x 10(4) to 1.6 x 10(5) M(-1). Thermal denaturation and viscosity data suggest DNA groove binding nature of the complexes. The complexes do not show any "chemical nuclease" activity in dark in the presence of 3-mercaptopropionic acid or H2O2. The dpq and dppz complexes are efficient photocleavers of plasmid DNA in UV-A (365 nm) and red light (676 nm) via singlet oxygen pathway. The dppz complexes exhibit photocytotoxicity in HeLa cancer cells giving IC50 values of 15.4 microM for 3 and 17.5 microM for 6 in visible light while being non-toxic in dark giving IC50 values of >100 microM. PMID:20563340

  16. Computational redesign of endonuclease DNA binding and cleavage specificity

    NASA Astrophysics Data System (ADS)

    Ashworth, Justin; Havranek, James J.; Duarte, Carlos M.; Sussman, Django; Monnat, Raymond J.; Stoddard, Barry L.; Baker, David

    2006-06-01

    The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein-DNA recognition, and has considerable practical relevance for biotechnology and medicine. Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI using a physically realistic atomic-level forcefield. Using an in silico screen, we identified single base-pair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling. A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site ~10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific endonucleases for gene therapy and other applications.

  17. DNA cleavage by Type ISP Restriction–Modification enzymes is initially targeted to the 3′-5′ strand

    PubMed Central

    van Aelst, Kara; Šišáková, Eva; Szczelkun, Mark D.

    2013-01-01

    The mechanism by which a double-stranded DNA break is produced following collision of two translocating Type I Restriction–Modification enzymes is not fully understood. Here, we demonstrate that the related Type ISP Restriction–Modification enzymes LlaGI and LlaBIII can cooperate to cleave DNA following convergent translocation and collision. When one of these enzymes is a mutant protein that lacks endonuclease activity, DNA cleavage of the 3′-5′ strand relative to the wild-type enzyme still occurs, with the same kinetics and at the same collision loci as for a reaction between two wild-type enzymes. The DNA nicking activity of the wild-type enzyme is still activated by a protein variant entirely lacking the Mrr nuclease domain and by a helicase mutant that cannot translocate. However, the helicase mutant cannot cleave the DNA despite the presence of an intact nuclease domain. Cleavage by the wild-type enzyme is not activated by unrelated protein roadblocks. We suggest that the nuclease activity of the Type ISP enzymes is activated following collision with another Type ISP enzyme and requires adenosine triphosphate binding/hydrolysis but, surprisingly, does not require interaction between the nuclease domains. Following the initial rapid endonuclease activity, additional DNA cleavage events then occur more slowly, leading to further processing of the initial double-stranded DNA break. PMID:23221632

  18. HMGB1/2 can target DNA for illegitimate cleavage by the RAG1/2 complex

    PubMed Central

    Zhang, Ming; Swanson, Patrick C

    2009-01-01

    Background V(D)J recombination is initiated in antigen receptor loci by the pairwise cleavage of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins via a nick-hairpin mechanism. The RSS contains highly conserved heptamer (consensus: 5'-CACAGTG) and nonamer (consensus: 5'-ACAAAAACC) motifs separated by either 12- or 23-base pairs of poorly conserved sequence. The high mobility group proteins HMGB1 and HMGB2 (HMGB1/2) are highly abundant architectural DNA binding proteins known to promote RAG-mediated synapsis and cleavage of consensus recombination signals in vitro by facilitating RSS binding and bending by the RAG1/2 complex. HMGB1/2 are known to recognize distorted DNA structures such as four-way junctions, and damaged or modified DNA. Whether HMGB1/2 can promote RAG-mediated DNA cleavage at sites lacking a canonical RSS by targeting or stabilizing structural distortions is unclear, but is important for understanding the etiology of chromosomal translocations involving antigen receptor genes and proto-oncogene sequences that do not contain an obvious RSS-like element. Results Here we identify a novel DNA breakpoint site in the plasmid V(D)J recombination substrate pGG49 (bps6197) that is cleaved by the RAG proteins via a nick-hairpin mechanism. The bps6197 sequence lacks a recognizable heptamer at the breakpoint (5'-CCTGACG-3') but contains a nonamer-like element (5'-ACATTAACC-3') 30 base pairs from the cleavage site. We find that RAG-mediated bps6197 cleavage is promoted by HMGB1/2, requiring both HMG-box domains to be intact to facilitate RAG-mediated cleavage, and is stimulated by synapsis with a 12-RSS. A dyad-symmetric inverted repeat sequence lying 5' to the breakpoint is implicated as a target for HMGB1/2 activity. Conclusion We have identified a novel DNA sequence, called bps6197, that supports standard V(D)J-type cleavage despite the absence of an apparent heptamer motif. Efficient RAG-mediated bps6197 cleavage requires the presence of

  19. Cleavage patterns of Drosophila melanogaster satellite DNA by restriction enzymes.

    PubMed Central

    Shen, C J; Wiesehahn, G; Hearst, J E

    1976-01-01

    The five satellite DNAs of Drosophila melanogaster have been isolated by the combined use of different equilibrium density gradients and hydrolyzed by seven different restriction enzymes; Hae III, Hind II + Hind III, Hinf, Hpa II, EcoR I and EcoR II. The 1.705 satellite is not hydrolyzed by any of the enzymes tested. Hae III is the only restriction enzyme that cuts the 1.672 and 1.686 satellites. The cleavage products from either of these reactions has a heterogeneous size distribution. Part of the 1.688 satellite is cut by Hae III and by Hinf into three discrete fragments with M.W. that are multiples of 2.3 X 10(5) daltons (approximately 350 base pairs). In addition, two minor bands are detected in the 1.688-Hinf products. The mole ratios of the trimer, dimer and monomer are: 1:6.30 : 63.6 for 1.688-Hae III and 1 : 22.0 : 403 for 1.688-Hinf. Circular mitochondrial DNA (rho = 1.680) is cut into discrete fragments by all of the enzymes tested and molecular weights of these fragments have been determined. Images PMID:818625

  20. Preferential sites of early DNA cleavage in apoptosis and the pathway of nuclear damage.

    PubMed

    Krystosek, A

    1999-04-01

    We have tested the specific hypothesis that the pathway of nuclear collapse in apoptosis is governed by the early attack on active chromatin at spatially restricted nuclear sites. Cell death in PC12 pheochromocytoma cells deprived of serum growth factors, in HL-60 leukemic cells treated with inhibitors of protein or RNA biosynthesis, and in U937 histiocytic lymphoma cells exposed to the cytokine tumor necrosis factor alpha showed a common mechanism in the targeting of DNA for degradation. An incorporation assay with labeled nucleotide revealed an early selective nicking in peripheral nuclear chromatin with concomitant diminution in the amount of immunoreactive lamin B protein. This was followed by a phase of more extensive cleavages, continued nuclear protein loss, chromatin collapse, and fragmentation of nuclei. The spatial restriction of early cleavages is similar to the nicking obtained by the application of exogenous DNase I to fixed nuclei of normal cells and to that obtained in the activation of the endogenous endonuclease of liver nuclei by Ca2+. These similarities suggest that, in apoptosis, activation of an endonuclease preferentially recognizing a specific chromatin configuration, such as that of active (DNase I-sensitive) genes, underlies the early spatial demarcation of cleavages. PMID:10219626

  1. Site-specific DNA cleavage by artificial zinc finger-type nuclease with cerium-binding peptide

    SciTech Connect

    Nakatsukasa, Takako; Shiraishi, Yasuhisa; Negi, Shigeru; Imanishi, Miki; Futaki, Shiroh; Sugiura, Yukio . E-mail: sugiura@scl.kyoto-u.ac.jp

    2005-04-29

    The addition of a new function to native proteins is one of the most attractive protein-based designs. In this study, we have converted a C{sub 2}H{sub 2}-type zinc finger as a DNA-binding motif into a novel zinc finger-type nuclease by connecting two distinct zinc finger proteins (Sp1 and GLI) with a functional linker possessing DNA cleavage activity. As a DNA cleavage domain, we chose an analogue of the metal-binding loop (12 amino acid residues), peptide P1, which has been reported to exhibit a strong binding affinity for a lanthanide ion and DNA cleavage ability in the presence of Ce(IV). Our newly designed nucleases, Sp1(P1)GLI and Sp1(P1G)GLI, can strongly bind to a lanthanide ion and show a unique DNA cleavage pattern, in which certain positions between the two DNA-binding sites are specifically cleaved. The present result provides useful information for expanding the design strategy for artificial nucleases.

  2. DNA cleavage during ethanol metabolism: Role of superoxide radicals and catalytic iron

    SciTech Connect

    Rajasinghe, H.; Jayatilleke, E.; Shaw, S. )

    1990-01-01

    The generation of superoxide and related free radicals and the mobilization of catalytic iron due to ethanol metabolism have been suggested as mechanisms of alcohol-induced liver injury as well as of the increased risk of cancer observed in alcoholics. Cleavage of double stranded DNA is produced by both free radicals as well as by catalytic iron. The effects of ethanol metabolism on DNA cleavage were therefore studied in vitro as well as in vivo in isolated hepatocytes. Intactness of double stranded DNA was studied by measuring ethidium bromide fluorescence after DNA electrophoresis. In vitro, the metabolism of acetaldehyde by aldehyde oxidase caused cleavage of lambda phage DNA. Cleavage was inhibited by both superoxide dismutase and desferrioxamine indicating the role of superoxide radicals and catalytic iron respectively. Studies with HIND III digests of the lambda phage indicate a lack of specificity in the breaks with respect to nucleotide sequences. Addition of EDTA greatly enhanced cleavage. In vivo, ethanol metabolism caused minimal breakage in hepatocyte DNA and addition of acetaldehyde markedly enhanced cleavage; all cleavage was inhibited by desferrioxamine.

  3. Effects of Secondary Metabolites from the Fungus Septofusidium berolinense on DNA Cleavage Mediated by Human Topoisomerase IIα

    PubMed Central

    Vann, Kendra R.; Ekiz, Güner; Zencir, Sevil; Bedir, Erdal; Topcu, Zeki; Osheroff, Neil

    2016-01-01

    Two metabolites from the ascomycete fungus Septofusidium berolinense were recently identified as having antineoplastic activity [Ekiz, et al. (2015) J. Antibiot. (Tokyo)]. However, the basis for this activity is not known. One of the compounds [3,6-dihydroxy-2-propylbenzaldehyde (GE-1)] is a hydroquinone and the other [2-hydroxymethyl-3-propylcyclohexa-2,5-diene-1,4-dione (GE-2)] is a quinone. Because some hydroquinones and quinones act as topoisomerase II poisons, the effects of GE-1 and GE-2 on DNA cleavage mediated by human topoisomerase IIα were assessed. GE-2 enhanced DNA cleavage ~4–fold and induced scission with a site specificity similar to that of the anticancer drug etoposide. Similar to other quinone-based topoisomerase II poisons, GE-2 displayed several hallmark characteristics of covalent topoisomerase II poisons, including: 1) the inability to poison a topoisomerase IIα construct that lacks the N-terminal domain; 2) the inhibition of DNA cleavage when the compound was incubated with the enzyme prior to the addition of plasmid, and 3) the loss of poisoning activity in the presence of a reducing agent. In contrast to GE-2, GE-1 did not enhance DNA cleavage mediated by topoisomerase IIα except at very high concentrations. However, the activity and potency of the metabolite were dramatically enhanced under oxidizing conditions. Results suggest that topoisomerase IIα may play a role in mediating the cytotoxic effects of these fungal metabolites. PMID:26894873

  4. Synthesis, characterization, DNA binding, cleavage activity, cytotoxicity and molecular docking of new nano water-soluble [M(5-CH₂PPh₃-3,4-salpyr)](ClO₄)₂ (M = Ni, Zn) complexes.

    PubMed

    Mandegani, Zeinab; Asadi, Zahra; Asadi, Mozaffar; Karbalaei-Heidari, Hamid Reza; Rastegari, Banafsheh

    2016-04-21

    Some new water soluble complexes [N,N'-bis{5-[(triphenyl phosphonium chloride)-methyl]salicylidine}-3,4-diaminopyridine] M(ii), which are formulated as nano-[Zn(5-CH2PPh3-3,4-salpyr)](ClO4)2 (), [Zn(5-CH2PPh3-3,4-salpyr)](ClO4)2 (), nano-[Ni(5-CH2PPh3-3,4-salpyr)](ClO4)2 (), [Ni(5-CH2PPh3-3,4-salpyr)](ClO4)2 (), and [N,N'-bis{5-[(triphenyl phosphonium chloride)-methyl]salicylidine}-2,3-diaminopyridine]Ni(ii) [Ni(5-CH2PPh3-2,3-salpyr)](ClO4)2 () have been isolated and characterized by elemental analysis, FT-IR, (1)H NMR, (13)C NMR, (31)P NMR, and UV-vis spectroscopy. The morphology and size of the nano complexes were determined using FE-SEM and TEM. In vitro DNA binding studies were investigated by UV-vis absorption spectroscopy, viscosity measurements, CD spectroscopy, cyclic voltammetry, emission spectra and gel electrophoresis, which suggest that the metal complexes act as efficient DNA binders. The absorption spectroscopy of the compounds with DNA reveals that the DNA binding affinity (Kb) has this order: > > > > > Ligand. The metal complexes show DNA binding stronger than the ligand, which is expected due to the nature of the metal. The nano complexes display DNA binding stronger than the other complexes which is related to the effect of size on binding affinity and the Ni(ii) complexes reveal DNA binding stronger than the corresponding Zn(ii) analogues, which is expected due to their z* effect and geometry. The prominent double strand DNA cleavage abilities of compound are observed in the absence of H2O2 with efficiencies of more than 50% even at 70 μM complex concentration. Surprisingly, Zn(ii) complexes (compounds & ) exhibit a higher cytotoxicity (IC50: 7.3 & 10.9 μM at 24 h; IC50: 4.6 & 8.7 μM at 48 h) against human hepatoma (HepG2) and HeLa cell lines than the Ni(ii) complexes (compounds , & ) and 5-fluorouracil as control in spite of their inability to cleave DNA. Finally, DNA binding interactions were performed by docking studies. Density functional

  5. DNA binding, DNA cleavage, antioxidant and cytotoxicity studies on ruthenium(II) complexes of benzaldehyde 4-methyl-3-thiosemicarbazones

    NASA Astrophysics Data System (ADS)

    Sampath, Krishnan; Sathiyaraj, Subbaiyan; Jayabalakrishnan, Chinnasamy

    2013-03-01

    Four new ruthenium(II) complexes with N(4)-methyl thiosemicarbazone ligands, (E)-2-(2-chlorobenzylidene)-N-methylhydrazinecarbothioamide (HL1) and (E)-N-methyl-2-(2-nitrobenzylidene)hydrazinecarbothioamide (HL2), were prepared and fully characterized by various spectro-analytical techniques. The Schiff bases act as bidentate, monobasic chelating ligands with S and N as the donor sites and are preferably found in the thiol form in all the complexes studied. The molecular structure of HL1 and HL2 were determined by single crystal X-ray diffraction method. DNA binding of the compounds was investigated by absorption spectroscopy which indicated that the complexes bind to DNA via intercalation. The oxidative cleavage of the complexes with CT-DNA inferred that the effects of cleavage are dose dependent. Antioxidant studies of the ligands and complexes showed the significant antioxidant activity against DPPH radical. In addition, the in vitro cytotoxicity of the ligands and complexes against MCF-7 cell line was assayed which showed higher cytotoxic activity with the lower IC50 values indicating their efficiency in killing the cancer cells even at low concentrations.

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

    PubMed

    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. PMID:24260216

  7. Copper(II) complexes with 4-hydroxyacetophenone-derived acylhydrazones: Synthesis, characterization, DNA binding and cleavage properties

    NASA Astrophysics Data System (ADS)

    Gup, Ramazan; Gökçe, Cansu; Aktürk, Selçuk

    2015-01-01

    Two new Cu(II) complexes of Schiff base-hydrazone ligands, hydroxy-N‧-[(1Z)-1-(4-hydroxyphenyl)ethylidene]benzohydrazide [H3L1] and ethyl 2-(4-(1-(2-(4-(2-ethoxy-2-oxoethoxy)benzoyl)hydrazono)ethyl)phenoxy)acetate (HL2) have been synthesized and then characterized by microcopy and spectral studies. X-ray powder diffraction illustrates that [Cu(L2)2] complex is crystalline in nature whereas [Cu(H2L1)2]·2H2O has an amorphous structure. Binding of the copper complexes with Calf thymus DNA (CT-DNA) has been investigated by UV-visible spectra, exhibiting non-covalent binding to CT-DNA. DNA cleavage experiments have been also investigated by agarose gel electrophoresis in the presence and absence of an oxidative agent (H2O2). The effect of complex concentration on the DNA cleavage reaction has been also studied. Both copper complexes show nuclease activity, which significantly depends on concentrations of the complexes, in the presence of H2O2 through oxidative mechanism whereas they slightly cleavage DNA in the absence an oxidative agent.

  8. HMG-box domain stimulation of RAG1/2 cleavage activity is metal ion dependent

    PubMed Central

    Kriatchko, Aleksei N; Bergeron, Serge; Swanson, Patrick C

    2008-01-01

    Background RAG1 and RAG2 initiate V(D)J recombination by assembling a synaptic complex with a pair of antigen receptor gene segments through interactions with their flanking recombination signal sequence (RSS), and then introducing a DNA double-strand break at each RSS, separating it from the adjacent coding segment. While the RAG proteins are sufficient to mediate RSS binding and cleavage in vitro, these activities are stimulated by the architectural DNA binding and bending factors HMGB1 and HMGB2. Two previous studies (Bergeron et al., 2005, and Dai et al., 2005) came to different conclusions regarding whether only one of the two DNA binding domains of HMGB1 is sufficient to stimulate RAG-mediated binding and cleavage of naked DNA in vitro. Here we test whether this apparent discrepancy is attributed to the choice of divalent metal ion and the concentration of HMGB1 used in the cleavage reaction. Results We show here that single HMG-box domains of HMGB1 stimulate RAG-mediated RSS cleavage in a concentration-dependent manner in the presence of Mn2+, but not Mg2+. Interestingly, the inability of a single HMG-box domain to stimulate RAG-mediated RSS cleavage in Mg2+ is overcome by the addition of partner RSS to promote synapsis. Furthermore, we show that mutant forms of HMGB1 which otherwise fail to stimulate RAG-mediated RSS cleavage in Mg2+ can be substantially rescued when Mg2+ is replaced with Mn2+. Conclusion The conflicting data published previously in two different laboratories can be substantially explained by the choice of divalent metal ion and abundance of HMGB1 in the cleavage reaction. The observation that single HMG-box domains can promote RAG-mediated 23-RSS cleavage in Mg2+ in the presence, but not absence, of partner RSS suggests that synaptic complex assembly in vitro is associated with conformational changes that alter how the RAG and/or HMGB1 proteins bind and bend DNA in a manner that functionally replaces the role of one of the HMG-box domains

  9. Mapping small DNA ligand hydroxyl radical footprinting and affinity cleavage products for capillary electrophoresis.

    PubMed

    He, Gaofei; Vasilieva, Elena; Bashkin, James K; Dupureur, Cynthia M

    2013-08-15

    The mapping of DNA footprints and affinity cleavage sites for small DNA ligands is affected by the choice of sequencing chemistry and end label, and the potential for indexing errors can be significant when mapping small ligand-DNA interactions. Described here is a mechanism for avoiding such errors based on a summary of standard labeling, cleavage, and indexing chemistries and a comparison among them for analysis of these interactions by capillary electrophoresis. The length dependence of the difference between Sanger and Maxam-Gilbert indexing is examined for a number of duplexes of mixed sequence. PMID:23608054

  10. Mapping Small DNA Ligand Hydroxyl Radical Footprinting and Affinity Cleavage Products for Capillary Electrophoresis

    PubMed Central

    He, Gaofei; Vasilieva, Elena; Bashkin, James K.; Dupureur, Cynthia M.

    2013-01-01

    The mapping of DNA footprints and affinity cleavage sites for small DNA ligands is affected by the choice of sequencing chemistry and end label, and the potential for indexing errors can be significant when mapping small ligand-DNA interactions. Described here is a mechanism for avoiding such errors based on a summary of standard labeling, cleavage and indexing chemistries and a comparison among them for analysis of these interactions by capillary electrophoresis. The length dependence of the difference between Sanger and Maxam-Gilbert indexing is examined for a number of duplexes of mixed sequence. PMID:23608054

  11. Synthesis, DNA recognition and cleavage studies of novel tetrapeptide complexes, Cu(II)/Zn(II)-Ala-Pro-Ala-Pro

    NASA Astrophysics Data System (ADS)

    Arjmand, Farukh; Jamsheera, A.; Mohapatra, D. K.

    2013-05-01

    New tetrapeptide complexes Cu(II)·Ala-Pro-Ala-Pro (1) and Zn(II)·Ala-Pro-Ala-Pro (2) were synthesized from the reaction of tetrapeptide, Ala-Pro-Ala-Pro and CuCl2/ZnCl2 and were thoroughly characterized by elemental analysis, IR,1H and 13C NMR (in case of 2), ESI-MS, UV and molar conductance measurements. The solution stability study was carried out employing UV-vis absorption titrations over a broad range of pH which suggested the stability of the complexes in solution. In vitro interaction of complexes 1 and 2 with CT-DNA was studied employing UV-vis, fluorescence, circular dichroic and viscometry studies. To throw insight into molecular binding event at the target site, UV-vis titrations of 1 and 2 with mononucleotides of interest viz.; 5'-GMP and 5'-TMP were carried out. Cleavage activity of the complexes with pBR322 plasmid DNA was evaluated by agarose gel electrophoresis and, the electrophoresis pattern demonstrated that both the complexes 1 and 2 are efficient cleavage agents. Further, the Cu(II) complex displayed efficient oxidative cleavage of supercoiled DNA while various reactive oxygen species are responsible for the cleavage in Zn(II) complex.

  12. Evaluation of DNA Binding, Cleavage, and Cytotoxic Activity of Cu(II), Co(II), and Ni(II) Schiff Base Complexes of 1-Phenylindoline-2,3-dione with Isonicotinohydrazide

    PubMed Central

    Gomathi, Ramadoss; Ramu, Andy; Murugan, Athiappan

    2014-01-01

    One new series of Cu(II), Co(II), and Ni(II) Schiff base complexes was prepared through the condensation reaction between 1-phenylindoline-2,3-dione with isonicotinohydrazide followed by metalation, respectively. The Schiff base ligand(L), (E)-N′-(2-oxo-1-phenylindolin-3-lidene)isonicotinohydrazide, and its complexes were found soluble in DMF and DMSO solvents and characterized by using the modern analytical and spectral techniques such as elemental analysis, conductivity, magnetic moments, IR, NMR, UV-visible, Mass, CV, and EPR. The elemental analysis data of ligand and their complexes were well agreed with their calculated values in which metal and ligand stoichiometry ratio 1 : 2 was noted. Molar conductance values indicated that all the complexes were found to be nonelectrolytes. All the complexes showed octahedral geometry around the central metal ions. Herein, we better characterized DNA binding with the complexes by UV-visible and CD spectroscopy and cyclic voltammetry techniques. The DNA cleavage experiments were carried out by Agarose gel electrophoresis method and the cytotoxicity experiments by MTT assay method. Based on the DNA binding, cleavage, and cytotoxicity studies, Cu and Ni complexes were found to be good anticancer agents against AGS-human gastric cancer cell line. PMID:24744691

  13. Transsynaptic signaling by activity-dependent cleavage of neuroligin-1.

    PubMed

    Peixoto, Rui T; Kunz, Portia A; Kwon, Hyungbae; Mabb, Angela M; Sabatini, Bernardo L; Philpot, Benjamin D; Ehlers, Michael D

    2012-10-18

    Adhesive contact between pre- and postsynaptic neurons initiates synapse formation during brain development and provides a natural means of transsynaptic signaling. Numerous adhesion molecules and their role during synapse development have been described in detail. However, once established, the mechanisms of adhesive disassembly and its function in regulating synaptic transmission have been unclear. Here, we report that synaptic activity induces acute proteolytic cleavage of neuroligin-1 (NLG1), a postsynaptic adhesion molecule at glutamatergic synapses. NLG1 cleavage is triggered by NMDA receptor activation, requires Ca2+ /calmodulin-dependent protein kinase, and is mediated by proteolytic activity of matrix metalloprotease 9 (MMP9). Cleavage of NLG1 occurs at single activated spines, is regulated by neural activity in vivo, and causes rapid destabilization of its presynaptic partner neurexin-1β (NRX1β). In turn, NLG1 cleavage depresses synaptic transmission by abruptly reducing presynaptic release probability. Thus, local proteolytic control of synaptic adhesion tunes synaptic transmission during brain development and plasticity. PMID:23083741

  14. Modeling Radial Holoblastic Cleavage: A Laboratory Activity for Developmental Biology.

    ERIC Educational Resources Information Center

    Ellis, Linda K.

    2000-01-01

    Introduces a laboratory activity designed for an undergraduate developmental biology course. Uses Play-Doh (plastic modeling clay) to build a multicellular embryo in order to provide a 3-D demonstration of cleavage. Includes notes for the instructor and student directions. (YDS)

  15. Sequence-specific cleavage of single-stranded DNA: oligodeoxynucleotide-EDTA X Fe(II).

    PubMed Central

    Dreyer, G B; Dervan, P B

    1985-01-01

    The synthesis of a DNA hybridization probe 19 nucleotides in length, equipped with the metal chelator EDTA at C-5 of thymidine in position 10 (indicated by T*) is described. DNA-EDTA 1 has the sequence 5'-T-A-A-C-G-C-A-G-T*-C-A-G-G-C-A-C-C-G-T-3', which is complementary to a 19-nucleotide sequence in the plasmid pBR322. In the presence of Fe(II), O2, and dithiothreitol, DNA-EDTA 1 affords specific cleavage (25 degrees C, pH 7.4, 60 min) at its complementary sequence in a heat-denatured 167-base-pair restriction fragment. Cleavage occurs over a range of 16 nucleotides at the site of hybridization of 1, presumably due to a diffusible reactive species. No other cleavage sites are observed in the 167-base-pair restriction fragment. The procedure used to synthesize DNA-EDTA probes is based on the incorporation of a thymidine modified at C-5 with the triethyl ester of EDTA. By using routine phosphoramidite procedures, thymidine-EDTA can be incorporated into oligodeoxynucleotides of any desired length and sequence. Because the efficiency of the DNA cleavage reaction is dependent on the addition of both Fe(II) and reducing agent (dithiothreitol), the initiation of the cleavage reaction can be controlled. These DNA-EDTA X Fe(II) probes should be useful for the sequence-specific cleavage of single-stranded DNA (and most likely RNA) under mild conditions. Images PMID:3919391

  16. Cleavage and activation of human factor IX by serine proteases

    SciTech Connect

    Enfield, D.L.; Thompson, A.R.

    1984-10-01

    Human factor IX circulates as a single-chain glycoprotein. Upon activation in vitro, it is cleaved into disulfide-linked light and heavy chains and an activation peptide. After reduction of activated /sup 125/I-factor IX, the heavy and light chains are readily identified by gel electrophoresis. A direct, immunoradiometric assay for factor IXa was developed to assess activation of factor IX for proteases that cleaved it. The assay utilized radiolabeled antithrombin III with heparin to identify the active site and antibodies to distinguish factor IX. After cleavage of factor IX by factor XIa, factor VIIa-tissue thromboplastin complex, or the factor X-activating enzyme from Russell's viper venom, antithrombin III bound readily to factor IXa. Cleavage of /sup 125/I-factor IX by trypsin, chymotrypsin, and granulocyte elastase in the presence of calcium yielded major polypeptide fragments of the sizes of the factor XIa-generated light and heavy chains. When the immunoradiometric assay was used to assess trypsin-cleaved factor IX, the product bound antithrombin III, but not maximally. After digesting with insolubilized trypsin, clotting activity confirmed activation. In evaluating activation of factor IX, physical evidence of activation cleavages does not necessarily correlate with generation of an active site.

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

    PubMed

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

    2016-05-19

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

  18. DNA cleavage enzymes for treatment of persistent viral infections: Recent advances and the pathway forward

    SciTech Connect

    Weber, Nicholas D.; Aubert, Martine; Dang, Chung H.; Stone, Daniel; Jerome, Keith R.

    2014-04-15

    Treatment for most persistent viral infections consists of palliative drug options rather than curative approaches. This is often because long-lasting viral DNA in infected cells is not affected by current antivirals, providing a source for viral persistence and reactivation. Targeting latent viral DNA itself could therefore provide a basis for novel curative strategies. DNA cleavage enzymes can be used to induce targeted mutagenesis of specific genes, including those of exogenous viruses. Although initial in vitro and even in vivo studies have been carried out using DNA cleavage enzymes targeting various viruses, many questions still remain concerning the feasibility of these strategies as they transition into preclinical research. Here, we review the most recent findings on DNA cleavage enzymes for human viral infections, consider the most relevant animal models for several human viral infections, and address issues regarding safety and enzyme delivery. Results from well-designed in vivo studies will ideally provide answers to the most urgent remaining questions, and allow continued progress toward clinical application. - Highlights: • Recent in vitro and in vivo results for DNA cleavage enzymes targeting persistent viral infections. • Analysis of the best animal models for testing enzymes for HBV, HSV, HIV and HPV. • Challenges facing in vivo delivery of therapeutic enzymes for persistent viral infections. • Safety issues to be addressed with proper animal studies.

  19. Shape Transformation Following Reduction-Sensitive PEG Cleavage of Polymer/DNA Nanoparticles

    PubMed Central

    Williford, John-Michael; Ren, Yong; Huang, Kevin; Pan, Deng; Mao, Hai-Quan

    2014-01-01

    PEGylated polycation/DNA micellar nanoparticles have been developed that can undergo shape transformation upon cleavage of the PEG grafts in response to an environmental cue. As a proof-of-principle, DNA nanoparticles with higher PEG grafting density adopting long, worm- and rod-like morphologies, transition to more condensed nanoparticles with spherical and short-rod morphologies upon cleavage of a fraction of the PEG grafts from the copolymer. This shape transformation leads to increased surface charges, correlating with improved transfection efficiency. PMID:25530853

  20. Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

    PubMed Central

    van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D.

    2015-01-01

    The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1–2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ∼30 bp. PMID:26507855

  1. Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations.

    PubMed

    van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D

    2015-12-01

    The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1-2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ∼30 bp. PMID:26507855

  2. Bifunctional alkylating agent-mediated MGMT-DNA cross-linking and its proteolytic cleavage in 16HBE cells.

    PubMed

    Cheng, Jin; Ye, Feng; Dan, Guorong; Zhao, Yuanpeng; Wang, Bin; Zhao, Jiqing; Sai, Yan; Zou, Zhongmin

    2016-08-15

    Nitrogen mustard (NM), a bifunctional alkylating agent (BAA), contains two alkyl arms and can act as a cross-linking bridge between DNA and protein to form a DNA-protein cross-link (DPC). O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme for alkyl adducts removal, is found to enhance cell sensitivity to BAAs and to promote damage, possibly due to its stable covalent cross-linking with DNA mediated by BAAs. To investigate MGMT-DNA cross-link (mDPC) formation and its possible dual roles in NM exposure, human bronchial epithelial cell line 16HBE was subjected to different concentrations of HN2, a kind of NM, and we found mDPC was induced by HN2 in a concentration-dependent manner, but the mRNA and total protein of MGMT were suppressed. As early as 1h after HN2 treatment, high mDPC was achieved and the level maintained for up to 24h. Quick total DPC (tDPC) and γ-H2AX accumulation were observed. To evaluate the effect of newly predicted protease DVC1 on DPC cleavage, we applied siRNA of MGMT and DVC1, MG132 (proteasome inhibitor), and NMS-873 (p97 inhibitor) and found that proteolysis plays a role. DVC1 was proven to be more important in the cleavage of mDPC than tDPC in a p97-dependent manner. HN2 exposure induced DVC1 upregulation, which was at least partially contributed to MGMT cleavage by proteolysis because HN2-induced mDPC level and DNA damage was closely related with DVC1 expression. Homologous recombination (HR) was also activated. Our findings demonstrated that MGMT might turn into a DNA damage promoter by forming DPC when exposed to HN2. Proteolysis, especially DVC1, plays a crucial role in mDPC repair. PMID:27342729

  3. The modulation of topoisomerase I-mediated DNA cleavage and the induction of DNA–topoisomerase I crosslinks by crotonaldehyde-derived DNA adducts

    PubMed Central

    Dexheimer, Thomas S.; Kozekova, Albena; Rizzo, Carmelo J.; Stone, Michael P.; Pommier, Yves

    2008-01-01

    Crotonaldehyde is a representative α,β-unsaturated aldehyde endowed of mutagenic and carcinogenic properties related to its propensity to react with DNA. Cyclic crotonaldehyde-derived deoxyguanosine (CrA-PdG) adducts can undergo ring opening in duplex DNA to yield a highly reactive aldehydic moiety. Here, we demonstrate that site-specifically modified DNA oligonucleotides containing a single CrA-PdG adduct can form crosslinks with topoisomerase I (Top1), both directly and indirectly. Direct covalent complex formation between the CrA-PdG adduct and Top1 is detectable after reduction with sodium cyanoborohydride, which is consistent with the formation of a Schiff base between Top1 and the ring open aldehyde form of the adduct. In addition, we show that the CrA-PdG adduct alters the cleavage and religation activities of Top1. It suppresses Top1 cleavage complexes at the adduct site and induces both reversible and irreversible cleavage complexes adjacent to the CrA-PdG adduct. The formation of stable DNA–Top1 crosslinks and the induction of Top1 cleavage complexes by CrA-PdG are mutually exclusive. Lastly, we found that crotonaldehyde induces the formation of DNA–Top1 complexes in mammalian cells, which suggests a potential relationship between formation of DNA–Top1 crosslinks and the mutagenic and carcinogenic properties of crotonaldehyde. PMID:18550580

  4. Efficient plasmid DNA cleavage by a mononuclear copper(II) complex.

    PubMed

    Sissi, Claudia; Mancin, Fabrizio; Gatos, Maddalena; Palumbo, Manlio; Tecilla, Paolo; Tonellato, Umberto

    2005-04-01

    The Cu(II) complex of the ligand all-cis-2,4,6-triamino-1,3,5-trihydroxycyclohexane (TACI) is a very efficient catalyst of the cleavage of plasmid DNA in the absence of any added cofactor. The maximum rate of degradation of the supercoiled plasmid DNA form, obtained at pH 8.1 and 37 degrees C, in the presence of 48 microM TACI.Cu(II), is 2.3 x 10(-3) s(-1), corresponding to a half-life time of only 5 min for the cleavage of form I (supercoiled) to form II (relaxed circular). The dependence of the rate of plasmid DNA cleavage from the TACI.Cu(II) complex concentration follows an unusual and very narrow bell-like profile, which suggests an high DNA affinity of the complexes but also a great tendency to form unreactive dimers. The reactivity of the TACI.Cu(II) complexes is not affected by the presence of several scavengers for reactive oxygen species or when measured under anaerobic conditions. Moreover, no degradation of the radical reporter Rhodamine B is observed in the presence of such complexes. These results are consistent with the operation of a prevailing hydrolytic pathway under the normal conditions used, although the failure to obtain enzymatic religation of the linearized DNA does not allow one to rule out the occurrence of a nonhydrolytic oxygen-independent cleavage. A concurrent oxidative mechanism becomes competitive upon addition of reductants or in the presence of high levels of molecular oxygen: under such conditions, in fact, a remarkable increase in the rate of DNA cleavage is observed. PMID:15792466

  5. Topoisomerase I-Mediated DNA Cleavage Induced by the Minor Groove-Directed Binding of Bibenzimidazoles to a Distal Site

    PubMed Central

    Khan, Qasim A.; Pilch, Daniel S.

    2007-01-01

    Summary Many agents (e.g., camptothecins, indolocarbazoles, indenoisoquinolines, and dibenzonaphthyridines) stimulate topoisomerase I-mediated DNA cleavage (a behavior termed topoisomerase I poisoning) by interacting with both the DNA and the enzyme at the site of cleavage (typically by intercalation between the −1 and +1 base pairs). The bibenzimidazoles, which include Hoechst 33258 and 33342, are a family of DNA minor groove-directed agents that also stimulate topoisomerase I-mediated DNA cleavage. However, the molecular mechanism by which these ligands poison TOP1 is poorly understood. Toward this goal, we have used a combination of mutational, footprinting, and DNA binding affinity analyses to define the DNA binding site for Hoechst 33258 and a related derivative that results in optimal induction of TOP1-mediated DNA cleavage. We show that this DNA binding site is located downstream from the site of DNA cleavage, encompassing the base pairs from position +4 to +8. The distal nature of this binding site relative to the site of DNA cleavage suggests that minor groove-directed agents like the bibenzimidazoles poison TOP1 via a mechanism distinct from compounds like the camptothecins, which interact at the site of cleavage. PMID:17095016

  6. Differential reaction kinetics, cleavage complex formation, and nonamer binding domain dependence dictate the structure-specific and sequence-specific nuclease activity of RAGs.

    PubMed

    Naik, Abani Kanta; Raghavan, Sathees C

    2012-01-20

    During V(D)J recombination, RAG (recombination-activating gene) complex cleaves DNA based on sequence specificity. Besides its physiological function, RAG has been shown to act as a structure-specific nuclease. Recently, we showed that the presence of cytosine within the single-stranded region of heteroduplex DNA is important when RAGs cleave on DNA structures. In the present study, we report that heteroduplex DNA containing a bubble region can be cleaved efficiently when present along with a recombination signal sequence (RSS) in cis or trans configuration. The sequence of the bubble region influences RAG cleavage at RSS when present in cis. We also find that the kinetics of RAG cleavage differs between RSS and bubble, wherein RSS cleavage reaches maximum efficiency faster than bubble cleavage. In addition, unlike RSS, RAG cleavage at bubbles does not lead to cleavage complex formation. Finally, we show that the "nonamer binding region," which regulates RAG cleavage on RSS, is not important during RAG activity in non-B DNA structures. Therefore, in the current study, we identify the possible mechanism by which RAG cleavage is regulated when it acts as a structure-specific nuclease. PMID:22119487

  7. DNA CLEAVAGE AND DETECTION OF DNA RADICALS FORMED FROM HYDRALAZINE AND COPPER (II) BY ESR AND IMMUNO-SPIN TRAPPING

    PubMed Central

    Sinha, Birandra K.; Leinisch, Fabian; Bhattacharjee, Suchandra; Mason, Ronald P.

    2014-01-01

    Metal ion-catalyzed oxidation of hydrazine and its derivatives leads to the formation of the hydrazyl radical and subsequently to oxy-radicals in the presence of molecular oxygen. Here we have examined the role of Cu2+-catalyzed oxidation of hydralazine in the induction of DNA damage. Neither 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) nor dimethyl sulfoxide (DMSO) were effective in inhibiting hydralazine-Cu2+-induced DNA damage. Singlet oxygen did not appear to participate in this DNA cleavage. The one-electron oxidation of hydralazine also leads to the formation of DNA radicals as confirmed by immuno-spin trapping with 5, 5-dimethyl-1-pyrroline-N-oxide. Electron spin resonance (ESR) and spin trapping studies further confirmed the formation of DNA radicals; predominantly 2′-deoxyadenosine radical adducts were detected, while some radicals were also detected with other nucleosides. Our results suggest that free hydroxyl radicals may not be the main damaging species causing DNA cleavage, and possibly, Cu-peroxide complexes, formed from Cu+-H2O2, areresponsible for this hydralazine-Cu2+-induced DNA cleavage. PMID:24502259

  8. DNA cleavage by oxymyoglobin and cysteine-introduced metmyoglobin.

    PubMed

    Deshpande, Megha Subhash; Junedi, Sendy; Prakash, Halan; Nagao, Satoshi; Yamanaka, Masaru; Hirota, Shun

    2014-12-11

    Double stranded DNA was cleaved oxidatively by incubation with oxygenated myoglobin, and Lys96Cys sperm whale myoglobin in its stable ferric form functioned as an artificial nuclease under air by formation of an oxygenated species, owing to electron transfer from the SH group of the introduced cysteine to the heme. PMID:25327831

  9. Dynamics of Bleomycin Interaction with a Strongly Bound Hairpin DNA Substrate, and Implications for Cleavage of the Bound DNA

    PubMed Central

    Bozeman, Trevor C.; Nanjunda, Rupesh; Tang, Chenhong; Liu, Yang; Segerman, Zachary J.; Zaleski, Paul A.; Wilson, W. David; Hecht, Sidney M.

    2013-01-01

    Recent studies involving DNAs bound strongly by bleomycins have documented that such DNAs are degraded by the antitumor antibiotic with characteristics different from those observed when studying the cleavage of randomly chosen DNAs in the presence of excess Fe•BLM. In the present study, surface plasmon resonance has been used to characterize the dynamics of BLM B2 binding to a strongly bound hairpin DNA, to define the effects of Fe3+, salt and temperature on BLM–DNA interaction. One strong primary DNA binding site, and at least one much weaker site was documented. In contrast, more than one strong cleavage site was found, an observation also made for two other hairpin DNAs. Evidence is presented for BLM equilibration between the stronger and weaker binding sites in a way that renders BLM unavailable to other, less strongly bound DNAs. Thus enhanced binding to a given site does not necessarily result in increased DNA degradation at that site, i.e. for strongly bound DNAs, the facility of DNA cleavage must involve parameters in addition to the intrinsic rate of C-4′ H atom abstraction from DNA sugars. PMID:23072568

  10. DNA binding, DNA cleavage and cytotoxicity studies of a new water soluble copper(II) complex: The effect of ligand shape on the mode of binding

    NASA Astrophysics Data System (ADS)

    Kashanian, Soheila; Khodaei, Mohammad Mehdi; Roshanfekr, Hamideh; Shahabadi, Nahid; Mansouri, Ghobad

    2012-02-01

    The interaction of native calf thymus DNA (CT-DNA) with [Cu(ph 2phen)(phen-dione)Cl]Cl was studied at physiological pH by spectrophotometric, spectrofluorometric, circular dichroism, and viscometric techniques. Considerable hypochromicity and red shift are observed in the UV absorption band of the Cu complex. Binding constants ( Kb) of DNA with the complex were calculated at different temperatures. Thermodynamic parameters, enthalpy and entropy changes were calculated according to Van't Hoff equation, which indicated that reaction is predominantly enthalpically driven. All these results indicate that Cu(II) complex interacts with CT-DNA via intercalative mode. Also, this new complex induced cleavage in pUC18 plasmid DNA as indicated in gel electrophoresis and showed excellent antitumor activity against K562 (human chronic myeloid leukemia) and human T lymphocyte carcinoma-Jurkat cell lines.

  11. CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus

    PubMed Central

    Ramanan, Vyas; Shlomai, Amir; Cox, David B.T.; Schwartz, Robert E.; Michailidis, Eleftherios; Bhatta, Ankit; Scott, David A.; Zhang, Feng; Rice, Charles M.; Bhatia, Sangeeta N.

    2015-01-01

    Chronic hepatitis B virus (HBV) infection is prevalent, deadly, and seldom cured due to the persistence of viral episomal DNA (cccDNA) in infected cells. Newly developed genome engineering tools may offer the ability to directly cleave viral DNA, thereby promoting viral clearance. Here, we show that the CRISPR/Cas9 system can specifically target and cleave conserved regions in the HBV genome, resulting in robust suppression of viral gene expression and replication. Upon sustained expression of Cas9 and appropriately chosen guide RNAs, we demonstrate cleavage of cccDNA by Cas9 and a dramatic reduction in both cccDNA and other parameters of viral gene expression and replication. Thus, we show that directly targeting viral episomal DNA is a novel therapeutic approach to control the virus and possibly cure patients. PMID:26035283

  12. Cell cycle-specific cleavage of Scc2 regulates its cohesin deposition activity

    PubMed Central

    Woodman, Julie; Fara, Tyler; Dzieciatkowska, Monika; Trejo, Michael; Luong, Nancy; Hansen, Kirk C.; Megee, Paul C.

    2014-01-01

    Sister chromatid cohesion (SCC), efficient DNA repair, and the regulation of some metazoan genes require the association of cohesins with chromosomes. Cohesins are deposited by a conserved heterodimeric loading complex composed of the Scc2 and Scc4 proteins in Saccharomyces cerevisiae, but how the Scc2/Scc4 deposition complex regulates the spatiotemporal association of cohesin with chromosomes is not understood. We examined Scc2 chromatin association during the cell division cycle and found that the affinity of Scc2 for chromatin increases biphasically during the cell cycle, increasing first transiently in late G1 phase and then again later in G2/M. Inactivation of Scc2 following DNA replication reduces cellular viability, suggesting that this post S-phase increase in Scc2 chromatin binding affinity is biologically relevant. Interestingly, high and low Scc2 chromatin binding levels correlate strongly with the presence of full-length or amino-terminally cleaved forms of Scc2, respectively, and the appearance of the cleaved Scc2 species is promoted in vitro either by treatment with specific cell cycle-staged cellular extracts or by dephosphorylation. Importantly, Scc2 cleavage eliminates Scc2–Scc4 physical interactions, and an scc2 truncation mutant that mimics in vivo Scc2 cleavage is defective for cohesin deposition. These observations suggest a previously unidentified mechanism for the spatiotemporal regulation of cohesin association with chromosomes through cell cycle regulation of Scc2 cohesin deposition activity by Scc2 dephosphorylation and cleavage. PMID:24778232

  13. Small molecule activators of pre-mRNA 3′ cleavage

    PubMed Central

    Ryan, Kevin; Khleborodova, Asya; Pan, Jingyi; Ryan, Xiaozhou P.

    2009-01-01

    3′ Cleavage and polyadenylation are obligatory steps in the biogenesis of most mammalian pre-mRNAs. In vitro reconstitution of the 3′ cleavage reaction from human cleavage factors requires high concentrations of creatine phosphate (CP), though how CP activates cleavage is not known. Previously, we proposed that CP might work by competitively inhibiting a cleavage-suppressing serine/threonine (S/T) phosphatase. Here we show that fluoride/EDTA, a general S/T phosphatase inhibitor, activates in vitro cleavage in place of CP. Subsequent testing of inhibitors specific for different S/T phosphatases showed that inhibitors of the PPM family of S/T phosphatases, which includes PP2C, but not the PPP family, which includes PP1, PP2A, and PP2B, activated 3′ cleavage in vitro. In particular, NCI 83633, an inhibitor of PP2C, activated extensive 3′ cleavage at a concentration 50-fold below that required by fluoride or CP. The testing of structural analogs led to the identification of a more potent compound that activated 3′ cleavage at 200 μM. While testing CP analogs to understand the origin of its cleavage activation effect, we found phosphocholine to be a more effective activator than CP. The minimal structural determinants of 3′ cleavage activation by phosphocholine were identified. Our results describe a much improved small molecule activator of in vitro pre-mRNA cleavage, identify the molecular determinants of cleavage activation by phosphoamines such as phosphocholine, and suggest that a PPM family phosphatase is involved in the negative regulation of mammalian pre-mRNA 3′ cleavage. PMID:19155323

  14. Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes.

    PubMed

    Chand, Mahesh K; Nirwan, Neha; Diffin, Fiona M; van Aelst, Kara; Kulkarni, Manasi; Pernstich, Christian; Szczelkun, Mark D; Saikrishnan, Kayarat

    2015-11-01

    Production of endonucleolytic double-strand DNA breaks requires separate strand cleavage events. Although catalytic mechanisms for simple, dimeric endonucleases are known, there are many complex nuclease machines that are poorly understood. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide after convergent ATP-driven translocation. We report the 2.7-Å resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are located upstream of the direction of translocation, an observation inconsistent with simple nuclease-domain dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex in which the nuclease domains are distal. Sequencing of the products of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand-nicking events combine to produce DNA scission. PMID:26389736

  15. Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes

    PubMed Central

    Chand, Mahesh Kumar; Nirwan, Neha; Diffin, Fiona M.; van Aelst, Kara; Kulkarni, Manasi; Pernstich, Christian; Szczelkun, Mark D.; Saikrishnan, Kayarat

    2015-01-01

    Endonucleolytic double-strand DNA break production requires separate strand cleavage events. Although catalytic mechanisms for simple dimeric endonucleases are available, there are many complex nuclease machines which are poorly understood in comparison. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide following convergent ATP-driven translocation. We report the 2.7 Angstroms resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are unexpectedly located upstream of the direction of translocation, inconsistent with simple nuclease domain-dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex where the nuclease domains are distal. Sequencing of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand nicking events combine to produce DNA scission. PMID:26389736

  16. Photo-induced DNA cleavage and cytotoxicity of a ruthenium(II) arene anticancer complex.

    PubMed

    Brabec, Viktor; Pracharova, Jitka; Stepankova, Jana; Sadler, Peter J; Kasparkova, Jana

    2016-07-01

    We report DNA cleavage by ruthenium(II) arene anticancer complex [(η(6)-p-terp)Ru(II)(en)Cl](+) (p-terp=para-terphenyl, en=1,2-diaminoethane, complex 1) after its photoactivation by UVA and visible light, and the toxic effects of photoactivated 1 in cancer cells. It was shown in our previous work (T. Bugarcic et al., J. Med. Chem. 51 (2008) 5310-5319) that this complex exhibits promising toxic effects in several human tumor cell lines and concomitantly its DNA binding mode involves combined intercalative and monofunctional (coordination) binding modes. We demonstrate in the present work that when photoactivated by UVA or visible light, 1 efficiently photocleaves DNA, also in hypoxic media. Studies of the mechanism underlying DNA cleavage by photoactivated 1 reveal that the photocleavage reaction does not involve generation of reactive oxygen species (ROS), although contribution of singlet oxygen ((1)O2) to the DNA photocleavage process cannot be entirely excluded. Notably, the mechanism of DNA photocleavage by 1 appears to involve a direct modification of mainly those guanine residues to which 1 is coordinatively bound. As some tumors are oxygen-deficient and cytotoxic effects of photoactivated ruthenium compounds containing {Ru(η(6)-arene)}(2+) do not require the presence of oxygen, this class of ruthenium complexes may be considered potential candidate agents for improved photodynamic anticancer chemotherapy. PMID:26778426

  17. Effects of 2′-O-Methyl Nucleotide Substitution on EcoRI Endonuclease Cleavage Activities

    PubMed Central

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

    2013-01-01

    To investigate the effect of sugar pucker conformation on DNA-protein interactions, we used 2′-O-methyl nucleotide (2′-OMeN) to modify the EcoRI recognition sequence -TGAATTCT-, and monitored the enzymatic cleavage process using FRET method. The 2′-O-methyl nucleotide has a C3′-endo sugar pucker conformation different from the C2′-endo sugar pucker conformation of native DNA nucleotides. The initial reaction velocities were measured and the kinetic parameters, Km and Vmax were derived using Michaelis-Menten equation. Experimental results showed that 2′-OMeN substitutions for the EcoRI recognition sequence decreased the cleavage efficiency for A2, A3 and T4 substitutions significantly, and 2′-OMeN substitution for T5 residue inhibited the enzymatic activity completely. In contrast, substitutions for G1 and C6 could maintain the original activity. 2′-fluoro nucleic acid (2′-FNA) and locked nucleic acid (LNA) having similar C3′-endo sugar pucker conformation also demonstrated similar enzymatic results. This position-dependent enzymatic cleavage property might be attributed to the phosphate backbone distortion caused by the switch from C2′-endo to C3′-endo sugar pucker conformation, and was interpreted on the basis of the DNA-EcoRI structure. These 2′-modified nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property will have potential applications in genetic engineering and biomedicine. PMID:24194862

  18. Caspase-10 triggers Bid cleavage and caspase cascade activation in FasL-induced apoptosis.

    PubMed

    Milhas, Delphine; Cuvillier, Olivier; Therville, Nicole; Clavé, Patricia; Thomsen, Mogens; Levade, Thierry; Benoist, Hervé; Ségui, Bruno

    2005-05-20

    In contrast to caspase-8, controversy exists as to the ability of caspase-10 to mediate apoptosis in response to FasL. Herein, we have shown activation of caspase-10, -3, and -7 as well as B cell lymphoma-2-interacting domain (Bid) cleavage and cytochrome c release in caspase-8-deficient Jurkat (I9-2) cells treated with FasL. Apoptosis was clearly induced as illustrated by nuclear and DNA fragmentation. These events were inhibited by benzyloxycarbonyl-VAD-fluoromethyl ketone, a broad spectrum caspase inhibitor, indicating that caspases were functionally and actively involved. Benzyloxycarbonyl-AEVD-fluoromethyl ketone, a caspase-10 inhibitor, had a comparable effect. FasL-induced cell death was not completely abolished by caspase inhibitors in agreement with the existence of a cytotoxic caspase-independent pathway. In subpopulations of I9-2 cells displaying distinct caspase-10 expression levels, cell sensitivity to FasL correlated with caspase-10 expression. A robust caspase activation, Bid cleavage, and DNA fragmentation were observed in cells with high caspase-10 levels but not in those with low levels. In vitro, caspase-10, as well as caspase-8, could cleave Bid to generate active truncated Bid (p15). Altogether, our data strongly suggest that caspase-10 can serve as an initiator caspase in Fas signaling leading to Bid processing, caspase cascade activation, and apoptosis. PMID:15772077

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

    PubMed

    Li, Cuiping; Wang, Hailin

    2015-08-01

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

  20. Rates of Chemical Cleavage of DNA and RNA Oligomers Containing Guanine Oxidation Products

    PubMed Central

    2016-01-01

    The nucleobase guanine in DNA (dG) and RNA (rG) has the lowest standard reduction potential of the bases, rendering it a major site of oxidative damage in these polymers. Mapping the sites at which oxidation occurs in an oligomer via chemical reagents utilizes hot piperidine for cleaving oxidized DNA and aniline (pH 4.5) for cleaving oxidized RNA. In the present studies, a series of time-dependent cleavages of DNA and RNA strands containing various guanine lesions were examined to determine the strand scission rate constants. The guanine base lesions 8-oxo-7,8-dihydroguanine (OG), spiroiminodihydantoin (Sp), 5-guanidinohydantoin (Gh), 2,2,4-triamino-2H-oxazol-5-one (Z), and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) were evaluated in piperidine-treated DNA and aniline-treated RNA. These data identified wide variability in the chemical lability of the lesions studied in both DNA and RNA. Further, the rate constants for cleaving lesions in RNA were generally found to be significantly smaller than for lesions in DNA. The OG nucleotides were poorly cleaved in DNA and RNA; Sp nucleotides were slowly cleaved in DNA and did not cleave significantly in RNA; Gh and Z nucleotides cleaved in both DNA and RNA at intermediate rates; and 2Ih oligonucleotides cleaved relatively quickly in both DNA and RNA. The data are compared and contrasted with respect to future experimental design. PMID:25853314

  1. Methylene blue photosensitised strand cleavage of DNA: effects of dye binding and oxygen.

    PubMed Central

    OhUigin, C; McConnell, D J; Kelly, J M; van der Putten, W J

    1987-01-01

    It is shown that methylene blue (MB+) photosensitises DNA in either aerated or deaerated solutions, causing direct cleavage of phosphodiester bonds and rendering additional bonds labile to alkali. Evidence from unwinding and fluorimetric studies indicates that MB+ binds to DNA in at least 2 ways. Intercalation, which optimally induces helical unwinding of 24 degrees +/- 2 degrees per MB+, is markedly reduced upon neutralisation by Mg2+ of the DNA phosphates, while significant non-intercalative binding persists as shown by substantial fluorescence quenching at Mg2+ concentrations where there is little unwinding. MB+ induces photolysis at both low and high Mg2+ concentration - intercalation is apparently not required for photolysis. The quantum yield for strand breakage varies from 1-3 X 10(-7) under different conditions and is oxygen enhanced. The DNA cleavage is guanine specific. The 3' termini of the primary MB+-induced DNA photoproducts, unlike those generated by chemical sequencing retain an alkali labile adduct on the terminal phosphate. Images PMID:2821508

  2. The beyond 12/23 restriction is imposed at the nicking and pairing steps of DNA cleavage during V(D)J recombination.

    PubMed

    Drejer-Teel, Anna H; Fugmann, Sebastian D; Schatz, David G

    2007-09-01

    The beyond 12/23 (B12/23) rule ensures inclusion of a Dbeta gene segment in the assembled T-cell receptor (TCR) beta variable region exon and is manifest by a failure of direct Vbeta-to-Jbeta gene segment joining. The restriction is enforced during the DNA cleavage step of V(D)J recombination by the recombination-activating gene 1 and 2 (RAG1/2) proteins and the recombination signal sequences (RSSs) flanking the TCRbeta gene segments. Nothing is known about the step(s) at which DNA cleavage is defective or how TCRbeta locus sequences contribute to these defects. To address this, we examined the steps of DNA cleavage by the RAG proteins using TCRbeta locus V, D, and J RSS oligonucleotide substrates. The results demonstrate that the B12/23 rule is enforced through slow nicking of Jbeta substrates and to some extent through poor synapsis of Vbeta and Jbeta substrates. Nicking is controlled largely by the coding flank and, unexpectedly, the RSS spacer, while synapsis is controlled primarily by the RSS nonamer. The results demonstrate that different Jbeta substrates are crippled at different steps of cleavage by distinct combinations of defects in the various DNA elements and strongly suggest that the DNA nicking step of V(D)J recombination can be rate limiting in vivo. PMID:17636023

  3. The Beyond 12/23 Restriction Is Imposed at the Nicking and Pairing Steps of DNA Cleavage during V(D)J Recombination▿

    PubMed Central

    Drejer-Teel, Anna H.; Fugmann, Sebastian D.; Schatz, David G.

    2007-01-01

    The beyond 12/23 (B12/23) rule ensures inclusion of a Dβ gene segment in the assembled T-cell receptor (TCR) β variable region exon and is manifest by a failure of direct Vβ-to-Jβ gene segment joining. The restriction is enforced during the DNA cleavage step of V(D)J recombination by the recombination-activating gene 1 and 2 (RAG1/2) proteins and the recombination signal sequences (RSSs) flanking the TCRβ gene segments. Nothing is known about the step(s) at which DNA cleavage is defective or how TCRβ locus sequences contribute to these defects. To address this, we examined the steps of DNA cleavage by the RAG proteins using TCRβ locus V, D, and J RSS oligonucleotide substrates. The results demonstrate that the B12/23 rule is enforced through slow nicking of Jβ substrates and to some extent through poor synapsis of Vβ and Jβ substrates. Nicking is controlled largely by the coding flank and, unexpectedly, the RSS spacer, while synapsis is controlled primarily by the RSS nonamer. The results demonstrate that different Jβ substrates are crippled at different steps of cleavage by distinct combinations of defects in the various DNA elements and strongly suggest that the DNA nicking step of V(D)J recombination can be rate limiting in vivo. PMID:17636023

  4. Red-light photosensitized cleavage of DNA by (l-lysine)(phenanthroline base)copper(II) complexes.

    PubMed

    Patra, Ashis K; Nethaji, Munirathinam; Chakravarty, Akhil R

    2005-08-21

    Ternary copper(II) complexes [Cu(l-lys)B(ClO4)](ClO4)(1-4), where B is a heterocyclic base, viz. 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 3) and dipyrido[3,2-a:2',3'-c]phenazene (dppz, 4), are prepared and their DNA binding and photo-induced DNA cleavage activity studied (l-lys =l-lysine). Complex 2, structurally characterized by X-ray crystallography, shows a square-pyramidal (4 + 1) coordination geometry in which the N,O-donor l-lysine and N,N-donor heterocyclic base bind at the basal plane and the perchlorate ligand is bonded at the elongated axial site. The crystal structure shows the presence of a pendant cationic amine moiety -(CH2)4NH3+ of l-lysine. The one-electron paramagnetic complexes display a d-d band in the range of 598-762 nm in DMF and exhibit cyclic voltammetric response due to Cu(II)/Cu(I) couple in the range of 0.07 to -0.20 V vs. SCE in DMF-Tris-HCl buffer. The complexes having phenanthroline bases display good binding propensity to the calf thymus DNA giving an order: 4 (dppz) > 3 (dpq) > 2 (phen)> 1 (bpy). Control cleavage experiments using pUC19 supercoiled DNA and distamycin suggest major groove binding for the dppz and minor groove binding for the other complexes. Complexes 2-4 show efficient DNA cleavage activity on UV (365 nm) or visible light (694 nm ruby laser) irradiation via a mechanistic pathway involving formation of singlet oxygen as the reactive species. The amino acid l-lysine bound to the metal shows photosensitizing effect at red light, while the heterocyclic bases are primarily DNA groove binders. The dpq and dppz ligands display red light-induced photosensitizing effects in copper-bound form. PMID:16075123

  5. Diastereoselective DNA Cleavage Recognition by Ni(II)•Gly-Gly-His Derived Metallopeptides

    PubMed Central

    Fang, Ya-Yin; Claussen, Craig A.; Lipkowitz, Kenny B.; Long, Eric C.

    2008-01-01

    Site-selective DNA cleavage by diastereoisomers of Ni(II)•Gly-Gly-His-derived metallopeptides was investigated through high-resolution gel analyses and molecular dynamics simulations. Ni(II)•L-Arg-Gly-His and Ni(II)•D-Arg-Gly-His (and their respective Lys analogues) targeted A/T-rich regions; however, the L-isomers consistently modified a sub-set of available nucleotides within a given minor groove site while the D-isomers differed in both their sites of preference and ability to target individual nucleotides within some sites. In comparison, Ni(II)•L-Pro-Gly-His and Ni(II)•D-Pro-Gly-His were unable to exhibit a similar diastereoselectivity. Simulations of the above systems, along with Ni(II)•Gly-Gly-His, indicated that the stereochemistry of the amino-terminal amino acid produces either an isohelical metallopeptide that associates stably at individual DNA sites (L-Arg or L-Lys) or, with D-Arg and D-Lys, a non-complementary metallopeptide structure that cannot fully employ its side chain nor amino-terminal amine as a positional stabilizing moiety. In contrast, amino-terminal Pro-containing metallopeptides of either stereochemistry, lacking an extended side chain directed toward the minor groove, did not exhibit a similar diastereoselectivity. While the identity and stereochemistry of amino acids located in the amino-terminal peptide position influenced DNA cleavage, metallopeptide diastereoisomers containing L- and D-Arg (or Lys) within the second peptide position did not exhibit diastereoselective DNA cleavage patterns; simulations indicated that a positively-charged amino acid in this location alters the interaction of the metallopeptide equatorial plane and the minor groove leading to an interaction similar to Ni(II)•Gly-Gly-His. PMID:16522100

  6. DNA cleavage by new oxovanadium(IV) complexes of N-salicylidene alpha-amino acids and phenanthroline bases in the photodynamic therapy window.

    PubMed

    Sasmal, Pijus K; Patra, Ashis K; Nethaji, Munirathinam; Chakravarty, Akhil R

    2007-12-24

    Oxovanadium(IV) complexes [VO(salmet)(B)] (1-3) and [VO(saltrp)(B)] (4-6), where salmet and saltrp are N-salicylidene-l-methionate and N-salicylidene-l-tryptophanate, respectively, and B is a N,N-donor heterocyclic base (viz. 1,10-phenanthroline (phen, 1, 4), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 2, 5), and dipyrido[3,2-a:2',3'-c]phenazine (dppz, 3, 6)) are prepared and characterized and their DNA binding and photoinduced DNA cleavage activity studied. Complexes 1, 2, and 4 are structurally characterized by single-crystal X-ray crystallography. The molecular structure shows the presence of a vanadyl group in the VO3N3 coordination geometry. The dianionic alpha-amino acid Schiff base acts as a tridentate O,N,O-donor ligand in a meridional binding mode. The N,N-donor heterocyclic base displays a chelating mode of bonding with a N-donor site trans to the oxo group. The complexes show a d-d band in the range of 680-710 nm in DMF with a shoulder near 840 nm. They exhibit an irreversible oxidative cyclic voltammetric response near 0.8 V assignable to the V(V)/V(IV) couple and a quasi-reversible V(IV)/V(III) redox couple near -1.1 V vs SCE in DMF-0.1 M TBAP. The complexes show good binding propensity to calf thymus DNA giving binding constant values in the range from 5.2 x 10(4) to 7.2 x 10(5) M(-1). The binding site size, thermal melting, and viscosity data suggest DNA surface and/or groove binding nature of the complexes. The complexes show poor "chemical nuclease" activity in the dark in the presence of 3-mercaptopropionic acid or hydrogen peroxide. The dpq and dppz complexes show efficient DNA cleavage activity on irradiation with UV-A light of 365 nm via a mechanistic pathway involving formation of singlet oxygen as the reactive species. They also show significant DNA cleavage activity on photoexcitation in red light (>750 nm) by (1)O2 species. Observation of red-light-induced cleavage of DNA is unprecedented in the vanadium chemistry. The DNA cleavage activity is

  7. Sox11 Reduces Caspase-6 Cleavage and Activity

    PubMed Central

    Waldron-Roby, Elaine; Hoerauf, Janine; Arbez, Nicolas; Zhu, Shanshan; Kulcsar, Kirsten; Ross, Christopher A.

    2015-01-01

    The apoptotic cascade is an orchestrated event, whose final stages are mediated by effector caspases. Regulatory binding proteins have been identified for caspases such as caspase-3, -7, -8, and -9. Many of these proteins belong to the inhibitor of apoptosis (IAP) family. By contrast, caspase-6 is not believed to be influenced by IAPs, and little is known about its regulation. We therefore performed a yeast-two-hybrid screen using a constitutively inactive form of caspase-6 for bait in order to identify novel regulators of caspase-6 activity. Sox11 was identified as a potential caspase-6 interacting protein. Sox11 was capable of dramatically reducing caspase-6 activity, as well as preventing caspase-6 self- cleavage. Several regions, including amino acids 117–214 and 362–395 within sox11 as well as a nuclear localization signal (NLS) all contributed to the reduction in caspase-6 activity. Furthermore, sox11 was also capable of decreasing other effector caspase activity but not initiator caspases -8 and -9. The ability of sox11 to reduce effector caspase activity was also reflected in its capacity to reduce cell death following toxic insult. Interestingly, other sox proteins also had the ability to reduce caspase-6 activity but to a lesser extent than sox11. PMID:26505998

  8. Synthesis, characterization, DNA binding and cleavage studies of chiral Ru(II) salen complexes

    NASA Astrophysics Data System (ADS)

    Khan, Noor-ul H.; Pandya, Nirali; Kureshy, Rukhsana I.; Abdi, Sayed H. R.; Agrawal, Santosh; Bajaj, Hari C.; Pandya, Jagruti; Gupte, Akashya

    2009-09-01

    Interaction of chiral Ru(II) salen complexes (S)-1 and (R)-1 with Calf Thymus DNA (CT-DNA) was studied by absorption spectroscopy, competitive binding study, viscosity measurements, CD measurements, thermal denaturation study and cleavage studies by agarose gel electrophoresis. The DNA binding affinity of (S)-1 (6.25 × 10 3 M -1) was found to be greater than (R)-1 (3.0 × 10 3 M -1). The antimicrobial studies of these complexes on five different gram (+)/(-) bacteria and three different fungal organisms showed selective inhibition of the growth of gram (+) bacteria and were not affective against gram (-) and fungal organisms. Further, the (S)-1 enantiomer inhibited the growth of organisms to a greater extent as compared to (R)-1 enantiomer.

  9. Sequence-specific interactions of drugs interfering with the topoisomerase-DNA cleavage complex.

    PubMed

    Palumbo, Manlio; Gatto, Barbara; Moro, Stefano; Sissi, Claudia; Zagotto, Giuseppe

    2002-07-18

    DNA-processing enzymes, such as the topoisomerases (tops), represent major targets for potent anticancer (and antibacterial) agents. The drugs kill cells by poisoning the enzymes' catalytic cycle. Understanding the molecular details of top poisoning is a fundamental requisite for the rational development of novel, more effective antineoplastic drugs. In this connection, sequence-specific recognition of the top-DNA complex is a key step to preferentially direct the action of the drugs onto selected genomic sequences. In fact, the (reversible) interference of drugs with the top-DNA complex exhibits well-defined preferences for DNA bases in the proximity of the cleavage site, each drug showing peculiarities connected to its structural features. A second level of selectivity can be observed when chemically reactive groups are present in the structure of the top-directed drug. In this case, the enzyme recognizes or generates a unique site for covalent drug-DNA binding. This will further subtly modulate the drug's efficiency in stimulating DNA damage at selected sites. Finally, drugs can discriminate not only among different types of tops, but also among different isoenzymes, providing an additional level of specific selection. Once the molecular basis for DNA sequence-dependent recognition has been established, the above-mentioned modes to generate selectivity in drug poisoning can be rationally exploited, alone or in combination, to develop tailor-made drugs targeted at defined loci in cancer cells. PMID:12084456

  10. Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage

    SciTech Connect

    Yuan,Y.; Pei, Y.; Ma, J.; Kuryavyi, V.; Zhadina, M.; Meister, G.; Chen, H.; Dauter, Z.; Tuschi, T.; Patel, D.

    2005-01-01

    Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5' phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by RNase H-type divalent cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent hAgo2 reinforce the role of the PIWI domain in 'slicer' activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct perspectives into the mechanism of guide strand-mediated mRNA cleavage within the RISC.

  11. Electrochemical DNA sensor-based strategy for sensitive detection of DNA demethylation and DNA demethylase activity.

    PubMed

    Shen, Qingming; Fan, Mengxing; Yang, Yin; Zhang, Hui

    2016-08-31

    DNA demethylation and demethylase activity play important roles in DNA self-repair, and their detection is key to early diagnosis of fatal diseases. Herein, a facile electrochemical DNA (E-DNA) sensor was developed for the sensitive detection of DNA demethylation and demethylase activity based on an enzyme cleavage strategy. The thiol modified hemi-methylated hairpin probe DNA (pDNA) was self-assembled on a Au electrode surface through the formation of AuS bonds. The hemi-methylated pDNA served as the substrate of DNA demethylase (using methyl-CpG-binding domain protein 2 (MBD2) as an example). Following demethylation, the hairpin stem was then recognized and cleaved by BstUI endonuclease. The ferrocene carboxylic acid (FcA)-tagged pDNA strands were released into the buffer solution from the electrode surface, resulting in a significant decrease of electrochemical signal and providing a means to observe DNA demethylation. The activity of DNA demethylase was analyzed in the concentration ranging from 0.5 to 500 ng mL(-1) with a limit of detection as low as 0.17 ng mL(-1). With high specificity and sensitivity, rapid response, and low cost, this simple E-DNA sensor provides a unique platform for the sensitive detection of DNA demethylation, DNA demethylase activity, and related molecular diagnostics and drug screening. PMID:27506345

  12. Ferromagnetic nanoparticles with peroxidase-like activity enhance the cleavage of biological macromolecules for biofilm elimination

    NASA Astrophysics Data System (ADS)

    GaoCurrent Address: University Of Pennsylvania, School Of Dental Medicine, Philadelphia, Pa 19104, Usa. E.-Mail: Gaoliz@Dental. Upenn. Edu, Lizeng; Giglio, Krista M.; Nelson, Jacquelyn L.; Sondermann, Holger; Travis, Alexander J.

    2014-02-01

    strategy for biofilm elimination, and other applications utilizing oxidative breakdown. Electronic supplementary information (ESI) available: Magnetic nanoparticles with peroxidase activity, cleavage details on DNA and BSA, killing of E. coli, and cell viability of Pseudomonas aeruginosa in biofilms. See DOI: 10.1039/c3nr05422e

  13. Metal-assisted red light-induced DNA cleavage by ternary L-methionine copper(II) complexes of planar heterocyclic bases.

    PubMed

    Patra, Ashis K; Dhar, Shanta; Nethaji, Munirathinam; Chakravarty, Akhil R

    2005-03-01

    Ternary copper(II) complexes [Cu(l-met)B(Solv)](ClO4) (1-4), where B is a N,N-donor heterocyclic base like 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 3) and dipyrido[3,2-a:2'],3'-c]phenazene (dppz, 4), are prepared and their DNA binding and photo-induced DNA cleavage activity studied (L-Hmet =L-methionine). Complex 2, structurally characterized by X-ray crystallography, shows a square pyramidal (4 + 1) coordination geometry in which the N,O-donor L-methionine and N,N-donor heterocyclic base bind at the basal plane and a solvent molecule is coordinated at the axial site. The complexes display a d-d band at approximately 600 nm in DMF and exhibit a cyclic voltammetric response due to the Cu(II)/Cu(I) couple near -0.1 V in DMF-Tris-HCl buffer. The complexes display significant binding propensity to the calf thymus DNA in the order: 4(dppz) > 3(dpq) > 2(phen> 1(bpy). Control cleavage experiments using pUC19 supercoiled DNA and distamycin suggest major groove binding for the dppz and minor groove binding for the other complexes. Complexes 2-4 show efficient DNA cleavage activity on UV (365 nm) or red light (632.8 nm) irradiation via a mechanistic pathway involving formation of singlet oxygen as the reactive species. The DNA cleavage activity of the dpq complex is found to be significantly more than its dppz and phen analogues. PMID:15726142

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

    PubMed Central

    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

  15. Detection of Strand Cleavage And Oxidation Damage Using Model DNA Molecules Captured in a Nanoscale Pore

    NASA Technical Reports Server (NTRS)

    Vercoutere, W.; Solbrig, A.; DeGuzman, V.; Deamer, D.; Akeson, M.

    2003-01-01

    We use a biological nano-scale pore to distinguish among individual DNA hairpins that differ by a single site of oxidation or a nick in the sugar-phosphate backbone. In earlier work we showed that the protein ion channel alpha-hemolysin can be used as a detector to distinguish single-stranded from double-stranded DNA, single base pair and single nucleotide differences. This resolution is in part a result of sensitivity to structural changes that influence the molecular dynamics of nucleotides within DNA. The strand cleavage products we examined here included a 5-base-pair (5-bp) hairpin with a 5-prime five-nucleotide overhang, and a complementary five-nucleotide oligomer. These produced predictable shoulder-spike and rapid near-full blockade signatures, respectively. When combined, strand annealing was monitored in real time. The residual current level dropped to a lower discrete level in the shoulder-spike blockade signatures, and the duration lengthened. However, these blockade signatures had a shorter duration than the unmodified l0bp hairpin. To test the pore sensitivity to nucleotide oxidation, we examined a 9-bp hairpin with a terminal 8-oxo-deoxyguanosine (8-oxo-dG), or a penultimate 8-oxo-dG. Each produced blockade signatures that differed from the otherwise identical control 9bp hairpins. This study showed that DNA structure is modified sufficiently by strand cleavage or oxidation damage at a single site to alter in a predictable manner the ionic current blockade signatures produced. This technique improves the ability to assess damage to DNA, and can provide a simple means to help characterize the risks of radiation exposure. It may also provide a method to test radiation protection.

  16. Retinoid-induced apoptosis and Sp1 cleavage occur independently of transcription and require caspase activation.

    PubMed Central

    Piedrafita, F J; Pfahl, M

    1997-01-01

    Vitamin A and its derivatives, the retinoids, are essential regulators of many important biological functions, including cell growth and differentiation, development, homeostasis, and carcinogenesis. Natural retinoids such as all-trans retinoic acid can induce cell differentiation and inhibit growth of certain cancer cells. We recently identified a novel class of synthetic retinoids with strong anti-cancer cell activities in vitro and in vivo which can induce apoptosis in several cancer cell lines. Using an electrophoretic mobility shift assay, we analyzed the DNA binding activity of several transcription factors in T cells treated with apoptotic retinoids. We found that the DNA binding activity of the general transcription factor Sp1 is lost in retinoid-treated T cells undergoing apoptosis. A truncated Sp1 protein is detected by immunoblot analysis, and cytosolic protein extracts prepared from apoptotic cells contain a protease activity which specifically cleaves purified Sp1 in vitro. This proteolysis of Sp1 can be inhibited by N-ethylmaleimide and iodoacetamide, indicating that a cysteine protease mediates cleavage of Sp1. Furthermore, inhibition of Sp1 cleavage by ZVAD-fmk and ZDEVD-fmk suggests that caspases are directly involved in this event. In fact, caspases 2 and 3 are activated in T cells after treatment with apoptotic retinoids. The peptide inhibitors also blocked retinoid-induced apoptosis, as well as processing of caspases and proteolysis of Sp1 and poly(ADP-ribose) polymerase in intact cells. Degradation of Sp1 occurs early during apoptosis and is therefore likely to have profound effects on the basal transcription status of the cell. Interestingly, retinoid-induced apoptosis does not require de novo mRNA and protein synthesis, suggesting that a novel mechanism of retinoid signaling is involved, triggering cell death in a transcriptional activation-independent, caspase-dependent manner. PMID:9343396

  17. Evolutionary tree for apes and humans based on cleavage maps of mitochondrial DNA.

    PubMed Central

    Ferris, S D; Wilson, A C; Brown, W M

    1981-01-01

    The high rate of evolution of mitochondrial DNA makes this molecule suitable for genealogical research on such closely related species as humans and apes. Because previous approaches failed to establish the branching order of the lineages leading to humans, gorillas, and chimpanzees, we compared human mitochondrial DNA to mitochondrial DNA from five species of ape (common chimpanzee, pygmy chimpanzee, gorilla, orangutan, and gibbon). About 50 restriction endonuclease cleavage sites were mapped in each mitochondrial DNA, and the six maps were aligned with respect to 11 invariant positions. Differences among the maps were evident at 121 positions. Both conserved and variable sites are widely dispersed in the mitochondrial genome. Besides site differences, ascribed to point mutations, there is evidence for one rearrangement: the gorilla map is shorter than the other owing to the deletion of 95 base pairs near the origin of replication. The parsimony method of deriving all six maps from a common ancestor produced a genealogical tree in which the common and pygmy chimpanzee maps are the most closely related pair; the closest relative of this pair is the gorilla map; most closely related to this trio is the human map. This tree is only slightly more parsimonious than some alternative trees. Although this study has given a magnified view of the genetic differences among humans and apes, the possibility of a three-way split among the lineages leading to humans, gorillas, and chimpanzees still deserves serious consideration. Images PMID:6264476

  18. Cleavage of Nuclear DNA into Oligonucleosomal Fragments during Cell Death Induced by Fungal Infection or by Abiotic Treatments.

    PubMed Central

    Ryerson, DE; Heath, MC

    1996-01-01

    It is often claimed that programmed cell death (pcd) exists in plants and that a form of pcd known as the hypersensitive response is triggered as a defense mechanism by microbial pathogens. However, in contrast to animals, no feature in plants universally identifies or defines pcd. We have looked for a hallmark of pcd in animal cells, namely, DNA cleavage, in plant cells killed by infection with incompatible fungi or by abiotic means. We found that cell death triggered in intact leaves of two resistant cowpea cultivars by the cowpea rust fungus is accompanied by the cleavage of nuclear DNA into oligonucleosomal fragments (DNA laddering). Terminal deoxynucleotidyl transferase-mediated dUTP nick end in situ labeling of leaf sections showed that fungus-induced DNA cleavage occurred only in haustorium-containing cells and was detectable early in the degeneration process. Such cytologically detectable DNA cleavage was also observed in vascular tissue of infected and uninfected plants, but no DNA laddering was detected in the latter. DNA laddering was triggered by [greater than or equal to]100 mM KCN, regardless of cowpea cultivar, but not by physical cell disruption or by concentrations of H2O2, NaN3, CuSO4, or ZnCl2 that killed cowpea cells at a rate similar to that of ladder-inducing KCN concentrations. These and other results suggest that the hypersensitive response to microbial pathogens may involve a pcd with some of the characteristics of animal apoptosis and that DNA cleavage is a potential indicator of pcd in plants. PMID:12239388

  19. Synthesis, DNA-binding, cytotoxicity, photo cleavage, antimicrobial and docking studies of Ru(II) polypyridyl complexes.

    PubMed

    Srishailam, A; Kumar, Yata Praveen; Gabra, Nazar M D; Reddy, P Venkat; Deepika, N; Veerababu, Nageti; Satyanarayana, S

    2013-09-01

    Three Ruthenium(II) polypyridine complexes, [Ru(phen)2(mipc)](2+)(1), [Ru(bpy)2(mipc)](2+) (2) and [Ru(dmb)2(mipc)](2+)(3) [mipc = 2-(6-methyl-3-(1H-imidazo[4, 5-f][1,10]-phenanthroline-2-yl)-4H-chromene-4-one, phen = 1,10-phenanthroline,bpy = 2, 2'bipyridine,dmb = 4, 4'-dimethyl-2, 2'-bipyridine] have been synthesized and characterized by elemental analysis, IR, UV-Vis, (1)H& (13)C NMR and mass spectra. The DNA-binding properties of the Ruthenium(II) complexes were investigated by spectrophotometric methods, viscosity measurements and light switch studies. These three complexes have been focused on photo activated cleavage studies with pBR-322 and antimicrobial studies. Experimental results indicate that the three complexes intercalate into DNA base pairs and follows the order of 1 > 2 > 3 respectively. Molecular docking studies also support the DNA interactions with complexes through hydrogen bonding and vander Waal's interactions. Cytotoxicity studies with Hela cell lines has been revealing about anti tumor activity of these complexes. PMID:23553642

  20. Implications of caspase-dependent proteolytic cleavage of cyclin A1 in DNA damage-induced cell death

    SciTech Connect

    Woo, Sang Hyeok; Seo, Sung-Keum; An, Sungkwan; Choe, Tae-Boo; Hong, Seok-Il; Lee, Yun-Han; Park, In-Chul

    2014-10-24

    Highlights: • Caspase-1 mediates doxorubicin-induced downregulation of cyclin A1. • Active caspase-1 effectively cleaved cyclin A1 at D165. • Cyclin A1 expression is involved in DNA damage-induced cell death. - Abstract: Cyclin A1 is an A-type cyclin that directly binds to CDK2 to regulate cell-cycle progression. In the present study, we found that doxorubicin decreased the expression of cyclin A1 at the protein level in A549 lung cancer cells, while markedly downregulating its mRNA levels. Interestingly, doxorubicin upregulated caspase-1 in a concentration-dependent manner, and z-YAVD-fmk, a specific inhibitor of caspase-1, reversed the doxorubicin-induced decrease in cyclin A1 in A549 lung cancer and MCF7 breast cancer cells. Active caspase-1 effectively cleaved cyclin A1 at D165 into two fragments, which in vitro cleavage assays showed were further cleaved by caspase-3. Finally, we found that overexpression of cyclin A1 significantly reduced the cytotoxicity of doxorubicin, and knockdown of cyclin A1 by RNA interference enhanced the sensitivity of cells to ionizing radiation. Our data suggest a new mechanism for the downregulation of cyclin A1 by DNA-damaging stimuli that could be intimately involved in the cell death induced by DNA damage-inducing stimuli, including doxorubicin and ionizing radiation.

  1. A DNA enzyme with N-glycosylase activity

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  2. Peptide Nucleic Acid with a Lysine Side Chain at the β-Position: Synthesis and Application for DNA Cleavage.

    PubMed

    Sugiyama, Toru; Kuwata, Keiko; Imamura, Yasutada; Demizu, Yosuke; Kurihara, Masaaki; Takano, Masashi; Kittaka, Atsushi

    2016-01-01

    This paper reports the synthesis of new β-Lys peptide nucleic acid (PNA) monomers and their incorporation into a 10-residue PNA sequence. PNA containing β-Lys PNA units formed a stable hybrid duplex with DNA. However, incorporation of β-Lys PNA units caused destabilization of PNA-DNA duplexes to some extent. Electrostatic attractions between β-PNA and DNA could reduce this destabilization effect. Subsequently, bipyridine-conjugated β-Lys PNA was prepared and exhibited sequence selective cleavage of DNA. Based on the structures of the cleavage products and molecular modeling, we reasoned that bipyridine moiety locates within the minor groove of the PNA-DNA duplexes. The lysine side chain of β-PNA is a versatile handle for attaching various functional molecules. PMID:27373637

  3. Groove binding mediated structural modulation and DNA cleavage by quinoline appended chalcone derivative.

    PubMed

    Kumar, Himank; Devaraji, Vinod; Prasath, Rangaraj; Jadhao, Manojkumar; Joshi, Ritika; Bhavana, Purushothaman; Ghosh, Sujit Kumar

    2015-12-01

    The present study embodies the detail DNA binding interaction of a potential bioactive quinoline appended chalcone derivative (E)-3-(anthracen-10-yl)-1-(6,8-dibromo-2-methylquinolin-3-yl)prop-2-en-1-one (ADMQ) with calf thymus DNA (ctDNA) and its consequences by UV-Vis absorption, steady state fluorescence spectroscopy, fluorescence anisotropy, circular dichromism, helix melting, agarose gel electrophoresis, molecular docking, Induced Fit Docking (IFD) and molecular dynamics (MD) simulation. The UV-Vis absorption and fluorescence study reveal that the molecule undergoes considerable interaction with the nucleic acid. The control KI quenching experiment shows the lesser accessibility of ADMQ molecule to the ionic quencher (I(-)) in presence of ctDNA as compared to the bulk aqueous phase. Insignificant change in helix melting temperature as well as in circular dichromism (CD) spectra points toward non-covalent groove binding interaction. The moderate rotational confinement of this chalcone derivative (anisotropy=0.106) trapped in the nucleic acid environment, the comparative displacement assay with well-known minor groove binder Hoechst 33258 and intercalator Ethidium Bromide establishes the minor groove binding interactions of the probe molecule. Molecular docking, IFD and MD simulation reveal that the DNA undergoes prominent morphological changes in terms of helix unwinding and bending to accommodate ADMQ in a crescent shape at an angle of 110° in a sequence specific manner. During interaction, ADMQ rigidifies and bends the sugar phosphate backbone of the nucleic acid and thereby shortens its overall length by 3.02Å. Agarose gel electrophoresis experiment with plasmid pBR 322 reveals that the groove binded ADMQ result in a concentration dependent cleavage of plasmid DNA into its supercoiled and nicked circular form. The consolidated spectroscopic research described herein provides quantitative insight into the interaction of a heterocyclic chalcone derivative

  4. Salicylate, a catalytic inhibitor of topoisomerase II, inhibits DNA cleavage and is selective for the α isoform.

    PubMed

    Bau, Jason T; Kang, Zhili; Austin, Caroline A; Kurz, Ebba U

    2014-02-01

    Topoisomerase II (topo II) is a ubiquitous enzyme that is essential for cell survival through its role in regulating DNA topology and chromatid separation. Topo II can be poisoned by common chemotherapeutics (such as doxorubicin and etoposide), leading to the accumulation of cytotoxic enzyme-linked DNA double-stranded breaks. In contrast, nonbreak-inducing topo II catalytic inhibitors have also been described and have more limited use in clinical chemotherapy. These agents, however, may alter the efficacy of regimens incorporating topo II poisons. We previously identified salicylate, the primary metabolite of aspirin, as a novel catalytic inhibitor of topo II. We have now determined the mechanism by which salicylate inhibits topo II. As catalytic inhibitors can act at a number of steps in the topo II catalytic cycle, we used multiple independent, biochemical approaches to interrogate the catalytic cycle. Furthermore, as mammalian cells express two isoforms of topo II (α and β), we examined whether salicylate was isoform selective. Our results demonstrate that salicylate is unable to intercalate DNA, and does not prevent enzyme-DNA interaction, nor does it promote stabilization of topo IIα in closed clamps on DNA. Although salicylate decreased topo IIα ATPase activity in a dose-dependent noncompetitive manner, this was secondary to salicylate-mediated inhibition of DNA cleavage. Surprisingly, comparison of salicylate's effects using purified human topo IIα and topo IIβ revealed that salicylate selectively inhibits the α isoform. These findings provide a definitive mechanism for salicylate-mediated inhibition of topo IIα and provide support for further studies determining the basis for its isoform selectivity. PMID:24220011

  5. The dual topoisomerase inhibitor A35 preferentially and specially targets topoisomerase 2α by enhancing pre-strand and post-strand cleavage and inhibiting DNA religation

    PubMed Central

    Bi, Chongwen; Li, Yangbiao; Liu, Jingbo; Ye, Cheng; He, Hongwei; Li, Liang

    2015-01-01

    DNA topoisomerases play a key role in tumor proliferation. Chemotherapeutics targeting topoisomerases have been widely used in clinical oncology, but resistance and side effects, particularly cardiotoxicity, usually limit their application. Clinical data show that a decrease in topoisomerase (top) levels is the primary factor responsible for resistance, but in cells there is compensatory effect between the levels of top1 and top2α. Here, we validated cyclizing-berberine A35, which is a dual top inhibitor and preferentially targets top2α. The impact on the top2α catalytic cycle indicated that A35 could intercalate into DNA but did not interfere with DNA-top binding and top2α ATPase activity. A35 could facilitate DNA-top2α cleavage complex formation by enhancing pre-strand and post-strand cleavage and inhibiting religation, suggesting this compound can be a topoisomerase poison and had a district mechanism from other topoisomerase inhibitors. TARDIS and comet assays showed that A35 could induce cell DNA breakage and DNA-top complexes but had no effect on the cardiac toxicity inducer top2β. Silencing top1 augmented DNA break and silencing top2α decreased DNA break. Further validation in H9c2 cardiac cells showed A35 did not disturb cell proliferation and mitochondrial membrane potency. Additionally, an assay with nude mice further demonstrated A35 did not damage the heart. Our work identifies A35 as a novel skeleton compound dually inhibits topoisomerases, and predominantly and specially targets top2α by interfering with all cleavage steps and its no cardiac toxicity was verified by cardiac cells and mice heart. A35 could be a novel and effective targeting topoisomerase agent. PMID:26462155

  6. The tertiary structure of the four-way DNA junction affords protection against DNase I cleavage.

    PubMed Central

    Murchie, A I; Carter, W A; Portugal, J; Lilley, D M

    1990-01-01

    The accessibility of phosphodiester bonds in the DNA of four-way helical junctions has been probed with the nuclease DNase I. Regions of protection were observed on all four strands of the junctions, that tended to be longer on the strands that are exchanged between the coaxially stacked pairs of helices. The protected regions on the continuous strands of the stacked helices were not located exactly at the junction, but were displaced towards the 3' side of the strand. This is the region of backbone that becomes located in the major groove of the opposed helix in the non-crossed, right-handed structure for the junction, and might therefore be predicted to be protected against cleavage by an enzyme. However, the major grooves of the structure remain accessible to the much smaller probe dimethyl sulphate. Images PMID:2339051

  7. The Ser176 of T4 endonuclease IV is crucial for the restricted and polarized dC-specific cleavage of single-stranded DNA implicated in restriction of dC-containing DNA in host Escherichia coli

    PubMed Central

    Hirano, Nobutaka; Ohshima, Hiroyuki; Sakashita, Hidenori; Takahashi, Hideo

    2007-01-01

    Endonuclease (Endo) IV encoded by denB of bacteriophage T4 is an enzyme that cleaves single-stranded (ss) DNA in a dC-specific manner. Also the growth of dC-substituted T4 phage and host Escherichia coli cells is inhibited by denB expression presumably because of the inhibitory effect on replication of dC-containing DNA. Recently, we have demonstrated that an efficient cleavage by Endo IV occurs exclusively at the 5′-proximal dC (dC1) within a hexameric or an extended sequence consisting of dC residues at the 5′-proximal and the 3′-proximal positions (dCs tract), in which a third dC residue within the tract affects the polarized cleavage and cleavage rate. Here we isolate and characterize two denB mutants, denB(W88R) and denB(S176N). Both mutant alleles have lost the detrimental effect on the host cell. Endo IV(W88R) shows no enzymatic activity (<0.4% of that of wild-type Endo IV). On the other hand, Endo IV(S176N) retains cleavage activity (17.5% of that of wild-type Endo IV), but has lost the polarized and restricted cleavage of a dCs tract, indicating that the Ser176 residue of Endo IV is implicated in the polarized cleavage of a dCs tract which brings about a detrimental effect on the replication of dC-containing DNA. PMID:17913749

  8. Quantification of primary versus secondary C-H bond cleavage in alkane activation: Propane on Pt

    SciTech Connect

    Weinberg, W.H.; Sun, Yongkui )

    1991-08-02

    The trapping-mediated dissociative chemisorption of three isotopes of propane (C{sub 3}H{sub 8}, CH{sub 3}, CD{sub 2}CH{sub 3}, and C{sub 3}D{sub 8}) has been investigated on the Pt(110)-(1 {times} 2) surface, and both the apparent activation energies and the preexponential factors of the surface reaction rate coefficients have been measured. In addition, the probabilities of primary and secondary C-H bond cleavage for alkane activation on a surface were evaluated. The activation energy for primary C-H bond cleavage was 425 calories per mole greater than that of secondary C-H bond cleavage, and the two true activation energies that embody the single measured activation energy were determined for each of the three isotopes. Secondary C-H bond cleavage is also preferred on entropic grounds, and the magnitude of the effect was quantified.

  9. AgNP-DNA@GQDs hybrid: new approach for sensitive detection of H2O2 and glucose via simultaneous AgNP etching and DNA cleavage.

    PubMed

    Wang, Lili; Zheng, Jing; Li, Yinhui; Yang, Sheng; Liu, Changhui; Xiao, Yue; Li, Jishan; Cao, Zhong; Yang, Ronghua

    2014-12-16

    A growing body of evidence suggests that hydrogen peroxide (H2O2) plays an active role in the regulation of various physiological processes. Development of sensitive probes for H2O2 is an urgent work. In this study, we proposed a DNA-mediated silver nanoparticle and graphene quantum dot hybrid nanocomposite (AgNP-DNA@GQDs) for sensitive fluorescent detection of H2O2. The sensing mechanism is based on the etching effect of H2O2 to AgNPs and the cleavage of DNA by as-generated hydroxyl radicals (•OH). The formation of AgNP-DNA@GQDs nanocomposite can result in fluorescence quenching of GQDs by AgNPs through the resonance energy transfer. Upon H2O2 addition, the energy transfer between AgNPs and GQDs mediated by DNA was weakened and obvious fluorescence recovery of GQDs could be observed. It is worth noting that the reaction product •OH between H2O2 and AgNPs could cleave the DNA-bridge and result in the disassembly of AgNP-DNA@GQDs to achieve further signal enhancement. With optimal conditions, the approach achieves a low detection limit of 0.10 μM for H2O2. Moreover, this nanocomposite is further extended to the glucose sensing in human urine combining with glucose oxidase (GOx) for the oxidation of glucose and formation of H2O2. The glucose concentrations in human urine are detected with satisfactory recoveries of 94.6-98.8% which holds potential for ultrasensitive quantitative analysis of glucose and supplies valuable information for diabetes mellitus research and clinical diagnosis. PMID:25390796

  10. Caspase Activation and Specific Cleavage of Substrates after Coxsackievirus B3-Induced Cytopathic Effect in HeLa Cells

    PubMed Central

    Carthy, Christopher M.; Granville, David J.; Watson, Kathleen A.; Anderson, Daniel R.; Wilson, Janet E.; Yang, Decheng; Hunt, David W. C.; McManus, Bruce M.

    1998-01-01

    Coxsackievirus B3 (CVB3), an enterovirus in the family Picornaviridae, induces cytopathic changes in cell culture systems and directly injures multiple susceptible organs and tissues in vivo, including the myocardium, early after infection. Biochemical analysis of the cell death pathway in CVB3-infected HeLa cells demonstrated that the 32-kDa proform of caspase 3 is cleaved subsequent to the degenerative morphological changes seen in infected HeLa cells. Caspase activation assays confirm that the cleaved caspase 3 is proteolytically active. The caspase 3 substrates poly(ADP-ribose) polymerase, a DNA repair enzyme, and DNA fragmentation factor, a cytoplasmic inhibitor of an endonuclease responsible for DNA fragmentation, were degraded at 9 h following infection, yielding their characteristic cleavage fragments. Inhibition of caspase activation by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) did not inhibit the virus-induced cytopathic effect, while inhibition of caspase activation by ZVAD.fmk in control apoptotic cells induced by treatment with the porphyrin photosensitizer benzoporphyrin derivative monoacid ring A and visible light inhibited the apoptotic phenotype. Caspase activation and cleavage of substrates may not be responsible for the characteristic cytopathic effect produced by picornavirus infection yet may be related to late-stage alterations of cellular homeostatic processes and structural integrity. PMID:9696873

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

    PubMed Central

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

    2005-01-01

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

  12. Site-specific cleavage/packaging of herpes simplex virus DNA and the selective maturation of nucleocapsids containing full-length viral DNA

    PubMed Central

    Vlazny, Donald A.; Kwong, Ann; Frenkel, Niza

    1982-01-01

    Defective genomes present in serially passaged herpes simplex virus (HSV) stocks have been shown to consist of tandemly arranged repeat units containing limited sets of the standard virus DNA sequences. Invariably, the HSV defective genomes terminate with the right (S component) terminus of HSV DNA. Because the oligomeric forms can arise from a single repeat unit, it has been concluded that the defective genomes arise by a rolling circle mechanism of replication. We now report on our studies of defective genomes packaged in viral capsids accumulating in the nuclei and in mature virions (enveloped capsids) translocated into the cytoplasm of cells infected with serially passaged virus. These studies have revealed that, upon electrophoresis in agarose gels, the defective genomes prepared from cytoplasmic virions comigrated with nondefective standard virus DNA (Mr 100 × 106). In contrast, DNA prepared from capsids accumulating in nuclei consisted of both full-length defective virus DNA molecules and smaller DNA molecules of discrete sizes, ranging in Mr from 5.5 to 100 × 106. These smaller DNA species were shown to consist of different integral numbers (from 1 to approximately 18) of defective genome repeat units and to terminate with sequences corresponding to the right terminal sequences of HSV DNA. We conclude on the basis of these studies that (i) sequences from the right end of standard virus DNA contain a recognition signal for the cleavage and packaging of concatemeric viral DNA, (ii) the sequence-specific cleavage is either a prerequisite for or occurs during the entry of viral DNA into capsid structures, and (iii) DNA molecules significantly shorter than full-length standard viral DNA can become encapsidated within nuclear capsids provided they contain the cleavage/packaging signal. However, capsids containing DNA molecules significantly shorter than standard virus DNA are not translocated into the cytoplasm. Images PMID:6280181

  13. DNA cleavage by CgII and NgoAVII requires interaction between N- and R-proteins and extensive nucleotide hydrolysis.

    PubMed

    Zaremba, Mindaugas; Toliusis, Paulius; Grigaitis, Rokas; Manakova, Elena; Silanskas, Arunas; Tamulaitiene, Giedre; Szczelkun, Mark D; Siksnys, Virginijus

    2014-12-16

    The stress-sensitive restriction-modification (RM) system CglI from Corynebacterium glutamicum and the homologous NgoAVII RM system from Neisseria gonorrhoeae FA1090 are composed of three genes: a DNA methyltransferase (M.CglI and M.NgoAVII), a putative restriction endonuclease (R.CglI and R.NgoAVII, or R-proteins) and a predicted DEAD-family helicase/ATPase (N.CglI and N.NgoAVII or N-proteins). Here we report a biochemical characterization of the R- and N-proteins. Size-exclusion chromatography and SAXS experiments reveal that the isolated R.CglI, R.NgoAVII and N.CglI proteins form homodimers, while N.NgoAVII is a monomer in solution. Moreover, the R.CglI and N.CglI proteins assemble in a complex with R2N2 stoichiometry. Next, we show that N-proteins have ATPase activity that is dependent on double-stranded DNA and is stimulated by the R-proteins. Functional ATPase activity and extensive ATP hydrolysis (∼170 ATP/s/monomer) are required for site-specific DNA cleavage by R-proteins. We show that ATP-dependent DNA cleavage by R-proteins occurs at fixed positions (6-7 nucleotides) downstream of the asymmetric recognition sequence 5'-GCCGC-3'. Despite similarities to both Type I and II restriction endonucleases, the CglI and NgoAVII enzymes may employ a unique catalytic mechanism for DNA cleavage. PMID:25429977

  14. Evolutionary Relationships among Five Subspecies of MUS MUSCULUS Based on Restriction Enzyme Cleavage Patterns of Mitochondrial DNA

    PubMed Central

    Yonekawa, Hiromichi; Moriwaki, Kazuo; Gotoh, Osamu; Hayashi, Jun-Ichi; Watanabe, Junko; Miyashita, Nobumoto; Petras, Michael L.; Tagashira, Yusaku

    1981-01-01

    The intra- and intersubspecific genetic distances between five subspecies of Mus musculus were estimated from restriction enzyme cleavage patterns or maps of mitochondrial DNA (mtDNA). The European subspecies, M. m. domesticus and Asian subspecies, M. m. bactrianus, M. m. castaneus, M. m. molossinus and M. m. urbanus were examined. For each subspecies, except M. m. urbanus, at least two local races from widely separated localities were examined. Intrasubspecific heterogeneity was found in the mtDNA cleavage patterns of M. m. bactrianus and M. m. castaneus. M. m. molossinus and M. m. domesticus, however, revealed no intrasubspecific heterogeneity. Four of the subspecies had distinct cleavage patterns. The fifth, M. m. urbanus, had cleavage patterns identical to those of M. m. castaneus with several enzymes. Estimates of genetic distances between the various races and subspecies were obtained by comparing cleavage maps of the mtDNAs with various restriction enzymes. Nucleotide sequence divergences of mtDNA between local races were estimated to be less than 0.4% in M. m. bactrianus and less than 0.3% in M. m. castaneus. The times of divergence of both subspecies were calculated to be 0.1–0.2 x 106 years. These values suggest that the intrasubspecific divergence began some 0.1–0.2 x 106 years ago. On the other hand, nucleotide sequence divergences between European subspecies M. m. domesticus and Asian subspecies M. m. bactrianus and M. m. castaneus were 7.1% and 5.8%, respectively. The times of divergence were calculated to be 2.1–2.6 x 106 years. Further, the nucleotide sequence divergence and time of divergence between M. m. molossinus and the other two Asian subspecies were comparable to those between M. m. molossinus and M. m. domesticus (about 3% and 1 x 106 years, respectively). These results suggest that M. m. molossinus is situated in a unique evolutionary position among Asian subspecies. PMID:6277733

  15. Cleavage of a four-way DNA junction by a restriction enzyme spanning the point of strand exchange.

    PubMed Central

    Murchie, A I; Portugal, J; Lilley, D M

    1991-01-01

    The four-way DNA junction is believed to fold in the presence of metal ions into an X-shaped structure, in which there is pairwise coaxial stacking of helical arms. A restriction enzyme MboII has been used to probe this structure. A junction was constructed containing a recognition site for MboII in one helical arm, positioned such that stacking of arms would result in cleavage in a neighbouring arm. Strong cleavage was observed, at the sites expected on the basis of coaxial stacking. An additional cleavage was seen corresponding to the formation of an alternative stacking isomer, suggesting that the two isomeric forms are in dynamic equilibrium in solution. Images PMID:2001684

  16. Construction of HBV-specific ribozyme and its recombinant with HDV and their cleavage activity in vitro

    PubMed Central

    Wen, Shu-Juan; Xiang, Kai-Jun; Huang, Zhen-Hua; Zhou, Rong; Qi, Xue-Zhong

    2000-01-01

    AIM: To construct the recombinant of HDV cDNA and HBV-specific ribozyme gene by recombinant PCR in order to use HDV as a transporting vector carrying HBV-specific ribozyme into liver cells for inhibiting the replication of HBV. METHODS: We separately cloned the ribozyme (RZ) gene and recombinant DVRZ (comprising HDV cDNA and HBV-specific ribozyme gene) into the downstream of T7 promoter of pTAdv-T vector and studied the in vitro cleavage activity of their transcripts (rRZ, rDVRZ) on target RNA (rBVCF) from in vitro transcription of HBV C gene fragment(BVCF). RESULTS: Both the simple (rRZ) and the recombinant ribozyme rDVRZ could efficiently catalyze the cleavage of target RNA (rBVCF) under different temperatures (37 °C, 42 °C and 55 °C) and Mg2+ concentrations (10 mmol/L, 15 mmol/L and 20 mmol/L) and their catalytic activity tended to increase as the temperature was rising. But the activity of rRZ was evidently higher than that of rDVRZ. CONCLUSION: The recombinant of HDV cDNA and ribozyme gene had the potential of being further explored and used in gene therapy of HBV infection. PMID:11819602

  17. Tyrosyl-DNA phosphodiesterase I catalytic mutants reveal an alternative nucleophile that can catalyze substrate cleavage.

    PubMed

    Comeaux, Evan Q; Cuya, Selma M; Kojima, Kyoko; Jafari, Nauzanene; Wanzeck, Keith C; Mobley, James A; Bjornsti, Mary-Ann; van Waardenburg, Robert C A M

    2015-03-01

    Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3'-DNA adducts, such as the 3'-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (His(nuc)) that attacks DNA adducts to form a covalent 3'-phosphohistidyl intermediate and a general acid/base His (His(gab)), which resolves the Tdp1-DNA linkage. A His(nuc) to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of His(gab) to Arg. However, here we report that expression of the yeast His(nuc)Ala (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 His(gab) mutants, including H432N and the SCAN1-related H432R. Moreover, the His(nuc)Ala mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the His(nuc)Phe mutant was catalytically inactive and suppressed His(gab) mutant-induced toxicity. These data suggest that the activity of another nucleophile when His(nuc) is replaced with residues containing a small side chain (Ala, Asn, and Gln), but not with a bulky side chain. Indeed, genetic, biochemical, and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to His(nuc), can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate. PMID:25609251

  18. Synthesis, characterization, DNA binding and cleavage studies of Ru(II) complexes containing oxime ligands

    NASA Astrophysics Data System (ADS)

    Chitrapriya, Nataraj; Mahalingam, Viswanathan; Zeller, Matthias; Lee, Hyosun; Natarajan, Karuppannan

    2010-12-01

    The Ru(II) precursors, [RuHCl(CO)(EPh 3) 3] (E = P or As) when reacted with some well known monoxime and dioxime ligands in ethanolic solution afforded the new complexes of the types [RuCl(CO)(EPh 3) 2L1], [RuH(CO)(EPh 3) 2L2] and [RuCl(CO)(EPh 3) 2L3] ((H 1L1) = diacetylmonoxime, (H 1L2) = dimethylglyoxime and (H 2L3) = benzoiloxime). The ligands coordinated in a bidentate chelate mode forming a five membered chelate ring. The molecular structures of two of the complexes have been determined by single crystal X-ray diffraction study. The structural determination confirms the deprotonation of the oxime function. Examination of all the complexes by cyclic voltammetry showed the occurrence of some quasi-reversible redox reactions owing to changes in the oxidation state of the central metal atoms. Structural assignments are supported by combination of IR, UV-Vis, 1H NMR and elemental analyses. In addition, the DNA binding properties and cleavage efficiency of new complexes have been tested.

  19. Activation of the Kexin from Schizosaccharomyces pombe Requires Internal Cleavage of Its Initially Cleaved Prosequence

    PubMed Central

    Powner, Dale; Davey, John

    1998-01-01

    Members of the kexin family of processing enzymes are responsible for the cleavage of many proproteins during their transport through the secretory pathway. The enzymes themselves are made as inactive precursors, and we investigated the activation process by studying the maturation of Krp1, a kexin from the fission yeast Schizosaccharomyces pombe. Using a cell-free translation-translocation system prepared from Xenopus eggs, we found that Krp1 is made as a preproprotein that loses the presequence during translocation into the endoplasmic reticulum. The prosequence is also rapidly cleaved in a reaction that is autocatalytic and probably intramolecular and is inhibited by disruption of the P domain. Prosequence cleavage normally occurs at Arg-Tyr-Lys-Arg102↓ (primary cleavage site) but can occur at Lys-Arg82 (internal cleavage site) and/or Trp-Arg99 when the basic residues are removed from the primary site. Cleavage of the prosequence is necessary but not sufficient for activation, and Krp1 is initially unable to process substrates presented in trans. Full activation is achieved after further incubation in the extract and is coincident with the addition of O-linked sugars. O glycosylation is not, however, essential for activity, and the crucial event appears to be cleavage of the initially cleaved prosequence at the internal site. Our results are consistent with a model in which the cleaved prosequence remains noncovalently associated with the catalytic domain and acts as an autoinhibitor of the enzyme. Inhibition is then relieved by a second (internal) cleavage of the inhibitory prosequence. Further support for this model is provided by our finding that overexpression of a Krp1 prosequence lacking a cleavable internal site dramatically reduced the growth rate of otherwise wild-type S. pombe cells, an effect that was not seen after overexpression of the normal, internally cleavable, prosequence or prosequences that lack the Lys-Arg102 residues. PMID:9418887

  20. Molecular basis of the targeting of topoisomerase II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-forming oligonucleotides.

    PubMed

    Duca, Maria; Guianvarc'h, Dominique; Oussedik, Kahina; Halby, Ludovic; Garbesi, Anna; Dauzonne, Daniel; Monneret, Claude; Osheroff, Neil; Giovannangeli, Carine; Arimondo, Paola B

    2006-01-01

    Human topoisomerase II (topo II) is the cellular target for a number of widely used antitumor agents, such as etoposide (VP16). These agents 'poison' the enzyme and induce it to generate DNA breaks that are lethal to the cell. Topo II-targeted drugs show a limited sequence preference, triggering double-stranded breaks throughout the genome. Circumstantial evidence strongly suggests that some of these breaks induce chromosomal translocations that lead to specific types of leukaemia (called treatment-related or secondary leukaemia). Therefore, efforts are ongoing to decrease these secondary effects. An interesting option is to increase the sequence-specificity of topo II-targeted drugs by attaching them to triplex-forming oligonucleotides (TFO) that bind to DNA in a highly sequence-specific manner. Here five derivatives of VP16 were attached to TFOs. The active topo II poisons, once linked, induced cleavage 13-14 bp from the triplex end where the drug was attached. The use of triple-helical DNA structures offers an efficient strategy for targeting topo II-mediated cleavage to DNA specific sequences. Finally, drug-TFO conjugates are useful tools to investigate the mechanistic details of topo II poisoning. PMID:16598074

  1. Molecular basis of the targeting of topoisomerase II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-forming oligonucleotides

    PubMed Central

    Duca, Maria; Guianvarc'h, Dominique; Oussedik, Kahina; Halby, Ludovic; Garbesi, Anna; Dauzonne, Daniel; Monneret, Claude; Osheroff, Neil; Giovannangeli, Carine; Arimondo, Paola B.

    2006-01-01

    Human topoisomerase II (topo II) is the cellular target for a number of widely used antitumor agents, such as etoposide (VP16). These agents ‘poison’ the enzyme and induce it to generate DNA breaks that are lethal to the cell. Topo II-targeted drugs show a limited sequence preference, triggering double-stranded breaks throughout the genome. Circumstantial evidence strongly suggests that some of these breaks induce chromosomal translocations that lead to specific types of leukaemia (called treatment-related or secondary leukaemia). Therefore, efforts are ongoing to decrease these secondary effects. An interesting option is to increase the sequence-specificity of topo II-targeted drugs by attaching them to triplex-forming oligonucleotides (TFO) that bind to DNA in a highly sequence-specific manner. Here five derivatives of VP16 were attached to TFOs. The active topo II poisons, once linked, induced cleavage 13–14 bp from the triplex end where the drug was attached. The use of triple-helical DNA structures offers an efficient strategy for targeting topo II-mediated cleavage to DNA specific sequences. Finally, drug–TFO conjugates are useful tools to investigate the mechanistic details of topo II poisoning. PMID:16598074

  2. Carbon-carbon bond cleavage in activation of the prodrug nabumetone.

    PubMed

    Varfaj, Fatbardha; Zulkifli, Siti N A; Park, Hyoung-Goo; Challinor, Victoria L; De Voss, James J; Ortiz de Montellano, Paul R

    2014-05-01

    Carbon-carbon bond cleavage reactions are catalyzed by, among others, lanosterol 14-demethylase (CYP51), cholesterol side-chain cleavage enzyme (CYP11), sterol 17β-lyase (CYP17), and aromatase (CYP19). Because of the high substrate specificities of these enzymes and the complex nature of their substrates, these reactions have been difficult to characterize. A CYP1A2-catalyzed carbon-carbon bond cleavage reaction is required for conversion of the prodrug nabumetone to its active form, 6-methoxy-2-naphthylacetic acid (6-MNA). Despite worldwide use of nabumetone as an anti-inflammatory agent, the mechanism of its carbon-carbon bond cleavage reaction remains obscure. With the help of authentic synthetic standards, we report here that the reaction involves 3-hydroxylation, carbon-carbon cleavage to the aldehyde, and oxidation of the aldehyde to the acid, all catalyzed by CYP1A2 or, less effectively, by other P450 enzymes. The data indicate that the carbon-carbon bond cleavage is mediated by the ferric peroxo anion rather than the ferryl species in the P450 catalytic cycle. CYP1A2 also catalyzes O-demethylation and alcohol to ketone transformations of nabumetone and its analogs. PMID:24584631

  3. Carbon-Carbon Bond Cleavage in Activation of the Prodrug Nabumetone

    PubMed Central

    Varfaj, Fatbardha; Zulkifli, Siti N. A.; Park, Hyoung-Goo; Challinor, Victoria L.; De Voss, James J.

    2014-01-01

    Carbon-carbon bond cleavage reactions are catalyzed by, among others, lanosterol 14-demethylase (CYP51), cholesterol side-chain cleavage enzyme (CYP11), sterol 17β-lyase (CYP17), and aromatase (CYP19). Because of the high substrate specificities of these enzymes and the complex nature of their substrates, these reactions have been difficult to characterize. A CYP1A2-catalyzed carbon-carbon bond cleavage reaction is required for conversion of the prodrug nabumetone to its active form, 6-methoxy-2-naphthylacetic acid (6-MNA). Despite worldwide use of nabumetone as an anti-inflammatory agent, the mechanism of its carbon-carbon bond cleavage reaction remains obscure. With the help of authentic synthetic standards, we report here that the reaction involves 3-hydroxylation, carbon-carbon cleavage to the aldehyde, and oxidation of the aldehyde to the acid, all catalyzed by CYP1A2 or, less effectively, by other P450 enzymes. The data indicate that the carbon-carbon bond cleavage is mediated by the ferric peroxo anion rather than the ferryl species in the P450 catalytic cycle. CYP1A2 also catalyzes O-demethylation and alcohol to ketone transformations of nabumetone and its analogs. PMID:24584631

  4. Synthesis, structural characterization, fluorescence, antimicrobial, antioxidant and DNA cleavage studies of Cu(II) complexes of formyl chromone Schiff bases

    NASA Astrophysics Data System (ADS)

    Kavitha, P.; Saritha, M.; Laxma Reddy, K.

    2013-02-01

    Cu(II) complexes have been synthesized from different Schiff bases, such as 3-((2-hydroxy phenylimino)methyl)-4H-chromen-4-one (HL1), 2-((4-oxo-4H-chromen-3-yl)methylneamino) benzoicacid (HL2), 3-((3-hydroxypyridin-2-ylimino)methyl)-4H-chromen-4-one (HL3) and 3-((2-mercaptophenylimino)methyl)-4H-chromen-4-one (HL4). The complexes were characterized by analytical, molar conductance, IR, electronic, magnetic, ESR, thermal, powder XRD and SEM studies. The analytical data reveal that metal to ligand molar ratio is 1:2 in all the complexes. Molar conductivity data indicates that all the Cu(II) complexes are neutral. On the basis of magnetic and electronic spectral data, distorted octahedral geometry is proposed for all the Cu(II) complexes. Thermal behaviour of the synthesized complexes illustrates the presence of lattice water molecules in the complexes. X-ray diffraction studies reveal that all the ligands and their Cu(II) complexes have triclinic system with different unit cell parameters. Antimicrobial, antioxidant and DNA cleavage activities indicate that metal complexes exhibited greater activity as compared with ligands.

  5. Effect of Maternal Age on the Ratio of Cleavage and Mitochondrial DNA Copy Number in Early Developmental Stage Bovine Embryos

    PubMed Central

    TAKEO, Shun; GOTO, Hiroya; KUWAYAMA, Takehito; MONJI, Yasunori; IWATA, Hisataka

    2012-01-01

    Abstract Age-associated deterioration in both the quality and quantity of mitochondria occurs in older women. The main aim of this study was to examine the effect of age on mitochondrial DNA copy number (mtDNA number) in early developmental stage bovine embryos as well as the dynamics of mtDNA number during early embryo development. Real-time PCR was used to determine mtDNA number. In vitro-produced embryos 48 h after insemination derived from Japanese black cows, ranging in age from 25 to 209 months were categorized based on their cleavage status. There was an overall negative relationship between the age of the cow and cleavage status, to the extent that the ratio of embryos cleaved over the 4-cell stage was greater in younger cows. The mtDNA number did not differ among the cleaved status of embryos. In the next experiment, oocytes collected from each donor cow were divided into 2 groups containing 10 oocytes each, in order to compare the mtDNA number of mature oocytes and early developmental stage embryos within individuals. Upon comparing the mtDNA number between oocytes at the M2 stage and early developmental stage 48 h post insemination, mtDNA number was found to decrease in most cows, but was found to increase in some cows. In conclusion, age affects the cleaving ability of oocytes, and very old cows (> 180 months) tend to have lower mtDNA numbers in their oocytes. The change in mtDNA number during early development varied among individual cows, although overall, it showed a tendency to decrease. PMID:23269452

  6. Mixed ligand copper(II) complexes of 2,9-dimethyl-1,10-phenanthroline: tridentate 3N primary ligands determine DNA binding and cleavage and cytotoxicity.

    PubMed

    Ganeshpandian, Mani; Ramakrishnan, Sethu; Palaniandavar, Mallayan; Suresh, Eringathodi; Riyasdeen, Anvarbatcha; Akbarsha, Mohammad Abdulkadher

    2014-11-01

    A series of mononuclear mixed ligand copper(II) complexes of the type [Cu(L)(2,9-dmp)](ClO4)21-4, where L is a tridentate 3N ligand such as diethylenetriamine (L1) (1) or N-methyl-N'-(pyrid-2-yl-methyl)ethylenediamine (L2) (2) or di(2-picolyl)amine (L3) (3) or bis(pyrid-2-ylmethyl)-N-methylamine (L4) (4) and 2,9-dmp is 2,9-dimethyl-1,10-phenanthroline, has been isolated and characterized. The complexes 1 and 3 possess square-based pyramidal coordination geometry. Absorption spectral studies reveal that the intrinsic DNA binding affinity varies as 1>2>3>4. The higher DNA binding affinity of 1 arises from L1, which offers lower steric hindrance toward intercalation of 2,9-dmp co-ligand into DNA base pairs and is involved in hydrogen bonding interaction with DNA. Interestingly, all the complexes cleave pUC19 supercoiled DNA in the absence of an activating agent. They also exhibit oxidative (H2O2) DNA cleavage ability, which varies as 1>2>3>4, the highest cleavage efficiency of 1 being due to the largest amount of ROS it generates. The tryptophan emission-quenching experiment reveals that the stronger binding of 3 and 4 with bovine serum albumin (BSA) in the hydrophobic region, which is in line with DNA viscosity measurements. The IC50 values of 1-4 for MCF-7 breast cancer cell line are lower than that of cisplatin. Flow cytometry analysis reveals that 1 mediates the arrest of S and G2/M phases in the cell cycle progression at 24h harvesting time, which progresses into apoptosis. Hoechst 33258 staining studies indicate the higher potency of 1 to induce apoptosis. PMID:25151036

  7. Microwave assisted synthesis, spectroscopic, electrochemical and DNA cleavage studies of lanthanide(III) complexes with coumarin based imines

    NASA Astrophysics Data System (ADS)

    Kapoor, Puja; Fahmi, Nighat; Singh, R. V.

    2011-12-01

    The present work stems from our interest in the synthesis, characterization and biological evaluation of lanthanide(III) complexes of a class of coumarin based imines which have been prepared by the interaction of hydrated lanthanide(III) chloride with the sodium salts of 3-acetylcoumarin thiosemicarbazone (ACTSZH) and 3-acetylcoumarin semicarbazone (ACSZH) in 1:3 molar ratio using thermal as well as microwave method. Characterization of the ligands as well as the metal complexes have been carried out by elemental analysis, melting point determinations, molecular weight determinations, magnetic moment, molar conductance, IR, 1H NMR, 13C NMR, electronic, EPR, X-ray powder diffraction and mass spectral studies. Spectral studies confirm ligands to be monofunctional bidentate and octahedral environment around metal ions. The redox behavior of one of the synthesized metal complex was investigated by cyclic voltammetry. Further, free ligands and their metal complexes have been screened for their antimicrobial as well as DNA cleavage activity. The results of these findings have been presented and discussed.

  8. Herpes simplex virus amplicon: cleavage of concatemeric DNA is linked to packaging and involves amplification of the terminally reiterated a sequence.

    PubMed Central

    Deiss, L P; Frenkel, N

    1986-01-01

    Herpes simplex virus-infected cells contain large concatemeric DNA molecules arising from replication of the viral genome. The large concatemers are cleaved to generate unit-length molecules terminating at both ends with the a sequence. We have used constructed defective virus vectors (amplicons) derived from herpes simplex virus to study the mechanism of cleavage of viral DNA concatemers and the packaging of viral DNA into nucleocapsids. These studies revealed that (i) a 248-base-pair a sequence contained the signal(s) required for cleavage-packaging, (ii) the cleavage of viral DNA concatemers was coupled to packaging, (iii) the a sequence contained the information required for its own amplification, and (iv) cleavage-packaging occurred by a novel process involving the amplification of the a sequence. Images PMID:3005637

  9. Establishment of a non-radioactive cleavage assay to assess the DNA repair capacity towards oxidatively damaged DNA in subcellular and cellular systems and the impact of copper.

    PubMed

    Hamann, Ingrit; Schwerdtle, Tanja; Hartwig, Andrea

    2009-10-01

    Oxidative stress is involved in many diseases, and the search for appropriate biomarkers is one major focus in molecular epidemiology. 8-Oxoguanine (8-oxoG), a potentially mutagenic DNA lesion, is considered to be a sensitive biomarker for oxidative stress. Another approach consists in assessing the repair capacity towards 8-oxoG, mediated predominantly by the human 8-oxoguanine DNA glycosylase 1 (hOGG1). With respect to the latter, during the last few years so-called cleavage assays have been described, investigating the incision of (32)P-labelled and 8-oxoG damaged oligonucleotides by cell extracts. Within the present study, a sensitive non-radioactive test system based on a Cy5-labelled oligonucleotide has been established. Sources of incision activity are isolated proteins or extracts prepared from cultured cells and peripheral blood mononuclear cells (PBMC). After comparing different oligonucleotide structures, a hairpin-like structure was selected which was not degraded by cell extracts. Applying this test system the impact of copper on the activity of isolated hOGG1 and on hOGG activity in A549 cells was examined, showing a distinct inhibition of the isolated protein at low copper concentration as compared to a modest inhibition of hOGG activity in cells at beginning cytotoxic concentrations. For investigating PBMC, all reaction conditions, including the amounts of oligonucleotide and cell extract as well as the reaction time have been optimized. The incision activities of PBMC protein extracts obtained from different donors have been investigated, and inter-individual differences have been observed. In summary, the established method is as sensitive and even faster than the radioactive technique, and additionally, offers the advantage of reduced costs and low health risk. PMID:19505484

  10. Metal ion specificities for folding and cleavage activity in the Schistosoma hammerhead ribozyme

    PubMed Central

    Boots, Jennifer L.; Canny, Marella D.; Azimi, Ehsan; Pardi, Arthur

    2008-01-01

    The effects of various metal ions on cleavage activity and global folding have been studied in the extended Schistosoma hammerhead ribozyme. Fluorescence resonance energy transfer was used to probe global folding as a function of various monovalent and divalent metal ions in this ribozyme. The divalent metals ions Ca2+, Mg2+, Mn2+, and Sr2+ have a relatively small variation (less than sixfold) in their ability to globally fold the hammerhead ribozyme, which contrasts with the very large difference (>10,000-fold) in apparent rate constants for cleavage for these divalent metal ions in single-turnover kinetic experiments. There is still a very large range (>4600-fold) in the apparent rate constants for cleavage for these divalent metal ions measured in high salt (2 M NaCl) conditions where the ribozyme is globally folded. These results demonstrate that the identity of the divalent metal ion has little effect on global folding of the Schistosoma hammerhead ribozyme, whereas it has a very large effect on the cleavage kinetics. Mechanisms by which the identity of the divalent metal ion can have such a large effect on cleavage activity in the Schistosoma hammerhead ribozyme are discussed. PMID:18755844

  11. Functional domains within the a sequence involved in the cleavage-packaging of herpes simplex virus DNA.

    PubMed Central

    Deiss, L P; Chou, J; Frenkel, N

    1986-01-01

    Newly replicated herpes simplex virus (HSV) DNA consists of head-to-tail concatemers which are cleaved to generate unit-length genomes bounded by the terminally reiterated a sequence. Constructed defective HSV vectors (amplicons) containing a viral DNA replication origin and the a sequence are similarly replicated into large concatemers which are cleaved at a sequences punctuating the junctions between adjacent repeat units, concurrent with the packaging of viral DNA into nucleocapsids. In the present study we tested the ability of seed amplicons containing specific deletions in the a sequence to become cleaved and packaged and hence be propagated in virus stocks. These studies revealed that two separate signals, located within the Ub and Uc elements of the a sequence, were essential for amplicon propagation. No derivative defective genomes were recovered from seed constructs which lacked the Uc signal. In contrast, propagation of seed constructs lacking the Ub signal resulted in the selection of defective genomes with novel junctions, containing specific insertions of a sequences derived from the helper virus DNA. Comparison of published sequences of concatemeric junctions of several herpesviruses supported a uniform mechanism for the cleavage-packaging process, involving the measurement from two highly conserved blocks of sequences (pac-1 and pac-2) which were homologous to the required Uc and Ub sequences. These results form the basis for general models for the mechanism of cleavage-packaging of herpesvirus DNA. Images PMID:3016323

  12. Synthesis and crystal structure elucidation of new copper(II)-based chemotherapeutic agent coupled with 1,2-DACH and orthovanillin: Validated by in vitro DNA/HSA binding profile and pBR322 cleavage pathway.

    PubMed

    Zaki, Mehvash; Afzal, Mohd; Ahmad, Musheer; Tabassum, Sartaj

    2016-08-01

    New copper(II)-based complex (1) was synthesized and characterized by analytical, spectroscopic and single crystal X-ray diffraction. The in vitro binding studies of complex 1 with CT DNA and HSA have been investigated by employing biophysical techniques to examine the binding propensity of 1 towards DNA and HSA. The results showed that 1 avidly binds to CT DNA via electrostatic mode along with the hydrogen bonding interaction of NH2 and CN groups of Schiff base ligand with the base pairs of DNA helix, leads to partial unwinding and destabilization of the DNA double helix. Moreover, the CD spectral studies revealed that complex 1 binds through groove binding interaction that stabilizes the right-handed B-form of DNA. Complex 1 showed an impressive photoinduced nuclease activity generating single-strand breaks in comparison with the DNA cleavage activity in presence of visible light. The mechanistic investigation revealed the efficiency of 1 to cleave DNA strands by involving the generation of reactive oxygen species. Furthermore, the time dependent DNA cleavage activity showed that there was gradual increase in the amount of NC DNA on increasing the photoexposure time. However, the interaction of 1 and HSA showed that the change of intrinsic fluorescence intensity of HSA was induced by the microenvironment of Trp residue. PMID:27289445

  13. DNA cleavage and methylation specificity of the single polypeptide restriction–modification enzyme LlaGI

    PubMed Central

    Smith, Rachel M.; Diffin, Fiona M.; Savery, Nigel J.; Josephsen, Jytte; Szczelkun, Mark D.

    2009-01-01

    LlaGI is a single polypeptide restriction–modification enzyme encoded on the naturally-occurring plasmid pEW104 isolated from Lactococcus lactis ssp. cremoris W10. Bioinformatics analysis suggests that the enzyme contains domains characteristic of an mrr endonuclease, a superfamily 2 DNA helicase and a γ-family adenine methyltransferase. LlaGI was expressed and purified from a recombinant clone and its properties characterised. An asymmetric recognition sequence was identified, 5′-CTnGAyG-3′ (where n is A, G, C or T and y is C or T). Methylation of the recognition site occurred on only one strand (the non-degenerate dA residue of 5′-CrTCnAG-3′ being methylated at the N6 position). Double strand DNA breaks at distant, random sites were only observed when two head-to-head oriented, unmethylated copies of the site were present; single sites or pairs in tail-to-tail or head-to-tail repeat only supported a DNA nicking activity. dsDNA nuclease activity was dependent upon the presence of ATP or dATP. Our results are consistent with a directional long-range communication mechanism that is necessitated by the partial site methylation. In the accompanying manuscript [Smith et al. (2009) The single polypeptide restriction–modification enzyme LlaGI is a self-contained molecular motor that translocates DNA loops], we demonstrate that this communication is via 1-dimensional DNA loop translocation. On the basis of this data and that in the third accompanying manuscript [Smith et al. (2009) An Mrr-family nuclease motif in the single polypeptide restriction–modification enzyme LlaGI], we propose that LlaGI is the prototype of a new sub-classification of Restriction-Modification enzymes, named Type I SP (for Single Polypeptide). PMID:19808936

  14. Bacteriophage GIL01 gp7 interacts with host LexA repressor to enhance DNA binding and inhibit RecA-mediated auto-cleavage

    PubMed Central

    Fornelos, Nadine; Butala, Matej; Hodnik, Vesna; Anderluh, Gregor; Bamford, Jaana K.; Salas, Margarita

    2015-01-01

    The SOS response in Eubacteria is a global response to DNA damage and its activation is increasingly associated with the movement of mobile genetic elements. The temperate phage GIL01 is induced into lytic growth using the host's SOS response to genomic stress. LexA, the SOS transcription factor, represses bacteriophage transcription by binding to a set of SOS boxes in the lysogenic promoter P1. However, LexA is unable to efficiently repress GIL01 transcription unless the small phage-encoded protein gp7 is also present. We found that gp7 forms a stable complex with LexA that enhances LexA binding to phage and cellular SOS sites and interferes with RecA-mediated auto-cleavage of LexA, the key step in the initiation of the SOS response. Gp7 did not bind DNA, alone or when complexed with LexA. Our findings suggest that gp7 induces a LexA conformation that favors DNA binding but disfavors LexA auto-cleavage, thereby altering the dynamics of the cellular SOS response. This is the first account of an accessory factor interacting with LexA to regulate transcription. PMID:26138485

  15. Bacteriophage GIL01 gp7 interacts with host LexA repressor to enhance DNA binding and inhibit RecA-mediated auto-cleavage.

    PubMed

    Fornelos, Nadine; Butala, Matej; Hodnik, Vesna; Anderluh, Gregor; Bamford, Jaana K; Salas, Margarita

    2015-09-01

    The SOS response in Eubacteria is a global response to DNA damage and its activation is increasingly associated with the movement of mobile genetic elements. The temperate phage GIL01 is induced into lytic growth using the host's SOS response to genomic stress. LexA, the SOS transcription factor, represses bacteriophage transcription by binding to a set of SOS boxes in the lysogenic promoter P1. However, LexA is unable to efficiently repress GIL01 transcription unless the small phage-encoded protein gp7 is also present. We found that gp7 forms a stable complex with LexA that enhances LexA binding to phage and cellular SOS sites and interferes with RecA-mediated auto-cleavage of LexA, the key step in the initiation of the SOS response. Gp7 did not bind DNA, alone or when complexed with LexA. Our findings suggest that gp7 induces a LexA conformation that favors DNA binding but disfavors LexA auto-cleavage, thereby altering the dynamics of the cellular SOS response. This is the first account of an accessory factor interacting with LexA to regulate transcription. PMID:26138485

  16. The Structure of DNA-Bound Human Topoisomerase II Alpha: Conformational Mechanisms for Coordinating Inter-Subunit Interactions with DNA Cleavage

    PubMed Central

    Wendorff, Timothy J.; Schmidt, Bryan H.; Heslop, Pauline; Austin, Caroline A.; Berger, James M.

    2012-01-01

    Type II topoisomerases are required for the management of DNA superhelicity and chromosome segregation, and serve as frontline targets for a variety of small-molecule therapeutics. To better understand how these enzymes act in both contexts, we determined the 2.9-Å-resolution structure of the DNA cleavage core of human topoisomerase IIα (TOP2A) bound to a doubly nicked, 30-bp duplex oligonucleotide. In accord with prior biochemical and structural studies, TOP2A significantly bends its DNA substrate using a bipartite, nucleolytic center formed at an N-terminal dimerization interface of the cleavage core. However, the protein also adopts a global conformation in which the second of its two inter-protomer contact points, one at the C-terminus, has separated. This finding, together with comparative structural analyses, reveals that the principal site of DNA engagement undergoes highly quantized conformational transitions between distinct binding, cleavage, and drug-inhibited states that correlate with the control of subunit–subunit interactions. Additional consideration of our TOP2A model in light of an etoposide-inhibited complex of human topoisomerase IIβ (TOP2B) suggests possible modification points for developing paralog-specific inhibitors to overcome the tendency of topoisomerase II-targeting chemotherapeutics to generate secondary malignancies. PMID:22841979

  17. Phosphodiester and N-glycosidic bond cleavage in DNA induced by 4-15 eV electrons

    NASA Astrophysics Data System (ADS)

    Zheng, Yi; Cloutier, Pierre; Hunting, Darel J.; Wagner, J. Richard; Sanche, Léon

    2006-02-01

    Thin molecular films of the short single strand of DNA, GCAT, were bombarded under vacuum by electrons with energies between 4 and 15 eV. Ex vacuo analysis by high-pressure liquid chromatography of the samples exposed to the electron beam revealed the formation of a multitude of products. Among these, 12 fragments of GCAT were identified by comparison with reference compounds and their yields were measured as a function of electron energy. For all energies, scission of the backbone gave nonmodified fragments containing a terminal phosphate, with negligible amounts of fragments without the phosphate group. This indicates that phosphodiester bond cleavage by 4-15 eV electrons involves cleavage of the C-O bond rather than the P-O bond. The yield functions exhibit maxima at 6 and 10-12 eV, which are interpreted as due to the formation of transient anions leading to fragmentation. Below 15 eV, these resonances dominate bond dissociation processes. All four nonmodified bases are released from the tetramer, by cleavage of the N-glycosidic bond, which occurs principally via the formation of core-excited resonances located around 6 and 10 eV. The formation of the other nonmodified products leading to cleavage of the phosphodiester bond is suggested to occur principally via two different mechanisms: (1) the formation of a core-excited resonance on the phosphate unit followed by dissociation of the transient anion and (2) dissociation of the CO bond of the phosphate group formed by resonance electron transfer from the bases. In each case, phosphodiester bond cleavage leads chiefly to the formation of stable phosphate anions and sugar radicals with minimal amounts of alkoxyl anions and phosphoryl radicals.

  18. Initial activation of EpCAM cleavage via cell-to-cell contact

    PubMed Central

    2009-01-01

    Background Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is frequently over-expressed in simple epithelia, progenitors, embryonic and tissue stem cells, carcinoma and cancer-initiating cells. Besides functioning as a homophilic adhesion protein, EpCAM is an oncogenic receptor that requires regulated intramembrane proteolysis for activation of its signal transduction capacity. Upon cleavage, the extracellular domain EpEX is released as a soluble ligand while the intracellular domain EpICD translocates into the cytoplasm and eventually into the nucleus in combination with four-and-a-half LIM domains protein 2 (FHL2) and β-catenin, and drives cell proliferation. Methods EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were investigated under varying density conditions using confocal laser scanning microscopy, immunoblotting, cell counting, and conditional cell systems. Results EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were dependent on adequate cell-to-cell contact. If cell-to-cell contact was prohibited EpCAM did not provide growth advantages. If cells were allowed to undergo contact to each other, EpCAM transmitted proliferation signals based on signal transduction-related cleavage processes. Accordingly, the pre-cleaved version EpICD was not dependent on cell-to-cell contact in order to induce c-myc and cell proliferation, but necessitated nuclear translocation. For the case of contact-inhibited cells, although cleavage of EpCAM occurred, nuclear translocation of EpICD was reduced, as were EpCAM effects. Conclusion Activation of EpCAM's cleavage and oncogenic capacity is dependent on cellular interaction (juxtacrine) to provide for initial signals of regulated intramembrane proteolysis, which then support signalling via soluble EpEX (paracrine). PMID:19925656

  19. Synthesis, Characterization, Antimicrobial, DNA Cleavage, and Antioxidant Studies of Some Metal Complexes Derived from Schiff Base Containing Indole and Quinoline Moieties

    PubMed Central

    Karekal, Mahendra Raj; Biradar, Vivekanand; Bennikallu Hire Mathada, Mruthyunjayaswamy

    2013-01-01

    A new Schiff base of 5-chloro-3-phenyl-1H-indole-2-carboxyhydrazide and 3-formyl-2-hydroxy-1H-quinoline (HL), and its Cu(II), Co(II), Ni(II), Zn(II), Cd(II), and Hg(II) complexes have been synthesized and characterized in the light of microanalytical, IR, H1 NMR, UV-Vis, FAB-mass, ESR, XRD, and TGA spectral studies. The magnetic susceptibility measurements and low conductivity data provide evidence for monomeric and neutral nature of the complexes. On the basis of spectral studies and analytical data, it is evident that the Schiff base acts as tridentate ligand. The Cu(II), Co(II), and Ni(II) complexes were octahedral, whereas Zn(II), Cd(II), and Hg(II) complexes were tetrahedral in nature. The redox behavior of the Cu(II) complex was investigated by electrochemical method using cyclic voltammetry. In order to evaluate the effect of metal ions upon chelation, both the ligand and its metal complexes were screened for their antibacterial and antifungal activities by minimum inhibitory concentration (MIC) method. The DNA cleavage experiment performed using agarose gel electrophoresis method showed the cleavage of DNA by all the metal complexes. The free radical scavenging activity of newly synthesized compounds has been determined at a different concentration range by means of their interaction with the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). PMID:24194692

  20. Synthesis, spectral, crystal structure, thermal behavior, antimicrobial and DNA cleavage potential of two octahedral cadmium complexes: A supramolecular structure

    NASA Astrophysics Data System (ADS)

    Montazerozohori, M.; Musavi, S. A.; Masoudiasl, A.; Naghiha, A.; Dusek, M.; Kucerakova, M.

    2015-02-01

    Two new cadmium(II) complexes with the formula of CdL2(NCS)2 and CdL2(N3)2 (in which L is 2,2-dimethyl-N,N‧-bis-(3-phenyl-allylidene)-propane-1,3-diamine) have been synthesized and characterized by elemental analysis, molar conductivity measurements, FT/IR, UV-Visible, 1H and 13C NMR spectra and X-ray studies. The crystal structure analysis of CdL2(NCS)2 indicated that it crystallizes in orthorhombic system with space group of Pbca. Two Schiff base ligands are bonded to cadmium(II) ion as N2-donor chelate. Coordination geometry around the cadmium ion was found to be partially distorted octahedron. The Cd-Nimine bond distances are found in the range of 2.363(2)-2.427(2) Å while the Cd-Nisothiocyanate bond distances are 2.287(2) Å and 2.310(2) Å. The existence of C-H⋯π and C-H⋯S interactions in the CdL2(NCS)2 crystal leads to a supramolecular structure in its network. Then cadmium complexes were screened in vitro for their antibacterial and antifungal activities against two Gram-negative and two Gram-positive bacteria and also against Candida albicans as a fungus. Moreover, the compounds were subjected for DNA-cleavage potential by gel electrophoresis method. Finally thermo-gravimetric analysis of the complexes was applied for thermal behavior studies and then some thermo-kinetics activation parameters were evaluated.

  1. Spectroscopy: The study of DNA cleavage by newly synthesized polydentate macrocyclic ligand and its copper(II) complexes

    NASA Astrophysics Data System (ADS)

    Gupta, Lokesh Kumar; Chandra, Sulekh

    2008-11-01

    A novel hexadentate nitrogen donor [N 6] macrocyclic ligand, i.e. 2,6,12,16,21,22-hexaaza-3,5,13,15-tetramethyl-4,14-diethyl-tricyclo-[15.3.1.1(7-11)]docosane-1(21),2,5,7(22),8,10,12,15,17,19-decaene ( L), has been synthesized. Copper(II) complexes with this ligand have been prepared and subjected to elemental analyses, molar conductance measurements, magnetic susceptibility measurements, mass, 1H NMR (ligand), IR, electronic, and EPR spectral studies. On the basis of molar conductance the complexes may be formulated as [Cu(L)X 2] [X = Cl -, Br -, NO 3- and CH 3COO -] due to their nonelectrolytic nature in N, N'-dimethylformamide (DMF). All the complexes are of the high spin type and are six coordinated. On the basis of IR, electronic and EPR spectral studies tetragonal geometry has been assigned to the Cu(II) complexes. The interaction of these complexes with calf thymus DNA has been explored by using absorption, emission, viscosity measurements, electrochemical studies and DNA cleavage. All the experimental results suggest that the complexes bind to DNA and also promote the cleavage plasmid pBR 322, in the presence of H 2O 2 and ascorbic acid.

  2. Cleavage and activation of a Toll-like receptor by microbial proteases

    PubMed Central

    de Zoete, Marcel R.; Bouwman, Lieneke I.; Keestra, A. Marijke; van Putten, Jos P. M.

    2011-01-01

    Toll-like receptors (TLRs) are innate receptors that show high conservation throughout the animal kingdom. Most TLRs can be clustered into phylogenetic groups that respond to similar types of ligands. One exception is avian TLR15. This receptor does not categorize into one of the existing groups of TLRs and its ligand is still unknown. Here we report that TLR15 is a sensor for secreted virulence-associated fungal and bacterial proteases. Activation of TLR15 involves proteolytic cleavage of the receptor ectodomain and stimulation of NF-κB–dependent gene transcription. Receptor activation can be mimicked by the expression of a truncated TLR15 of which the entire ectodomain is removed, suggesting that receptor cleavage alleviates receptor inhibition by the leucine-rich repeat domain. Our results indicate TLR15 as a unique type of innate immune receptor that combines TLR characteristics with an activation mechanism typical for the evolutionary distinct protease-activated receptors. PMID:21383168

  3. Cleavage and activation of a Toll-like receptor by microbial proteases.

    PubMed

    de Zoete, Marcel R; Bouwman, Lieneke I; Keestra, A Marijke; van Putten, Jos P M

    2011-03-22

    Toll-like receptors (TLRs) are innate receptors that show high conservation throughout the animal kingdom. Most TLRs can be clustered into phylogenetic groups that respond to similar types of ligands. One exception is avian TLR15. This receptor does not categorize into one of the existing groups of TLRs and its ligand is still unknown. Here we report that TLR15 is a sensor for secreted virulence-associated fungal and bacterial proteases. Activation of TLR15 involves proteolytic cleavage of the receptor ectodomain and stimulation of NF-κB-dependent gene transcription. Receptor activation can be mimicked by the expression of a truncated TLR15 of which the entire ectodomain is removed, suggesting that receptor cleavage alleviates receptor inhibition by the leucine-rich repeat domain. Our results indicate TLR15 as a unique type of innate immune receptor that combines TLR characteristics with an activation mechanism typical for the evolutionary distinct protease-activated receptors. PMID:21383168

  4. Expression of a naturally occurring angiotensin AT(1) receptor cleavage fragment elicits caspase-activation and apoptosis.

    PubMed

    Cook, Julia L; Singh, Akannsha; DeHaro, Dawn; Alam, Jawed; Re, Richard N

    2011-11-01

    Several transmembrane receptors are documented to accumulate in nuclei, some as holoreceptors and others as cleaved receptor products. Our prior studies indicate that a population of the 7-transmembrane angiotensin type-1 receptor (AT(1)R) is cleaved in a ligand-augmented manner after which the cytoplasmic, carboxy-terminal cleavage fragment (CF) traffics to the nucleus. In the present report, we determine the precise cleavage site within the AT(1)R by mass spectrometry and Edman sequencing. Cleavage occurs between Leu(305) and Gly(306) at the junction of the seventh transmembrane domain and the intracellular cytoplasmic carboxy-terminal domain. To evaluate the function of the CF distinct from the holoreceptor, we generated a construct encoding the CF as an in-frame yellow fluorescent protein fusion. The CF accumulates in nuclei and induces apoptosis in CHO-K1 cells, rat aortic smooth muscle cells (RASMCs), MCF-7 human breast adenocarcinoma cells, and H9c2 rat cardiomyoblasts. All cell types show nuclear fragmentation and disintegration, as well as evidence for phosphotidylserine displacement in the plasma membrane and activated caspases. RASMCs specifically showed a 5.2-fold increase (P < 0.001) in CF-induced active caspases compared with control and a 7.2-fold increase (P < 0.001) in cleaved caspase-3 (Asp174). Poly(ADP-ribose)polymerase was upregulated 4.8-fold (P < 0.001) in CF expressing cardiomyoblasts and colocalized with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). CF expression also induces DNA laddering, the gold-standard for apoptosis in all cell types studied. CF-induced apoptosis, therefore, appears to be a general phenomenon as it is observed in multiple cell types including smooth muscle cells and cardiomyoblasts. PMID:21813711

  5. The role of chordin fragments generated by partial tolloid cleavage in regulating BMP activity.

    PubMed

    Troilo, Helen; Barrett, Anne L; Wohl, Alexander P; Jowitt, Thomas A; Collins, Richard F; Bayley, Christopher P; Zuk, Alexandra V; Sengle, Gerhard; Baldock, Clair

    2015-10-01

    Chordin-mediated regulation of bone morphogenetic protein (BMP) family growth factors is essential in early embryogenesis and adult homoeostasis. Chordin binds to BMPs through cysteine-rich von Willebrand factor type C (vWC) homology domains and blocks them from interacting with their cell surface receptors. These domains also self-associate and enable chordin to target related proteins to fine-tune BMP regulation. The chordin-BMP inhibitory complex is strengthened by the secreted glycoprotein twisted gastrulation (Tsg); however, inhibition is relieved by cleavage of chordin at two specific sites by tolloid family metalloproteases. As Tsg enhances this cleavage process, it serves a dual role as both promoter and inhibitor of BMP signalling. Recent developments in chordin research suggest that rather than simply being by-products, the cleavage fragments of chordin continue to play a role in BMP regulation. In particular, chordin cleavage at the C-terminus potentiates its anti-BMP activity in a type-specific manner. PMID:26517884

  6. The role of chordin fragments generated by partial tolloid cleavage in regulating BMP activity

    PubMed Central

    Troilo, Helen; Barrett, Anne L.; Wohl, Alexander P.; Jowitt, Thomas A.; Collins, Richard F.; Bayley, Christopher P.; Zuk, Alexandra V.; Sengle, Gerhard; Baldock, Clair

    2015-01-01

    Chordin-mediated regulation of bone morphogenetic protein (BMP) family growth factors is essential in early embryogenesis and adult homoeostasis. Chordin binds to BMPs through cysteine-rich von Willebrand factor type C (vWC) homology domains and blocks them from interacting with their cell surface receptors. These domains also self-associate and enable chordin to target related proteins to fine-tune BMP regulation. The chordin–BMP inhibitory complex is strengthened by the secreted glycoprotein twisted gastrulation (Tsg); however, inhibition is relieved by cleavage of chordin at two specific sites by tolloid family metalloproteases. As Tsg enhances this cleavage process, it serves a dual role as both promoter and inhibitor of BMP signalling. Recent developments in chordin research suggest that rather than simply being by-products, the cleavage fragments of chordin continue to play a role in BMP regulation. In particular, chordin cleavage at the C-terminus potentiates its anti-BMP activity in a type-specific manner. PMID:26517884

  7. Genomic DNA breakpoints in AML1/RUNX1 and ETO cluster with topoisomerase II DNA cleavage and DNase I hypersensitive sites in t(8;21) leukemia

    PubMed Central

    Zhang, Yanming; Strissel, Pamela; Strick, Reiner; Chen, Jianjun; Nucifora, Giuseppina; Le Beau, Michelle M.; Larson, Richard A.; Rowley, Janet D.

    2002-01-01

    The translocation t(8;21)(q22;q22) is one of the most frequent chromosome translocations in acute myeloid leukemia (AML). AML1/RUNX1 at 21q22 is involved in t(8;21), t(3;21), and t(16;21) in de novo and therapy-related AML and myelodysplastic syndrome as well as in t(12;21) in childhood B cell acute lymphoblastic leukemia. Although DNA breakpoints in AML1 and ETO (at 8q22) cluster in a few introns, the mechanisms of DNA recombination resulting in t(8;21) are unknown. The correlation of specific chromatin structural elements, i.e., topoisomerase II (topo II) DNA cleavage sites, DNase I hypersensitive sites, and scaffold-associated regions, which have been implicated in chromosome recombination with genomic DNA breakpoints in AML1 and ETO in t(8;21) is unknown. The breakpoints in AML1 and ETO were clustered in the Kasumi 1 cell line and in 31 leukemia patients with t(8;21); all except one had de novo AML. Sequencing of the breakpoint junctions revealed no common DNA motif; however, deletions, duplications, microhomologies, and nontemplate DNA were found. Ten in vivo topo II DNA cleavage sites were mapped in AML1, including three in intron 5 and seven in intron 7a, and two were in intron 1b of ETO. All strong topo II sites colocalized with DNase I hypersensitive sites and thus represent open chromatin regions. These sites correlated with genomic DNA breakpoints in both AML1 and ETO, thus implicating them in the de novo 8;21 translocation. PMID:11867721

  8. Chromium(VI) reduction by catechol(amine)s results in DNA cleavage in vitro: relevance to chromium genotoxicity.

    PubMed

    Pattison, D I; Davies, M J; Levina, A; Dixon, N E; Lay, P A

    2001-05-01

    Catechols are found extensively in nature both as essential biomolecules and as the byproducts of normal oxidative damage of amino acids and proteins. They are also present in cigarette smoke and other atmospheric pollutants. Here, the interactions of reactive species generated in Cr(VI)/catechol(amine) mixtures with plasmid DNA have been investigated to model a potential route to Cr(VI)-induced genotoxicity. Reduction of Cr(VI) by 3,4-dihydroxyphenylalanine (DOPA) (1), dopamine (2), or adrenaline (3) produces species that cause extensive DNA damage, but the products of similar reactions with catechol (4) or 4-tert-butylcatechol (5) do not damage DNA. The Cr(VI)/catechol(amine) reactions have been studied at low added H(2)O(2) concentrations, which lead to enhanced DNA cleavage with 1 and induce DNA cleavage with 4. The Cr(V) and organic intermediates generated by the reactions of Cr(VI) with 1 or 4 in the presence of H(2)O(2) were characterized by EPR spectroscopy. The detected signals were assigned to Cr(V)-catechol, Cr(V)-peroxo, and mixed Cr(V)-catechol-peroxo complexes. Oxygen consumption during the reactions of Cr(VI) with 1, 2, 4, and 5 was studied, and H(2)O(2) production was quantified. Reactions of Cr(VI) with 1 and 2, but not 4 and 5, consume considerable amounts of dissolved O(2), and give extensive H(2)O(2) production. Extents of oxygen consumption and H(2)O(2) production during the reaction of Cr(VI) with enzymatically generated 1 and N-acetyl-DOPA (from the reaction of Tyr and N-acetyl-Tyr with tyrosinase, respectively) were correlated with the DNA cleaving abilities of the products of these reactions. The reaction of Cr(VI) with enzymatically generated 1 produced significant amounts of H(2)O(2) and caused significant DNA damage, but the N-acetyl-DOPA did not. The extent of in vitro DNA damage is reduced considerably by treatment of the Cr(VI)/catechol(amine) mixtures with catalase, which shows that the DNA damage is H(2)O(2)-dependent and that the

  9. Internal cleavages of the autoinhibitory prodomain are required for membrane type 1 matrix metalloproteinase activation, although furin cleavage alone generates inactive proteinase.

    PubMed

    Golubkov, Vladislav S; Cieplak, Piotr; Chekanov, Alexei V; Ratnikov, Boris I; Aleshin, Alexander E; Golubkova, Natalya V; Postnova, Tatiana I; Radichev, Ilian A; Rozanov, Dmitri V; Zhu, Wenhong; Motamedchaboki, Khatereh; Strongin, Alex Y

    2010-09-01

    The functional activity of invasion-promoting membrane type 1 matrix metalloproteinase (MT1-MMP) is elevated in cancer. This elevated activity promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intradomain cleavage of the PGD/L(50) site initiates the MT1-MMP activation, whereas the (108)RRKR(111)/Y(112) cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP. PMID:20605791

  10. Internal Cleavages of the Autoinhibitory Prodomain Are Required for Membrane Type 1 Matrix Metalloproteinase Activation, although Furin Cleavage Alone Generates Inactive Proteinase*

    PubMed Central

    Golubkov, Vladislav S.; Cieplak, Piotr; Chekanov, Alexei V.; Ratnikov, Boris I.; Aleshin, Alexander E.; Golubkova, Natalya V.; Postnova, Tatiana I.; Radichev, Ilian A.; Rozanov, Dmitri V.; Zhu, Wenhong; Motamedchaboki, Khatereh; Strongin, Alex Y.

    2010-01-01

    The functional activity of invasion-promoting membrane type 1 matrix metalloproteinase (MT1-MMP) is elevated in cancer. This elevated activity promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intradomain cleavage of the PGD↓L50 site initiates the MT1-MMP activation, whereas the 108RRKR111↓Y112 cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP. PMID:20605791

  11. The active site of RNA polymerase II participates in transcript cleavage within arrested ternary complexes.

    PubMed Central

    Rudd, M D; Izban, M G; Luse, D S

    1994-01-01

    RNA polymerase II may become arrested during transcript elongation, in which case the ternary complex remains intact but further RNA synthesis is blocked. To relieve arrest, the nascent transcript must be cleaved from the 3' end. RNAs of 7-17 nt are liberated and transcription continues from the newly exposed 3' end. Factor SII increases elongation efficiency by strongly stimulating the transcript cleavage reaction. We show here that arrest relief can also occur by the addition of pyrophosphate. This generates the same set of cleavage products as factor SII, but the fragments produced with pyrophosphate have 5'-triphosphate termini. Thus, the active site of RNA polymerase II, in the presence of pyrophosphate, appears to be capable of cleaving phosphodiester linkages as far as 17 nt upstream of the original site of polymerization, leaving the ternary complex intact and transcriptionally active. Images PMID:8058756

  12. Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin.

    PubMed

    De Lorenzi, Valentina; Sarra Ferraris, Gian Maria; Madsen, Jeppe B; Lupia, Michela; Andreasen, Peter A; Sidenius, Nicolai

    2016-07-01

    Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process. PMID:27189837

  13. Detailed study of sequence-specific DNA cleavage of triplex-forming oligonucleotides linked to 1,10-phenanthroline.

    PubMed

    Shimizu, M; Inoue, H; Ohtsuka, E

    1994-01-18

    We introduced eight bases, including four base analogs, into 15-mer triplex-forming oligonucleotides (TFOs) [d-psTTTCTTTNTTTTCTT; ps = thiophosphate; N = A, G, C, T, 2'-deoxyinosine (I), 2'-deoxyxanthosine (X), 5-methyl-2'-deoxycytidine (m5C), or 5-bromo-2'-deoxyuridine(br5U)] to investigate the Hoogsteen-like hydrogen bonding to the base in the target 34-mer strand (d-TGAGTGAGTAAAGAAARAAAAGAATGAGTGCCAA.d-TTGGCACTCATTCTTTTYTTTCT TTACTCACTCA; RY = AT, GC, TA, or CG). We examined the thermal stability of 15-mer triplexes in buffer containing 100 mM sodium acetate and 1 M NaCl at pH 5.0. The triplexes with typical triplets of T.AT (51.3 degrees C), br5U.AT (52.4 degrees C), C+.GC (66.7 degrees C), and m5C+.GC (66.8 degrees C) at the central position showed relatively higher Tm values, as expected. The relatively high stability of the X.AT triplex (39.8 degrees C) was observed. Among the N.TA triplets, G.TA (44.8 degrees C) was thermally the most stable, and moreover, the data showed that the N.TA triplet was also stabilized by I in the N position (40.7 degrees C). Furthermore, the TFOs were converted to DNA-cleaving molecules by introducing a newly synthesized 1,10-phenanthroline (OP) derivative on the thiophosphate group at the 5' end. Cleavage reactions of the 32P-labeled DNA (34-mer) were carried out. The cleavage efficiencies were compared to the Tm values of triplexes with or without an OP derivative. Results showed that the increased cleavage yields reflect the higher thermal stability of the triplex formed in most cases, but a few exceptional cases existed. Especially, the G-containing TFO did not show the above correlation between thermal stability and cleavage yield.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8286392

  14. DNA cleavage within the MLL breakpoint cluster region is a specific event which occurs as part of higher-order chromatin fragmentation during the initial stages of apoptosis.

    PubMed Central

    Stanulla, M; Wang, J; Chervinsky, D S; Thandla, S; Aplan, P D

    1997-01-01

    A distinct population of therapy-related acute myeloid leukemia (t-AML) is strongly associated with prior administration of topoisomerase II (topo II) inhibitors. These t-AMLs display distinct cytogenetic alterations, most often disrupting the MLL gene on chromosome 11q23 within a breakpoint cluster region (bcr) of 8.3 kb. We recently identified a unique site within the MLL bcr that is highly susceptible to DNA double-strand cleavage by classic topo II inhibitors (e.g., etoposide and doxorubicin). Here, we report that site-specific cleavage within the MLL bcr can be induced by either catalytic topo II inhibitors, genotoxic chemotherapeutic agents which do not target topo II, or nongenotoxic stimuli of apoptotic cell death, suggesting that this site-specific cleavage is part of a generalized cellular response to an apoptotic stimulus. We also show that site-specific cleavage within the MLL bcr can be linked to the higher-order chromatin fragmentation that occurs during the initial stages of apoptosis, possibly through cleavage of DNA loops at their anchorage sites to the nuclear matrix. In addition, we show that site-specific cleavage is conserved between species, as specific DNA cleavage can also be demonstrated within the murine MLL locus. Lastly, site-specific cleavage during apoptosis can also be identified at the AML1 locus, a locus which is also frequently involved in chromosomal rearrangements present in t-AML patients. In conclusion, these results suggest the potential involvement of higher-order chromatin fragmentation which occurs as a part of a generalized apoptotic response in a mechanism leading to chromosomal translocation of the MLL and AML1 genes and subsequent t-AML. PMID:9199342

  15. Mercury Detoxification by Bacteria: Simulations of Transcription Activation and Mercury-Carbon Bond Cleavage

    SciTech Connect

    Guo, Hao-Bo; Parks, Jerry M; Johs, Alexander; Smith, Jeremy C

    2011-01-01

    In this chapter, we summarize recent work from our laboratory and provide new perspective on two important aspects of bacterial mercury resistance: the molecular mechanism of transcriptional regulation by MerR, and the enzymatic cleavage of the Hg-C bond in methylmercury by the organomercurial lyase, MerB. Molecular dynamics (MD) simulations of MerR reveal an opening-and-closing dynamics, which may be involved in initiating transcription of mercury resistance genes upon Hg(II) binding. Density functional theory (DFT) calculations on an active-site model of the enzyme reveal how MerB catalyzes the Hg-C bond cleavage using cysteine coordination and acid-base chemistry. These studies provide insight into the detailed mechanisms of microbial gene regulation and defense against mercury toxicity.

  16. Synthesis, characterization, in vitro antimicrobial and DNA cleavage studies of Co(II), Ni(II) and Cu(II) complexes with ONOO donor coumarin Schiff bases

    NASA Astrophysics Data System (ADS)

    Patil, Sangamesh A.; Unki, Shrishila N.; Kulkarni, Ajaykumar D.; Naik, Vinod H.; Badami, Prema S.

    2011-01-01

    A series of Co(II), Ni(II) and Cu(II) complexes have been synthesized with Schiff bases derived from 2-hydroxy-1-naphthaldehyde and 2-oxo-2H-chromene-3-carbohydrazide/6-bromo-2-oxo-2H-chromene-3-carbohydrazide. The chelation of the complexes has been proposed in the light of analytical, spectral (IR, UV-Vis, 1H NMR, ESR, FAB-mass and fluorescence), magnetic and thermal studies. The measured molar conductance values indicate that, the complexes are non-electrolytic in nature. The redox behavior of the complexes was investigated with electrochemical method by using cyclic voltammetry. The Schiff bases and their metal complexes have been screened for their in vitro antibacterial ( Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Salmonella typhi) and antifungal activities ( Candida albicans, Cladosporium and Aspergillus niger) by MIC method. The DNA cleavage is studied by agarose gel electrophoresis method.

  17. Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage.

    PubMed Central

    Anderson, E D; VanSlyke, J K; Thulin, C D; Jean, F; Thomas, G

    1997-01-01

    Activation of furin requires autoproteolytic cleavage of its 83-amino acid propeptide at the consensus furin site, Arg-Thr-Lys-Arg107/. This RER-localized cleavage is necessary, but not sufficient, for enzyme activation. Rather, full activation of furin requires exposure to, and correct routing within, the TGN/endosomal system. Here, we identify the steps in addition to the initial propeptide cleavage necessary for activation of furin. Exposure of membrane preparations containing an inactive RER-localized soluble furin construct to either: (i) an acidic and calcium-containing environment characteristic of the TGN; or (ii) mild trypsinization at neutral pH, resulted in the activation of the endoprotease. Taken together, these results suggest that the pH drop facilitates the removal of a furin inhibitor. Consistent with these findings, following cleavage in the RER, the furin propeptide remains associated with the enzyme and functions as a potent inhibitor of the endoprotease. Co-immunoprecipitation studies coupled with analysis by mass spectrometry show that release of the propeptide at acidic pH, and hence activation of furin, requires a second cleavage within the autoinhibitory domain at a site containing a P6 arginine (-Arg70-Gly-Val-Thr-Lys-Arg75/-). The significance of this cleavage in regulating the compartment-specific activation of furin, and the relationship of the furin activation pathway to those of other serine endoproteases are discussed. PMID:9130696

  18. Yeast SREBP cleavage activation requires the Golgi Dsc E3 ligase complex

    PubMed Central

    Stewart, Emerson V.; Nwosu, Christine C.; Tong, Zongtian; Roguev, Assen; Cummins, Timothy D.; Kim, Dong-Uk; Hayles, Jacqueline; Park, Han-Oh; Hoe, Kwang-Lae; Powell, David W.; Krogan, Nevan J.; Espenshade, Peter J.

    2011-01-01

    SUMMARY Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here, we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1–4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2 conjugating enzyme Ubc4, the Dsc1 RING E3 ligase and the proteasome. dsc mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation. PMID:21504829

  19. NMR structure of the active conformation of the Varkud satellite ribozyme cleavage site

    PubMed Central

    Hoffmann, Bernd; Mitchell, G. Thomas; Gendron, Patrick; Major, François; Andersen, Angela A.; Collins, Richard A.; Legault, Pascale

    2003-01-01

    Substrate cleavage by the Neurospora Varkud satellite (VS) ribozyme involves a structural change in the stem-loop I substrate from an inactive to an active conformation. We have determined the NMR solution structure of a mutant stem-loop I that mimics the active conformation of the cleavage site internal loop. This structure shares many similarities, but also significant differences, with the previously determined structures of the inactive internal loop. The active internal loop displays different base-pairing interactions and forms a novel RNA fold composed exclusively of sheared G-A base pairs. From chemical-shift mapping we identified two Mg2+ binding sites in the active internal loop. One of the Mg2+ binding sites forms in the active but not the inactive conformation of the internal loop and is likely important for catalysis. Using the structure comparison program mc-search, we identified the active internal loop fold in other RNA structures. In Thermus thermophilus 16S rRNA, this RNA fold is directly involved in a long-range tertiary interaction. An analogous tertiary interaction may form between the active internal loop of the substrate and the catalytic domain of the VS ribozyme. The combination of NMR and bioinformatic approaches presented here has identified a novel RNA fold and provides insights into the structural basis of catalytic function in the Neurospora VS ribozyme. PMID:12782785

  20. Nanoparticulate carbon black in cigarette smoke induces DNA cleavage and Th17-mediated emphysema

    PubMed Central

    You, Ran; Lu, Wen; Shan, Ming; Berlin, Jacob M; Samuel, Errol LG; Marcano, Daniela C; Sun, Zhengzong; Sikkema, William KA; Yuan, Xiaoyi; Song, Lizhen; Hendrix, Amanda Y; Tour, James M; Corry, David B; Kheradmand, Farrah

    2015-01-01

    Chronic inhalation of cigarette smoke is the major cause of sterile inflammation and pulmonary emphysema. The effect of carbon black (CB), a universal constituent of smoke derived from the incomplete combustion of organic material, in smokers and non-smokers is less known. In this study, we show that insoluble nanoparticulate carbon black (nCB) accumulates in human myeloid dendritic cells (mDCs) from emphysematous lung and in CD11c+ lung antigen presenting cells (APC) of mice exposed to smoke. Likewise, nCB intranasal administration induced emphysema in mouse lungs. Delivered by smoking or intranasally, nCB persisted indefinitely in mouse lung, activated lung APCs, and promoted T helper 17 cell differentiation through double-stranded DNA break (DSB) and ASC-mediated inflammasome assembly in phagocytes. Increasing the polarity or size of CB mitigated many adverse effects. Thus, nCB causes sterile inflammation, DSB, and emphysema and explains adverse health outcomes seen in smokers while implicating the dangers of nCB exposure in non-smokers. DOI: http://dx.doi.org/10.7554/eLife.09623.001 PMID:26437452

  1. An integrated system for enzymatic cleavage and electrostretching of freely-suspended single DNA molecules.

    PubMed

    Lam, Liza; Sakakihara, Shouichi; Ishizuka, Koji; Takeuchi, Shoji; Noji, Hiroyuki

    2007-12-01

    A novel polyacrylamide gel-based femtolitre microchamber system for performing single-molecule restriction enzyme assay on freely-suspended DNA molecules and subsequent DNA electrostretching by applying an alternating electric field has been developed. We attempted the integration by firstly initiating restriction enzyme reaction on a fluorescent-stained lambdaDNA molecule, encapsulated in a microchamber, using magnesium as an external trigger. Upon complete digestion, the cleaved DNA fragments were electrostretched to analyze the DNA lengths optically. The critical parameters for electrostretching of encapsulated DNA were investigated and optimum stretching was achieved by using 1.5 kHz pulses with electric field strength in the order of 10(3) V cm(-1) in 7% linear polyacrylamide (LPA) solution. LPA was adopted to minimize the adverse effects of ionic thermal agitation on molecular dielectrophoretic elongation in the microchamber. In our experiments, as the fragments were not immobilized throughout the entire protocol, it was found from repeated tests that digestion always occurred, producing the expected number of cleaved fragments. This versatile microchamber approach realized direct observation of these biological reactions on real-time basis at a single-molecule level. Furthermore, with the employment of porous polyacrylamide gel, the effective manipulation of DNA assays and the ability to combine conventionally independent bioanalytical processes have been demonstrated. PMID:18030395

  2. Anticancer potential of a photoactivated transplatin derivative containing the methylazaindole ligand mediated by ROS generation and DNA cleavage.

    PubMed

    Pracharova, Jitka; Radosova Muchova, Tereza; Dvorak Tomastikova, Eva; Intini, Francesco P; Pacifico, Concetta; Natile, Giovanni; Kasparkova, Jana; Brabec, Viktor

    2016-08-16

    The limitations associated with the clinical utility of conventional platinum anticancer drugs have stimulated research leading to the design of new metallodrugs with improved pharmacological properties, particularly with increased selectivity for cancer cells. Very recent research has demonstrated that photoactivation or photopotentiation of platinum drugs can be one of the promising approaches to tackle this challenge. This is so because the application of irradiation can be targeted exclusively to the tumor tissue so that the resulting effects could be much more selective and targeted to the tumor. We show in this work that the presence of 1-methyl-7-azaindole in trans-[PtCl2(NH3)(L)] (L = 1-methyl-7-azaindole, compound 1) markedly potentiated the DNA binding ability of 1 when irradiated by UVA light in a cell-free medium. Concomitantly, the formation of cytotoxic bifunctional cross-links was markedly enhanced. In addition, 1, when irradiated with UVA, was able to effectively cleave the DNA backbone also in living cells. The incorporation of 1-methyl-7-azaindole moiety had also a profound effect on the photophysical properties of 1, which can generate singlet oxygen responsible for the DNA cleavage reaction. Finally, we found that 1, upon irradiation with UVA light, exhibited a pronounced dose-dependent decrease in viability of A2780 cells whereas it was markedly less cytotoxic if the cells were treated in the absence of light. Hence, it is possible to conclude that 1 is amenable to photodynamic therapy. PMID:27396365

  3. DNA interactions and photocatalytic strand cleavage by artificial nucleases based on water-soluble gold(III) porphyrins.

    PubMed

    Haeubl, Martin; Reith, Lorenz Michael; Gruber, Bernadette; Karner, Uwe; Müller, Norbert; Knör, Günther; Schoefberger, Wolfgang

    2009-09-01

    The novel gold porphyrin complex (5,10,15-tris(N-methylpyridinium-4-yl)-20-(1-pyrenyl)-porphyrinato)gold(III) chloride, [Au(III)(TMPy3Pyr1P)]Cl4, was prepared and characterized by optical spectroscopy, high-resolution nuclear magnetic resonance (NMR), and electrospray mass spectrometry. This cationic multichromophore compound exhibits excellent water solubility and does not form aggregates under physiological conditions. Binding interactions of this complex and related model compounds with nucleic acid substrates have been studied and characterized by NMR and circular dichroism spectroscopy. The photoreactivity of [Au(III)(TMPy3Pyr1P)]Cl4 was investigated under anaerobic and aerobic conditions in the presence of an excess of purine nucleoside, guanosine, and plasmid DNA. Photocatalytic oxidative degradation of guanosine and the change from supercoiled to circular plasmid DNA upon monochromatic irradiation and polychromatic blue-light exposure with a maximum at 420 nm was explored. The potential of the novel water-soluble cationic metallointercalator complex [Au(III)(TMPy3Pyr1P)]Cl4 to serve as a catalytic photonuclease for the cleavage of DNA has been demonstrated. PMID:19471974

  4. Synthesis, Characterization, Antimicrobial, DNA Cleavage, and In Vitro Cytotoxic Studies of Some Metal Complexes of Schiff Base Ligand Derived from Thiazole and Quinoline Moiety

    PubMed Central

    Yernale, Nagesh Gunvanthrao; Bennikallu Hire Mathada, Mruthyunjayaswamy

    2014-01-01

    A novel Schiff base ligand N-(4-phenylthiazol-2yl)-2-((2-thiaxo-1,2-dihydroquinolin-3-yl)methylene)hydrazinecarboxamide (L) obtained by the condensation of N-(4-phenylthiazol-2-yl)hydrazinecarboxamide with 2-thioxo-1,2-dihydroquinoline-3-carbaldehyde and its newly synthesized Cu(II), Co(II), Ni(II), and Zn(II) complexes have been characterized by elemental analysis and various spectral studies like FT-IR, 1H NMR, ESI mass, UV-Visible, ESR, TGA/DTA, and powder X-ray diffraction studies. The Schiff base ligand (L) behaves as tridentate ONS donor and forms the complexes of type [ML(Cl)2] with square pyramidal geometry. The Schiff base ligand (L) and its metal complexes have been screened in vitro for their antibacterial and antifungal activities by minimum inhibitory concentration (MIC) method. The DNA cleavage activity of ligand and its metal complexes were studied using plasmid DNA pBR322 as a target molecule by gel electrophoresis method. The brine shrimp bioassay was also carried out to study the in vitro cytotoxicity properties for the ligand and its metal complexes against Artemia salina. The results showed that the biological activities of the ligand were found to be increased on complexation. PMID:24729778

  5. Initiation of Apoptosis by Granzyme B Requires Direct Cleavage of Bid, but Not Direct Granzyme B–Mediated Caspase Activation

    PubMed Central

    Sutton, Vivien R.; Davis, Joanne E.; Cancilla, Michael; Johnstone, Ricky W.; Ruefli, Astrid A.; Sedelies, Karin; Browne, Kylie A.; Trapani, Joseph A.

    2000-01-01

    The essential upstream steps in granzyme B–mediated apoptosis remain undefined. Herein, we show that granzyme B triggers the mitochondrial apoptotic pathway through direct cleavage of Bid; however, cleavage of procaspases was stalled when mitochondrial disruption was blocked by Bcl-2. The sensitivity of granzyme B–resistant Bcl-2–overexpressing FDC-P1 cells was restored by coexpression of wild-type Bid, or Bid with a mutation of its caspase-8 cleavage site, and both types of Bid were cleaved. However, Bid with a mutated granzyme B cleavage site remained intact and did not restore apoptosis. Bid with a mutation preventing its interaction with Bcl-2 was cleaved but also failed to restore apoptosis. Rapid Bid cleavage by granzyme B (<2 min) was not delayed by Bcl-2 overexpression. These results clearly placed Bid cleavage upstream of mitochondrial Bcl-2. In granzyme B–treated Jurkat cells, endogenous Bid cleavage and loss of mitochondrial membrane depolarization occurred despite caspase inactivation with z-Val-Ala-Asp-fluoromethylketone or Asp-Glu-Val-Asp-fluoromethylketone. Initial partial processing of procaspase-3 and -8 was observed irrespective of Bcl-2 overexpression; however, later processing was completely abolished by Bcl-2. Overall, our results indicate that mitochondrial perturbation by Bid is necessary to achieve a lethal threshold of caspase activity and cell death due to granzyme B. PMID:11085743

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

    PubMed Central

    Rodrigo, María J.; Alquézar, Berta; Al-Babili, Salim

    2013-01-01

    Citrus is the first tree crop in terms of fruit production. The colour of Citrus fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C30 apocarotenoids, mainly β-citraurin (3-hydroxy-β-apo-8′-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in Citrus fruits, little is known about the formation of C30 apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C30 apocarotenoids. In silico data mining revealed a new family of five CCD4-type genes in Citrus. One gene of this family, CCD4b1, was expressed in reproductive and vegetative tissues of different Citrus species in a pattern correlating with the accumulation of C30 apocarotenoids. Moreover, developmental processes and treatments which alter Citrus fruit peel pigmentation led to changes of β-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in β-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and β-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7′,8′ double bond in zeaxanthin and β-cryptoxanthin, confirming its role in C30 apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7′,8′ double bond of cyclic C40 carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C30 apocarotenoids in Citrus which are key pigments in fruit coloration. PMID:24006419

  7. Oxidative cleavage of DNA by pentamethine carbocyanine dyes irradiated with long-wavelength visible light.

    PubMed

    Mapp, Carla T; Owens, Eric A; Henary, Maged; Grant, Kathryn B

    2014-01-01

    Here we report the synthesis of seven symmetrical carbocyanine dyes in which two nitrogen-substituted benz[e]indolium rings are joined by a pentamethine bridge that is meso-substituted with chlorine or bromine versus hydrogen. The heteroatom of benz[e]indolium is modified with a phenylpropyl, methyl, or cationic quaternary ammonium group. In reactions containing micro molar concentrations of halogenated dye, irradiation at 575, 588, 623, or 700nm produces good photocleavage of plasmid DNA. UV-visible spectra show that the carbocyanines are in their H-aggregated and monomeric forms. Scavenger experiments point to the involvement of singlet oxygen and hydroxyl radicals in DNA photocleavage. PMID:24332091

  8. Flow Cytometric Assays for Interrogating LAGLIDADG Homing Endonuclease DNA-Binding and Cleavage Properties

    PubMed Central

    Baxter, Sarah K.; Lambert, Abigail R.; Scharenberg, Andrew M.; Jarjour, Jordan

    2014-01-01

    A fast, easy, and scalable method to assess the properties of site-specific nucleases is crucial to understanding their in cellulo behavior in genome engineering or population-level gene drive applications. Here we describe an analytical platform that enables high-throughput, semiquantitative interrogation of the DNA-binding and catalytic properties of LAGLIDADG homing endonucleases (LHEs). Using this platform, natural or engineered LHEs are expressed on the surface of Saccharomyces cerevisiae yeast where they can be rapidly evaluated against synthetic DNA target sequences using flow cytometry. PMID:23423888

  9. SODs, DNA binding and cleavage studies of new Mn(III) complexes with 2-((3-(benzyloxy)pyridin-2-ylimino)methyl)phenol

    NASA Astrophysics Data System (ADS)

    Shivakumar, L.; Shivaprasad, K.; Revanasiddappa, Hosakere D.

    2013-04-01

    Newly synthesized ligand [2-((3-(benzyloxy)pyridin-2-ylimino)methyl)phenol] (Bpmp) react with manganese(II) to form mononuclear complexes [Mn(phen)(Bpmp)(CH3COO)(H2O)]·4H2O (1), (phen = 1,10-phenanthroline) and [Mn(Bpmp)2(CH3COO)(H2O)]·5H2O (2). These complexes were characterized by elemental analysis, IR, 1H NMR, Mass, UV-vis spectral studies. Molar conductance and thermogravimetric analysis of these complexes were also recorded. The in vitro SOD mimic activity of Mn(III) complexes were carried out and obtained with good result. The DNA-binding properties of the complexes 1 and 2 were investigated by UV-spectroscopy, fluorescence spectroscopy and viscosity measurements. The spectral results suggest that the complexes 1 and 2 can bind to Calf thymus DNA by intercalation mode. The cleavage properties of these complexes with super coiled pUC19 have been studied using the gel electrophoresis method, wherein both complexes 1 and 2 displayed chemical nuclease activity in the absence and presence of H2O2via an oxidative mechanism. All the complexes inhibit the growth of both Gram positive and Gram negative bacteria to competent level. The MIC was determined by microtiter method.

  10. SODs, DNA binding and cleavage studies of new Mn(III) complexes with 2-((3-(benzyloxy)pyridin-2-ylimino)methyl)phenol.

    PubMed

    Shivakumar, L; Shivaprasad, K; Revanasiddappa, Hosakere D

    2013-04-15

    Newly synthesized ligand [2-((3-(benzyloxy)pyridin-2-ylimino)methyl)phenol] (Bpmp) react with manganese(II) to form mononuclear complexes [Mn(phen)(Bpmp)(CH3COO)(H2O)]·4H2O (1), (phen=1,10-phenanthroline) and [Mn(Bpmp)2(CH3COO)(H2O)]·5H2O (2). These complexes were characterized by elemental analysis, IR, (1)H NMR, Mass, UV-vis spectral studies. Molar conductance and thermogravimetric analysis of these complexes were also recorded. The in vitro SOD mimic activity of Mn(III) complexes were carried out and obtained with good result. The DNA-binding properties of the complexes 1 and 2 were investigated by UV-spectroscopy, fluorescence spectroscopy and viscosity measurements. The spectral results suggest that the complexes 1 and 2 can bind to Calf thymus DNA by intercalation mode. The cleavage properties of these complexes with super coiled pUC19 have been studied using the gel electrophoresis method, wherein both complexes 1 and 2 displayed chemical nuclease activity in the absence and presence of H2O2 via an oxidative mechanism. All the complexes inhibit the growth of both Gram positive and Gram negative bacteria to competent level. The MIC was determined by microtiter method. PMID:23429055

  11. Stimulation of topoisomerase II-mediated DNA cleavage by three DNA-intercalating plant alkaloids: cryptolepine, matadine, and serpentine.

    PubMed

    Dassonneville, L; Bonjean, K; De Pauw-Gillet, M C; Colson, P; Houssier, C; Quetin-Leclercq, J; Angenot, L; Bailly, C

    1999-06-15

    Cryptolepine, matadine, and serpentine are three indoloquinoline alkaloids isolated from the roots of African plants: Cryptolepis sanguinolenta, Strychnos gossweileri, and Rauwolfia serpentina, respectively. For a long time, these alkaloids have been used in African folk medicine in the form of plant extracts for the treatment of multiple diseases, in particular as antimalarial drugs. To date, the molecular basis for their diverse biological effects remains poorly understood. To elucidate their mechanism of action, we studied their interaction with DNA and their effects on topoisomerase II. The strength and mode of binding to DNA of the three alkaloids were investigated by spectroscopy. The alkaloids bind tightly to DNA and behave as typical intercalating agents. All three compounds stabilize the topoisomerase II-DNA covalent complex and stimulate the cutting of DNA by topoisomerase II. The poisoning effect is more pronounced with cryptolepine than with matadine and serpentine, but none of the drugs exhibit a preference for cutting at a specific base. Cryptolepine which binds 10-fold more tightly to DNA than the two related alkaloids proves to be much more cytotoxic toward B16 melanoma cells than matadine and serpentine. The cellular consequences of the inhibition of topoisomerase II by cryptolepine were investigated using the HL60 leukemia cell line. The flow cytometry analysis shows that the drug alters the cell cycle distribution, but no sign of drug-induced apoptosis was detected when evaluating the internucleosomal fragmentation of DNA in cells. Cryptolepine-treated cells probably die via necrosis rather than via apoptosis. The results provide evidence that DNA and topoisomerase II are the primary targets of cryptolepine, matadine, and serpentine. PMID:10387011

  12. Structure, DNA binding and cleavage of a new Zn(II)Mn(II) macrocyclic complex

    NASA Astrophysics Data System (ADS)

    Zhou, Jing-Jing; Mei, Yu; Pan, Zhiquan; Zhou, Hong

    2012-12-01

    A new heterodinuclear complex of an unsymmetrical macrocycle [ZnMnL(CH3O)2]·H2O has been synthesized by the cyclocondensation between N,N'-bis(3-formyl-5-chlorosalicylidene)ethylenediimine and 2-hydroxyl-1,3-propanediamine in the presence of the metal ions, and characterized by elemental analyses, IR spectra and X-ray determination. The interactions of the complex with DNA have been investigated by UV absorption, fluorescence spectroscopy, viscosity measurements and electrochemical studies. Absorption spectroscopic investigation reveals that the complex has good binding propensity to calf thymus DNA by intercalation with a binding constant of 2.52 × 105 M-1. Fluorescence spectroscopy shows that the complex can displace ethidium bromide and bind to DNA, with a quenching constant of 4.37 × 103 M-1. The agarose gel electrophoresis studies show that pBR322 plasmid DNA can be transformed to nicked form and linear form in air by the complex.

  13. Bacteriophage T4 Mutants Hypersensitive to an Antitumor Agent That Induces Topoisomerase-DNA Cleavage Complexes

    PubMed Central

    Woodworth, D. L.; Kreuzer, K. N.

    1996-01-01

    Many antitumor agents and antibiotics affect cells by interacting with type II topoisomerases, stabilizing a covalent enzyme-DNA complex. A pathway of recombination can apparently repair this DNA damage. In this study, transposon mutagenesis was used to identify possible components of the repair pathway in bacteriophage T4. Substantial increases in sensitivity to the antitumor agent m-AMSA [4'-(9-acridinyl-amino) methanesulfon-m-anisidide] were found with transposon insertion mutations that inactivate any of six T4-encoded proteins: UvsY (DNA synaptase accessory protein), UvsW (unknown function), Rnh (RNase H and 5' to 3' DNA exonuclease), α-gt (α-glucosyl transferase), gp47.1 (uncharacterized), and NrdB (β subunit of ribonucleotide reductase). The role of the rnh gene in drug sensitivity was further characterized. First, an in-frame rnh deletion mutation was constructed and analyzed, providing evidence that the absence of Rnh protein causes hypersensitivity to m-AMSA. Second, the m-AMSA sensitivity of the rnh-deletion mutant was shown to require a drug-sensitive T4 topoisomerase. Third, analysis of double mutants suggested that uvsW and rnh mutations impair a common step in the recombinational repair pathway for m-AMSA-induced damage. Finally, the rnh-deletion mutant was found to be hypersensitive to UV, implicating Rnh in recombinational repair of UV-induced damage. PMID:8807283

  14. DNA Interaction and DNA Cleavage Studies of a New Platinum(II) Complex Containing Aliphatic and Aromatic Dinitrogen Ligands

    PubMed Central

    Shahabadi, Nahid; Kashanian, Soheila; Mahdavi, Maryam; Sourinejad, Noorkaram

    2011-01-01

    A new Pt(II) complex, [Pt(DIP)(LL)](NO3)2 (in which DIP is 4,7-diphenyl-1,10-phenanthroline and LL is the aliphatic dinitrogen ligand, N,N-dimethyl-trimethylenediamine), was synthesized and characterized using different physico-chemical methods. The interaction of this complex with calf thymus DNA (CT-DNA) was investigated by absorption, emission, circular dichroism (CD), and viscosity measurements. The complex binds to CT-DNA in an intercalative mode. The calculated binding constant, Kb, was 6.6 × 104 M−1. The enthalpy and entropy changes of the reaction between the complex and CT-DNA showed that the van der Waals interactions and hydrogen bonds are the main forces in the interaction with CT-DNA. In addition, CD study showed that phenanthroline ligand insert between the base pair stack of double helical structure of DNA. It is remarkable that this complex has the ability to cleave the supercoiled plasmid. PMID:22235195

  15. RNA cleavage and chain elongation by Escherichia coli DNA-dependent RNA polymerase in a binary enzyme.RNA complex.

    PubMed Central

    Altmann, C R; Solow-Cordero, D E; Chamberlin, M J

    1994-01-01

    In the absence of DNA, Escherichia coli RNA polymerase (EC 2.7.7.6) can bind RNA to form an equimolar binary complex with the concomitant release of the sigma factor. We show now that E. coli RNA polymerase binds at a region near the 3' terminus of the RNA and that an RNA in such RNA.RNA polymerase complexes undergoes reactions previously thought to be unique to nascent RNA in ternary complexes with DNA. These include GreA/GreB-dependent cleavage of the RNA and elongation by 3'-terminal addition of NMP from NTP. Both of these reactions are inhibited by rifampicin. Hence, by several criteria, the RNA in binary complexes is bound to the polymerase in a manner quite similar to that in ternary complexes. These findings can be explained by a model for the RNA polymerase ternary complex in which the RNA is bound at the 3' terminus through two protein binding sites located up to 10 nt apart. In this model, the stability of RNA binding to the polymerase in the ternary complex is due primarily to its interaction with the protein. Images PMID:7513426

  16. Synthesis, characterization, DNA-binding and cleavage studies of polypyridyl copper(II) complexes

    NASA Astrophysics Data System (ADS)

    Gubendran, Ammavasi; Rajesh, Jegathalaprathaban; Anitha, Kandasamy; Athappan, Periyakaruppan

    2014-10-01

    Six new mixed-ligand copper(II) complexes were synthesized namely [Cu(phen)2OAc]ClO4ṡH2O(1), [Cu(bpy)2OAc]ClO4ṡH2O(2), [Cu(o-ampacac)(phen)]ClO4(3), [Cu(o-ampbzac)(phen)]ClO4(4), [Cu(o-ampacac)(bpy)]ClO4(5), and [Cu(o-ampbzac)(bpy)]ClO4(6) (phen = 1,10-phenanthroline, bpy = 2, 2‧-bipyridine, o-ampacac = (Z)-4-(2-hydroxylamino)pent-3-ene-2-one,o-ampbzac = (Z)-4-(2-hydroxylamino)-4-phenylbut-3-ene-2-one)and characterized by UV-Vis, IR, EPR and cyclic voltammetry. Ligands were characterized by NMR spectra. Single crystal X-ray studies of the complex 1 shows Cu(II) ions are located in a highly distorted octahedral environment. Absorption spectral studies reveal that the complexes 1-6 exhibit hypochromicity during the interaction with DNA and binding constant values derived from spectral and electrochemical studies indicate that complexes 1, 2 and 3 bind strongly with DNA possibly by an intercalative mode. Electrochemical studies reveal that the complexes 1-4 prefer to bind with DNA in Cu(I) rather than Cu(II) form. The shift in the formal potentials E1/2 and CD spectral studies suggest groove or electrostatic binding mode for the complexes 4-6. Complex 1 can cleave supercoiled (SC) pUC18 DNA efficiently into nicked form II under photolytic conditions and into an open circular form (form II) and linear form (form III) in the presence of H2O2 at pH 8.0 and 37 °C, while the complex 2 does not cleave DNA under similar conditions.

  17. Differential inhibition of restriction enzyme cleavage by chromophore-modified analogues of the antitumour antibiotics mithramycin and chromomycin reveals structure-activity relationships.

    PubMed

    Mansilla, Sylvia; Garcia-Ferrer, Irene; Méndez, Carmen; Salas, José A; Portugal, José

    2010-05-15

    Differential cleavage at three restriction enzyme sites was used to determine the specific binding to DNA of the antitumour antibiotics mithramycin A (MTA), chromomycin A(3) (CRO) and six chromophore-modified analogues bearing shorter side chains attached at C-3, instead of the pentyl chain. All these antibiotics were obtained through combinatorial biosynthesis in the producer organisms. MTA, CRO and their six analogues showed differences in their capacity for inhibiting the rate of cleavage by restriction enzymes that recognize C/G-rich tracts. Changes in DNA melting temperature produced by these molecules were also analyzed, as well as their antiproliferative activities against a panel of colon, ovarian and prostate human carcinoma cell lines. Moreover, the cellular uptake of several analogues was examined to identify whether intracellular retention was related to cytotoxicity. These experimental approaches provided mutually consistent evidence of a seeming correlation between the strength of binding to DNA and the antiproliferative activity of the chromophore-modified molecules. Four of the analogues (mithramycin SK, mithramycin SDK, chromomycin SK and chromomycin SDK) showed promising biological profiles. PMID:20093108

  18. Use of Plasmon Coupling to Reveal the Dynamics of DNA Bending andCleavage by Single EcoRV Restriction Enzymes

    SciTech Connect

    Reinhard, Bjorn; Sheikholeslami, Sassan; Mastroianni, Alexander; Alivisatos, A. Paul; Liphardt, Jan

    2006-09-06

    Pairs of Au nanoparticles have recently been proposed asplasmon rulers based on the dependence of their light scattering on theinterparticle distance. Preliminary work has suggested that plasmonrulers can be used to measure and monitor dynamic distance changes overthe 1 to 100nm length scale in biology. Here, we substantiate thatplasmon rulers can be used to effectively measure dynamical biophysicalprocesses by applying the ruler to a system that has been investigatedextensively using ensemble kinetic measurements: the cleavage of DNA bythe restriction enzyme EcoRV. Temporal resolutions of up to 240 Hz wereobtained, and the end-to-end extension of up to 1000 individual dsDNAenzyme substrates could be monitored in parallel for hours. The singlemolecule cleavage trajectories acquired here agree well with valuesobtained in bulk through other methods, and confirm well-known featuresof the cleavage process, such as the fact that the DNA is bent prior tocleavage. New dynamical information is revealed as well, for instance,the degree of softening of the DNA just prior to cleavage. The unlimitedlife time, high temporal resolution, and high signal/noise make theplasmon ruler an excellent tool for studying macromolecular assembliesand conformational changes at the single molecule level.

  19. Cleavage Activation of Human-adapted Influenza Virus Subtypes by Kallikrein-related Peptidases 5 and 12*

    PubMed Central

    Hamilton, Brian S.; Whittaker, Gary R.

    2013-01-01

    A critical step in the influenza virus replication cycle is the cleavage activation of the HA precursor. Cleavage activation of influenza HA enables fusion with the host endosome, allowing for release of the viral genome into the host cell. To date, studies have determined that HA activation is driven by trypsin-like host cell proteases, as well as yet to be identified bacterial proteases. Although the number of host proteases that can activate HA is growing, there is still uncertainty regarding which secreted proteases are able to support multicycle replication of influenza. In this study, we have determined that the kallikrein-related peptidases 5 and 12 are secreted from the human respiratory tract and have the ability to cleave and activate HA from the H1, H2, and H3 subtypes. Each peptidase appears to have a preference for particular influenza subtypes, with kallikrein 5 cleaving the H1 and H3 subtypes most efficiently and kallikrein 12 cleaving the H1 and H2 subtypes most efficiently. Cleavage analysis using HA cleavage site peptide mimics revealed that the amino acids neighboring the arginine cleavage site affect cleavage efficiency. Additionally, the thrombolytic zymogens plasminogen, urokinase, and plasma kallikrein have all been shown to cleave and activate influenza but are found circulating mainly as inactive precursors. Kallikrein 5 and kallikrein 12 were examined for their ability to activate the thrombolytic zymogens, and both resulted in activation of each zymogen, with kallikrein 12 being a more potent activator. Activation of the thrombolytic zymogens may therefore allow for both direct and indirect activation of the HA of human-adapted influenza viruses by kallikrein 5 and kallikrein 12. PMID:23612974

  20. Inhibition of thrombin activity with DNA-aptamers.

    PubMed

    Dobrovolsky, A B; Titaeva, E V; Khaspekova, S G; Spiridonova, V A; Kopylov, A M; Mazurov, A V

    2009-07-01

    The effects of two DNA aptamers (oligonucleotides) 15TBA and 31TBA (15- and 31-mer thrombin-binding aptamers, respectively) on thrombin activity were studied. Both aptamers added to human plasma dose-dependently increased thrombin time (fibrin formation upon exposure to exogenous thrombin), prothrombin time (clotting activation by the extrinsic pathway), and activated partial thromboplastin time (clotting activation by the intrinsic pathway). At the same time, these aptamers did not modify amidolytic activity of thrombin evaluated by cleavage of synthetic chromogenic substrate. Aptamers also inhibited thrombin-induced human platelet aggregation. The inhibitory effects of 31TBA manifested at lower concentrations than those of 15TBA in all tests. These data indicate that the studied antithrombin DNA aptamers effectively suppress its two key reactions, fibrin formation and stimulation of platelet aggregation, without modifying active center of the thrombin molecule. PMID:19902090

  1. DNA cleavage, antibacterial, antifungal and anthelmintic studies of Co(II), Ni(II) and Cu(II) complexes of coumarin Schiff bases: Synthesis and spectral approach

    NASA Astrophysics Data System (ADS)

    Patil, Sangamesh A.; Prabhakara, Chetan T.; Halasangi, Bhimashankar M.; Toragalmath, Shivakumar S.; Badami, Prema S.

    2015-02-01

    The metal complexes of Co(II), Ni(II) and Cu(II) have been synthesized from 6-formyl-7,8-dihydroxy-4-methylcoumarin with o-toluidine/3-aminobenzotrifluoride. The synthesized Schiff bases and their metal complexes were structurally characterized based on IR, 1H NMR, 13C NMR, UV-visible, ESR, magnetic, thermal, fluorescence, mass and ESI-MS studies. The molar conductance values indicate that complexes are non-electrolytic in nature. Elemental analysis reveals ML2·2H2O [M = Co(II), Ni(II) and Cu(II)] stoichiometry, where 'L' stands for a singly deprotonated ligand. The presence of co-ordinated water molecules were confirmed by thermal studies. The spectroscopic studies suggest the octahedral geometry. Redox behavior of the complexes were confirmed by cyclic voltammetry. All the synthesized compounds were screened for their antibacterial (Escherichia coli, Pseudomonas auregenosa, klebsiella, Proteus, Staphylococcus aureus and salmonella) antifungal (Candida, Aspergillus niger and Rhizopus), anthelmintic (Pheretima posthuma) and DNA cleavage (Calf Thymus DNA) activity.

  2. Synthesis, spectroscopic, antimicrobial, DNA binding and cleavage studies of some metal complexes involving symmetrical bidentate N, N donor Schiff base ligand

    NASA Astrophysics Data System (ADS)

    Arish, D.; Nair, M. Sivasankaran

    2011-11-01

    The Schiff base ligand, N, N'-bis-(4-isopropylbenzaldimine)-1,2-diaminoethane (L), obtained by the condensation of 4-isopropylbenzaldehyde and 1,2-diaminoethane, has been used to synthesize the complexes of the type [ML 2X 2] [M = Co(II), Ni(II) and Zn(II); X = Cl and OAc]. The newly synthesized ligand (L) and its complexes have been characterized on the basis of elemental analyses, mass, 1H and 13C-NMR, molar conductance, IR, UV-vis, magnetic moment, CV and thermal analyses, powder XRD and SEM. IR spectral data show that the ligand is coordinated to the metal ions in a bidentate manner. The geometrical structures of these complexes are found to be octahedral. Interestingly, reaction with Cu(II) ion with this ligand undergoes hydrolytic cleavage to form ethylenediamine copper(II) complex and the corresponding aldehyde. The antimicrobial results indicate that the chloro complexes exhibit more activity than the acetato complexes. The complexes bind to CT-DNA by intercalation modes. Novel chloroform soluble ZnL 2Cl 2 complex exhibits tremendous antimicrobial, DNA binding and cleaving properties.

  3. Ferromagnetic nanoparticles with peroxidase-like activity enhance the cleavage of biological macromolecules for biofilm elimination

    PubMed Central

    Gao, Lizeng; Giglio, Krista M.; Nelson, Jacquelyn L.; Sondermann, Holger; Travis, Alexander J.

    2014-01-01

    Hydrogen peroxide (H2O2) is a “green chemical” that has various cleaning and disinfectant uses, including as an anti-bacterial agent for hygienic and medical treatments. However, its efficacy is limited against biofilm-producing bacteria, because of poor penetration of the protective, organic matrix. Here we show new applications for ferromagnetic nanoparticles (Fe3O4, MNP) with peroxidase-like activity in potentiating the efficacy of H2O2 in biofilm degradation and prevention. Our data show that MNP enhanced oxidative cleavage of biofilm components (model nucleic acids, proteins, and oligosaccharides) in the presence of H2O2. When challenged with live, biofilm-producing bacteria, the MNP-H2O2 system efficiently broke down existing biofilm and prevented new biofilm from forming, killing both planktonic bacteria and those within biofilm. By enhancing oxidative cleavage of various substrates, the MNP-H2O2 system provides a novel strategy for biofilm elimination, and other applications utilizing oxidative breakdown. PMID:24468900

  4. Synthesis, spectroscopic, molecular orbital calculation, cytotoxic, molecular docking of DNA binding and DNA cleavage studies of transition metal complexes with N-benzylidene-N'-salicylidene-1,1-diaminopropane

    NASA Astrophysics Data System (ADS)

    Al-Mogren, Muneerah M.; Alaghaz, Abdel-Nasser M. A.; Elbohy, Salwa A. H.

    2013-10-01

    Eight mononuclear chromium(III), manganese(II), iron(III), cobalt(II), nickel(II), copper(II), zinc(II) and cadmium(II) complexes of Schiff's base ligand were synthesized and determined by different physical techniques. The complexes are insoluble in common organic solvents but soluble in DMF and DMSO. The measured molar conductance values in DMSO indicate that the complexes are non-electrolytic in nature. All the eight metal complexes have been fully characterized with the help of elemental analyses, molecular weights, molar conductance values, magnetic moments and spectroscopic data. The analytical data helped to elucidate the structure of the metal complexes. The Schiff base is found to act as tridentate ligand using N2O donor set of atoms leading to an octahedral geometry for the complexes around all the metal ions. Quantum chemical calculations were performed with semi-empirical method to find the optimum geometry of the ligand and its complexes. Additionally in silico, the docking studies and the calculated pharmacokinetic parameters show promising futures for application of the ligand and complexes as high potency agents for DNA binding activity. The interaction of the complexes with calf thymus DNA (CT-DNA) has been investigated by UV absorption method, and the mode of CT-DNA binding to the complexes has been explored. Furthermore, the DNA cleavage activity by the complexes was performed. The Schiff base and their complexes have been screened for their antibacterial activity against bacterial strains [Staphylococcus aureus (RCMB010027), Staphylococcus epidermidis (RCMB010024), Bacillis subtilis (RCMB010063), Proteous vulgaris (RCMB 010085), Klebsiella pneumonia (RCMB 010093) and Shigella flexneri (RCMB 0100542)] and fungi [(Aspergillus fumigates (RCMB 02564), Aspergillus clavatus (RCMB 02593) and Candida albicans (RCMB05035)] by disk diffusion method. All the metal complexes have potent biocidal activity than the free ligand.

  5. Proteolytic Activation of the Human Epithelial Sodium Channel by Trypsin IV and Trypsin I Involves Distinct Cleavage Sites*

    PubMed Central

    Haerteis, Silke; Krappitz, Annabel; Krappitz, Matteus; Murphy, Jane E.; Bertog, Marko; Krueger, Bettina; Nacken, Regina; Chung, Hyunjae; Hollenberg, Morley D.; Knecht, Wolfgang; Bunnett, Nigel W.; Korbmacher, Christoph

    2014-01-01

    Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK178), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases. PMID:24841206

  6. The Varicella-Zoster Virus Portal Protein Is Essential for Cleavage and Packaging of Viral DNA

    PubMed Central

    Visalli, Melissa A.; House, Brittany L.; Selariu, Anca; Zhu, Hua

    2014-01-01

    ABSTRACT The varicella-zoster virus (VZV) open reading frame 54 (ORF54) gene encodes an 87-kDa monomer that oligomerizes to form the VZV portal protein, pORF54. pORF54 was hypothesized to perform a function similar to that of a previously described herpes simplex virus 1 (HSV-1) homolog, pUL6. pUL6 and the associated viral terminase are required for processing of concatemeric viral DNA and packaging of individual viral genomes into preformed capsids. In this report, we describe two VZV bacterial artificial chromosome (BAC) constructs with ORF54 gene deletions, Δ54L (full ORF deletion) and Δ54S (partial internal deletion). The full deletion of ORF54 likely disrupted essential adjacent genes (ORF53 and ORF55) and therefore could not be complemented on an ORF54-expressing cell line (ARPE54). In contrast, Δ54S was successfully propagated in ARPE54 cells but failed to replicate in parental, noncomplementing ARPE19 cells. Transmission electron microscopy confirmed the presence of only empty VZV capsids in Δ54S-infected ARPE19 cell nuclei. Similar to the HSV-1 genome, the VZV genome is composed of a unique long region (UL) and a unique short region (US) flanked by inverted repeats. DNA from cells infected with parental VZV (VZVLUC strain) contained the predicted UL and US termini, whereas cells infected with Δ54S contained neither. This result demonstrates that Δ54S is not able to process and package viral DNA, thus making pORF54 an excellent chemotherapeutic target. In addition, the utility of BAC constructs Δ54L and Δ54S as tools for the isolation of site-directed ORF54 mutants was demonstrated by recombineering single-nucleotide changes within ORF54 that conferred resistance to VZV-specific portal protein inhibitors. IMPORTANCE Antivirals with novel mechanisms of action would provide additional therapeutic options to treat human herpesvirus infections. Proteins involved in the herpesviral DNA encapsidation process have become promising antiviral targets

  7. Photo-Fenton reaction of graphene oxide: a new strategy to prepare graphene quantum dots for DNA cleavage.

    PubMed

    Zhou, Xuejiao; Zhang, Yan; Wang, Chong; Wu, Xiaochen; Yang, Yongqiang; Zheng, Bin; Wu, Haixia; Guo, Shouwu; Zhang, Jingyan

    2012-08-28

    Graphene quantum dots (GQDs) are great promising in various applications owing to the quantum confinement and edge effects in addition to their intrinsic properties of graphene, but the preparation of the GQDs in bulk scale is challenging. We demonstrated in this work that the micrometer sized graphene oxide (GO) sheets could react with Fenton reagent (Fe(2+)/Fe(3+)/H(2)O(2)) efficiently under an UV irradiation, and, as a result, the GQDs with periphery carboxylic groups could be generated with mass scale production. Through a variety of techniques including atomic force microscopy, X-ray photoelectron spectroscopy, gas chromatography, ultraperformance liquid chromatography-mass spectrometry, and total organic carbon measurement, the mechanism of the photo-Fenton reaction of GO was elucidated. The photo-Fenton reaction of GO was initiated at the carbon atoms connected with the oxygen containing groups, and C-C bonds were broken subsequently, therefore, the reaction rate depends strongly on the oxidization extent of the GO. Given the simple and efficient nature of the photo-Fenton reaction of GO, this method should provide a new strategy to prepare GQDs in mass scale. As a proof-of-concept experiment, the novel DNA cleavage system using as-generated GQDs was constructed. PMID:22813062

  8. Effects of S1 Cleavage on the Structure, Surface Export, and Signaling Activity of Human Notch1 and Notch2

    PubMed Central

    Gordon, Wendy R.; Vardar-Ulu, Didem; L'Heureux, Sarah; Ashworth, Todd; Malecki, Michael J.; Sanchez-Irizarry, Cheryll; McArthur, Debbie G.; Histen, Gavin; Mitchell, Jennifer L.; Aster, Jon C.; Blacklow, Stephen C.

    2009-01-01

    Background Notch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia. Principal Findings The X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity. Conclusions/Significance S1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with

  9. Effects of S1 Cleavage on the Structure, Surface Export, and Signaling Activity of Human Notch1 and Notch2

    SciTech Connect

    Gordon, Wendy R.; Vardar-Ulu, Didem; L'Heureux, Sarah; Ashworth, Todd; Malecki, Michael J.; Sanchez-Irizarry, Cheryll; McArthur, Debbie G.; Histen, Gavin; Mitchell, Jennifer L.; Aster, Jon C.; Blacklow, Stephen C.

    2009-09-25

    Notch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia. The X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity. S1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with models for receptor activation in which ligand-binding or

  10. Release of cytochrome c and activation of pro-caspase-9 following lysosomal photodamage involves bid cleavage

    PubMed Central

    Reiners, JJ; Caruso, JA; Mathieu, P; Chelladurai, B; Yin, X-M; Kessel, D

    2015-01-01

    Photodynamic therapy (PDT) protocols employing lysosomal sensitizers induce apoptosis via a mechanism that causes cytochrome c release prior to loss of mitochondrial membrane potential (ΔΨm). The current study was designed to determine how lysosomal photodamage initiates mitochondrial-mediated apoptosis in murine hepatoma 1c1c7 cells. Fluorescence microscopy demonstrated that the photosensitizer N-aspartyl chlorin e6 (NPe6) localized to the lysosomes. Irradiation of cultures preloaded with NPe6 induced the rapid destruction of lysosomes, and subsequent cleavage/activation of Bid, pro-caspases-9 and -3. Pro-caspase-8 was not activated. Release of cytochrome c occurred at about the time of Bid cleavage and preceded the loss of ΔΨm. Extracts of purified lysosomes catalyzed the in vitro cleavage of cytosolic Bid, but not pro-caspase-3 activation. Pharmacological inhibition of cathepsin B, L and D activities did not suppress Bid cleavage or pro-caspases-9 and -3 activation. These studies demonstrate that photodamaged lysosomes trigger the mitochondrial apoptotic pathway by releasing proteases that activate Bid. PMID:12181744

  11. Kinetic isotope effects for RNA cleavage by 2'-O- transphosphorylation: Nucleophilic activation by specific base

    PubMed Central

    Harris, Michael E; Dai, Qing; Gu, Hong; Kellerman, Dan; Piccirilli, Joseph A; Anderson, Vernon E

    2010-01-01

    To better understand the interactions between catalysts and transition states during RNA strand cleavage, primary 18O kinetic isotope effects and solvent D2O isotope effects were measured to probe the mechanism of base-catalyzed 2'-O-transphosphorylation of the RNA dinucleotide 5'-UpG-3'. The observed 18O KIEs for the nucleophilic 2'-O and in the 5'-O leaving group at pH 14 are both large relative to reactions of phosphodiesters with good leaving groups, indicating that the reaction catalyzed by hydroxide has a transition state (TS) with advanced phosphorus-oxygen bond fission to the leaving group (18kLG = 1.034 ± 0.004) and phosphorous-nucleophile bond formation (18kNUC = 0.984 ± 0.004). A breakpoint in the pH dependence of the 2'-O-transphosphorylation rate to a pH independent phase above pH 13 has been attributed to the pKa of the 2'-OH nucleophile. A smaller nucleophile KIE is observed at pH 12 (18kNUC = 0.995 ± 0.004) that is interpreted as the combined effect of the equilibrium isotope effect (~1.02) on deprotonation of the 2′-hydroxyl nucleophile and the intrinsic KIE on the nucleophilic addition step (ca. 0.981). An alternative mechanism in which the hydroxide ion acts as a general base is considered unlikely given the lack of a solvent deuterium isotope effect above the breakpoint in the pH versus rate profile. These results represent the first direct analysis of the transition state for RNA strand cleavage. The primary 18O KIE results and the lack of a kinetic solvent deuterium isotope effect together provide strong evidence for a late transition state and 2'-O nucleophile activation by specific base catalysis. PMID:20669950

  12. Calcium-induced cleavage of DNA topoisomerase I involves the cytoplasmic-nuclear shuttling of calpain 2.

    PubMed

    Chou, Shang-Min; Huang, Ting-Hsiang; Chen, Hsiang-Chin; Li, Tsai-Kun

    2011-08-01

    Important to the function of calpains is temporal and spatial regulation of their proteolytic activity. Here, we demonstrate that cytoplasm-resident calpain 2 cleaves human nuclear topoisomerase I (hTOP1) via Ca(2+)-activated proteolysis and nucleoplasmic shuttling of proteases. This proteolysis of hTOP1 was induced by either ionomycin-caused Ca(2+) influx or addition of Ca(2+) in cellular extracts. Ca(2+) failed to induce hTOP1 proteolysis in calpain 2-knockdown cells. Moreover, calpain 2 cleaved hTOP1 in vitro. Furthermore, calpain 2 entered the nucleus upon Ca(2+) influx, and calpastatin interfered with this process. Calpain 2 cleavage sites were mapped at K(158) and K(183) of hTOP1. Calpain 2-truncated hTOP1 exhibited greater relaxation activity but remained able to interact with nucleolin and to form cleavable complexes. Interestingly, calpain 2 appears to be involved in ionomycin-induced protection from camptothecin-induced cytotoxicity. Thus, our data suggest that nucleocytoplasmic shuttling may serve as a novel type of regulation for calpain 2-mediated nuclear proteolysis. PMID:21086148

  13. Palladium-catalyzed oxidative arylalkylation of activated alkenes: dual C-H bond cleavage of an arene and acetonitrile.

    PubMed

    Wu, Tao; Mu, Xin; Liu, Guosheng

    2011-12-23

    Not one but two: The title reaction proceeds through the dual C-H bond cleavage of both aniline and acetonitrile. The reaction affords a variety of cyano-bearing indolinones in excellent yield. Mechanistic studies demonstrate that this reaction involves a fast arylation of the olefin and a rate-determining C-H activation of the acetonitrile. PMID:22076660

  14. Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage

    PubMed Central

    Ethier, Sylvain; Schmeing, T. Martin; Dostie, Josée; Pelletier, Jerry

    2014-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to 5′NGG3′ protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. In vitro analysis of target site recognition revealed that interactions between the 5′ end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data. PMID:25275497

  15. DNA cleavage, antimicrobial, spectroscopic and fluorescence studies of Co(II), Ni(II) and Cu(II) complexes with SNO donor coumarin Schiff bases

    NASA Astrophysics Data System (ADS)

    Patil, Sangamesh A.; Naik, Vinod H.; Kulkarni, Ajaykumar D.; Badami, Prema S.

    2010-01-01

    A series of Co(II), Ni(II) and Cu(II) complexes of the type ML 2 have been synthesized with Schiff bases derived from methylthiosemicarbazone and 5-formyl-6-hydroxy coumarin/8-formyl-7-Hydroxy-4-methylcoumarin. The complexes are insoluble in common organic solvents but soluble in DMF and DMSO. The measured molar conductance values in DMF indicate that, the complexes are non-electrolytes in nature. In view of analytical, spectral (IR, UV-vis, ESR, FAB-mass and fluorescence), magnetic and thermal studies, it has been concluded that, all the metal complexes possess octahedral geometry in which ligand is coordinated to metal ion through azomethine nitrogen, thione sulphur and phenolic oxygen atom via deprotonation. The redox behavior of the metal complexes was investigated by using cyclic voltammetry. The Schiff bases and their complexes have been screened for their antibacterial ( Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi) and antifungal activities ( Aspergillus niger, Aspergillus flavus and Cladosporium) by Minimum Inhibitory Concentration method. The DNA cleavage is studied by agarose gel electrophoresis method.

  16. ARTEMIS nuclease facilitates apoptotic chromatin cleavage.

    PubMed

    Britton, Sébastien; Frit, Philippe; Biard, Denis; Salles, Bernard; Calsou, Patrick

    2009-10-15

    One hallmark of apoptosis is DNA degradation that first appears as high molecular weight fragments followed by extensive internucleosomal fragmentation. During apoptosis, the DNA-dependent protein kinase (DNA-PK) is activated. DNA-PK is involved in the repair of DNA double-strand breaks (DSB) and its catalytic subunit is associated with the nuclease ARTEMIS. Here, we report that, on initiation of apoptosis in human cells by agents causing DNA DSB or by staurosporine or other agents, ARTEMIS binds to apoptotic chromatin together with DNA-PK and other DSB repair proteins. ARTEMIS recruitment to chromatin showed a time and dose dependency. It required DNA-PK protein kinase activity and was blocked by antagonizing the onset of apoptosis with a pan-caspase inhibitor or on overexpression of the antiapoptotic BCL2 protein. In the absence of ARTEMIS, no defect in caspase-3, poly(ADP-ribose) polymerase-1, and XRCC4 cleavage or in H2AX phosphorylation was observed and DNA-PK catalytic subunit was still phosphorylated on S2056 in response to staurosporine. However, DNA fragmentation including high molecular weight fragmentation was delayed in ARTEMIS-deficient cells compared with cells expressing ARTEMIS. In addition, ARTEMIS enhanced the kinetics of MLL gene cleavage at a breakage cluster breakpoint that is frequently translocated in acute or therapy-related leukemias. These results show a facilitating role for ARTEMIS at least in early, site-specific chromosome breakage during apoptosis. PMID:19808974

  17. Translation termination factor eRF3 is targeted for caspase-mediated proteolytic cleavage and degradation during DNA damage-induced apoptosis.

    PubMed

    Hashimoto, Yoshifumi; Hosoda, Nao; Datta, Pinaki; Alnemri, Emad S; Hoshino, Shin-ichi

    2012-12-01

    Polypeptide chain release factor eRF3 plays pivotal roles in translation termination and post-termination events including ribosome recycling and mRNA decay. It is not clear, however, if eRF3 is targeted for the regulation of gene expression. Here we show that DNA-damaging agents (UV and etoposide) induce the immediate cleavage and degradation of eRF3 in a caspase-dependent manner. The effect is selective since the binding partners of eRF3, eRF1 and PABP, and an unrelated control, GAPDH, were not affected. Point mutations of aspartate residues within overlapping DXXD motifs near the amino terminus of eRF3 prevented the appearance of the UV-induced cleavage product, identifying D32 as the major cleavage site. The cleavage and degradation occurred in a similar time-dependent manner to those of eIF4G, a previously established caspase-3 target involved in the inhibition of translation during apoptosis. siRNA-mediated knockdown of eRF3 led to inhibition of cellular protein synthesis, supporting the idea that the decrease in the amount of eRF3 caused by the caspase-mediated degradation contributes to the inhibition of translation during apoptosis. This is the first report showing that eRF3 could serve as a target in the regulation of gene expression. PMID:23054082

  18. Active DNA demethylation by DNA repair: Facts and uncertainties.

    PubMed

    Schuermann, David; Weber, Alain R; Schär, Primo

    2016-08-01

    Pathways that control and modulate DNA methylation patterning in mammalian cells were poorly understood for a long time, although their importance in establishing and maintaining cell type-specific gene expression was well recognized. The discovery of proteins capable of converting 5-methylcytosine (5mC) to putative substrates for DNA repair introduced a novel and exciting conceptual framework for the investigation and ultimate discovery of molecular mechanisms of DNA demethylation. Against the prevailing notion that DNA methylation is a static epigenetic mark, it turned out to be dynamic and distinct mechanisms appear to have evolved to effect global and locus-specific DNA demethylation. There is compelling evidence that DNA repair, in particular base excision repair, contributes significantly to the turnover of 5mC in cells. By actively demethylating DNA, DNA repair supports the developmental establishment as well as the maintenance of DNA methylation landscapes and gene expression patterns. Yet, while the biochemical pathways are relatively well-established and reviewed, the biological context, function and regulation of DNA repair-mediated active DNA demethylation remains uncertain. In this review, we will thus summarize and critically discuss the evidence that associates active DNA demethylation by DNA repair with specific functional contexts including the DNA methylation erasure in the early embryo, the control of pluripotency and cellular differentiation, the maintenance of cell identity, and the nuclear reprogramming. PMID:27247237

  19. Analysis of Carotenoid Isomerase Activity in a Prototypical Carotenoid Cleavage Enzyme, Apocarotenoid Oxygenase (ACO)*

    PubMed Central

    Sui, Xuewu; Kiser, Philip D.; Che, Tao; Carey, Paul R.; Golczak, Marcin; Shi, Wuxian; von Lintig, Johannes; Palczewski, Krzysztof

    2014-01-01

    Carotenoid cleavage enzymes (CCEs) constitute a group of evolutionarily related proteins that metabolize a variety of carotenoid and non-carotenoid substrates. Typically, these enzymes utilize a non-heme iron center to oxidatively cleave a carbon-carbon double bond of a carotenoid substrate. Some members also isomerize specific double bonds in their substrates to yield cis-apocarotenoid products. The apocarotenoid oxygenase from Synechocystis has been hypothesized to represent one such member of this latter category of CCEs. Here, we developed a novel expression and purification protocol that enabled production of soluble, native ACO in quantities sufficient for high resolution structural and spectroscopic investigation of its catalytic mechanism. High performance liquid chromatography and Raman spectroscopy revealed that ACO exclusively formed all-trans products. We also found that linear polyoxyethylene detergents previously used for ACO crystallization strongly inhibited the apocarotenoid oxygenase activity of the enzyme. We crystallized the native enzyme in the absence of apocarotenoid substrate and found electron density in the active site that was similar in appearance to the density previously attributed to a di-cis-apocarotenoid intermediate. Our results clearly demonstrated that ACO is in fact a non-isomerizing member of the CCE family. These results indicate that careful selection of detergent is critical for the success of structural studies aimed at elucidating structures of CCE-carotenoid/retinoid complexes. PMID:24648526

  20. DNA-binding and cleavage studies of a novel two-dimensional manganese(II) azide complex with N-methylimidazole.

    PubMed

    Chen, Fengjuan; Xu, Zhihong; Xi, Pinxian; Liu, Xiaohui; Zeng, Zhengzhi

    2009-03-01

    A new complex, manganese(II) azide complex with N-methylimidazole, has been synthesized and characterized by elemental analysis, IR spectra and single crystal X-ray studies. Absorption spectroscopy, emission spectroscopy, viscosity measurements and CD spectroscopy have been used to investigate the binding of the complex with calf thymus DNA (CTDNA). The intrinsic binding constant K(b) of the complex with DNA was obtained by electronic absorption titration; the value is consistent with the result by fluorescence titration method. The spectroscopic studies together with viscosity measurements support the claim that the title complex bonds to CT-DNA by a groove mode. Control cleavage experiments using pBR 322 plasmid DNA which suggest minor groove binding for the complex. PMID:19276591

  1. DNA cleavage, antimicrobial studies and a DFT-based QSAR study of new antimony(III) complexes as glutathione reductase inhibitor

    NASA Astrophysics Data System (ADS)

    Tunç, Turgay; Koç, Yasemin; Açık, Leyla; Karacan, Mehmet Sayım; Karacan, Nurcan

    2015-02-01

    New antimony(III) complexes, [Sb(2-aminopyridine)2Cl3] (1a), [Sb(2-aminopyridine)2Br3] (1b), [Sb(5-methyl-2-aminopyridine)2Cl3] (2a), [Sb(5-methyl-2-aminopyridine)2Br3] (2b), [Sb(2-aminopyrimidine)2Cl3] (3a), [Sb(2-aminopyrimidine)2Br3] (3b), [Sb(4,6-dimethoxy-2-aminopyrimidine)2Cl3] (4a), [Sb(4,6-dimethoxy-2-aminopyrimidine)2Br3] (4b), [Sb(2-amino-1,3,5-triazine)2Cl3] (5a), [Sb(2-amino-1,3,5-triazine)2Br3] (5b), [Sb(2-guanidinobenzimidazole) Cl3] (6a), [Sb(2-guanidinobenzimidazole)Br3] (6b) [Sb(2- benzyl-2-thiopseudeourea)2Cl3] (7a) and [Sb(2- benzyl-2-thiopseudeourea)2Br3] (7b) were synthesized. Their structures were characterized by elemental analysis, molecular conductivity, FT-IR, 1H NMR, LC-MS techniques. Glutathione reductase inhibitor activity, antimicrobial activity and DNA cleavage studies of the complexes were determined. The geometrical structures of the complexes were optimized by DFT/B3LYP method with LANL2DZ as basis set. Calculation results indicated that the equilibrium geometries of all complexes have square pyramidal shape. About 350 molecular descriptors (constitutional, topological, geometrical, electrostatic and quantum chemical parameters) of the complexes were calculated by DFT/B3LYP/LANL2DZ method with CODESSA software. Calculated molecular parameters were correlated to glutathione reductase inhibitory activity values (pIC50) of all complexes by Best Multi-Linear Regression (BMLR) method. Obtained two-parameter QSAR equation shows that increase in "maximum partial charge for a H atom" and decrease in HOMO-LUMO gap would be favorable for the glutathione reductase inhibitory activity.

  2. DNA cleavage, antimicrobial studies and a DFT-based QSAR study of new antimony(III) complexes as glutathione reductase inhibitor.

    PubMed

    Tunç, Turgay; Koç, Yasemin; Açık, Leyla; Karacan, Mehmet Sayım; Karacan, Nurcan

    2015-02-01

    New antimony(III) complexes, [Sb(2-aminopyridine)2Cl3] (1a), [Sb(2-aminopyridine)2Br3] (1b), [Sb(5-methyl-2-aminopyridine)2Cl3] (2a), [Sb(5-methyl-2-aminopyridine)2Br3] (2b), [Sb(2-aminopyrimidine)2Cl3] (3a), [Sb(2-aminopyrimidine)2Br3] (3b), [Sb(4,6-dimethoxy-2-aminopyrimidine)2Cl3] (4a), [Sb(4,6-dimethoxy-2-aminopyrimidine)2Br3] (4b), [Sb(2-amino-1,3,5-triazine)2Cl3] (5a), [Sb(2-amino-1,3,5-triazine)2Br3] (5b), [Sb(2-guanidinobenzimidazole) Cl3] (6a), [Sb(2-guanidinobenzimidazole)Br3] (6b) [Sb(2- benzyl-2-thiopseudeourea)2Cl3] (7a) and [Sb(2- benzyl-2-thiopseudeourea)2Br3] (7b) were synthesized. Their structures were characterized by elemental analysis, molecular conductivity, FT-IR, (1)H NMR, LC-MS techniques. Glutathione reductase inhibitor activity, antimicrobial activity and DNA cleavage studies of the complexes were determined. The geometrical structures of the complexes were optimized by DFT/B3LYP method with LANL2DZ as basis set. Calculation results indicated that the equilibrium geometries of all complexes have square pyramidal shape. About 350 molecular descriptors (constitutional, topological, geometrical, electrostatic and quantum chemical parameters) of the complexes were calculated by DFT/B3LYP/LANL2DZ method with CODESSA software. Calculated molecular parameters were correlated to glutathione reductase inhibitory activity values (pIC50) of all complexes by Best Multi-Linear Regression (BMLR) method. Obtained two-parameter QSAR equation shows that increase in "maximum partial charge for a H atom" and decrease in HOMO-LUMO gap would be favorable for the glutathione reductase inhibitory activity. PMID:25459701

  3. Enhancing cellular uptake of activable cell-penetrating peptide-doxorubicin conjugate by enzymatic cleavage.

    PubMed

    Shi, Nian-Qiu; Gao, Wei; Xiang, Bai; Qi, Xian-Rong

    2012-01-01

    The use of activable cell-penetrating peptides (ACPPs) as molecular imaging probes is a promising new approach for the visualization of enzymes. The cell-penetrating function of a polycationic cell-penetrating peptide (CPP) is efficiently blocked by intramolecular electrostatic interactions with a polyanionic peptide. Proteolysis of a proteinase-sensitive substrate present between the CPP and polyanionic peptide affords dissociation of both domains and enables the activated CPP to enter cells. This ACPP strategy could also be used to modify antitumor agents for tumor-targeting therapy. Here, we aimed to develop a conjugate of ACPP with antitumor drug doxorubicin (DOX) sensitive to matrix metalloproteinase-2 and -9 (MMP-2/9) for tumor-targeting therapy purposes. The ACPP-DOX conjugate was successfully synthesized. Enzymatic cleavage of ACPP-DOX conjugate by matrix metalloproteinase (MMP)-2/9 indicated that the activation of ACPP-DOX occurred in an enzyme concentration-dependent manner. Flow cytometry and laser confocal microscope studies revealed that the cellular uptake of ACPP-DOX was enhanced after enzymatic-triggered activation and was higher in HT-1080 cells (overexpressed MMPs) than in MCF-7 cells (under-expressed MMPs). The antiproliferative assay showed that ACPP had little toxicity and that ACPP-DOX effectively inhibited HT-1080 cell proliferation. These experiments revealed that the ACPP-DOX conjugate could be triggered by MMP-2/9, which enabled the activated CPP-DOX to enter cells. ACPP-DOX conjugate may be a potential prodrug delivery system used to carry antitumor drugs for MMP-related tumor therapy. PMID:22619516

  4. A caspase active site probe reveals high fractional inhibition needed to block DNA fragmentation.

    PubMed

    Méthot, Nathalie; Vaillancourt, John P; Huang, JingQi; Colucci, John; Han, Yongxin; Ménard, Stéphane; Zamboni, Robert; Toulmond, Sylvie; Nicholson, Donald W; Roy, Sophie

    2004-07-01

    Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, alphaII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [(125)I]M808, which acts as a caspase active site probe. [(125)I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [(125)I]M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis-induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies. PMID:15067000

  5. Structurally diverse low molecular weight activators of the mammalian pre-mRNA 3′ cleavage reaction

    PubMed Central

    Liu, Min Ting; Nagre, Nagaraja N.; Ryan, Kevin

    2014-01-01

    The 3′ end formation of mammalian pre-mRNA contributes to gene expression regulation by setting the downstream boundary of the 3′ untranslated region, which in many genes carries regulatory sequences. A large number of protein cleavage factors participate in this pre-mRNA processing step, but chemical tools to manipulate this process are lacking. Guided by a hypothesis that a PPM1 family phosphatase negatively regulates the 3′ cleavage reaction, we have found a variety of new small molecule activators of the in vitro reconstituted pre-mRNA 3′ cleavage reaction. New activators include a cyclic peptide PPM1D inhibitor, a dipeptide with modifications common to histone tails, abscisic acid and an improved L-arginine β-naphthylamide analog. The minimal concentration required for in vitro cleavage has been improved from 200 μM to the 200 nM-100 μM range. These compounds provide unexpected leads in the search for small molecule tools able to affect pre-mRNA 3′ end formation. PMID:24373842

  6. A DNA enzyme with Mg(2+)-Dependent RNA Phosphoesterase Activity

    NASA Technical Reports Server (NTRS)

    Breaker, Ronald R.; Joyce, Gerald F.

    1995-01-01

    Previously we demonstrated that DNA can act as an enzyme in the Pb(2+)-dependent cleavage of an RNA phosphoester. This is a facile reaction, with an uncatalyzed rate for a typical RNA phosphoester of approx. 10(exp -4)/ min in the presence of 1 mM Pb(OAc)2 at pH 7.0 and 23 C. The Mg(2+) - dependent reaction is more difficult, with an uncatalyzed rate of approx. 10(exp -7)/ min under comparable conditions. Mg(2+) - dependent cleavage has special relevance to biology because it is compatible with intracellular conditions. Using in vitro selection, we sought to develop a family of phosphoester-cleaving DNA enzymes that operate in the presence of various divalent metals, focusing particularly on the Mg(2+) - dependent reaction. Results: We generated a population of greater than 10(exp 13) DNAs containing 40 random nucleotides and carried out repeated rounds of selective amplification, enriching for molecules that cleave a target RNA phosphoester in the presence of 1 mM Mg(2+), Mn(2+), Zn(2+) or Pb(2+). Examination of individual clones from the Mg(2+) lineage after the sixth round revealed a catalytic motif comprised of a three-stem junction.This motif was partially randomized and subjected to seven additional rounds of selective amplification, yielding catalysts with a rate of 0.01/ min. The optimized DNA catalyst was divided into separate substrate and enzyme domains and shown to have a similar level of activity under multiple turnover conditions. Conclusions: We have generated a Mg(2+) - dependent DNA enzyme that cleaves a target RNA phosphoester with a catalytic rate approx. 10(exp 5) - fold greater than that of the uncatalyzed reaction. This activity is compatible with intracellular conditions, raising the possibility that DNA enzymes might be made to operate in vivo.

  7. Crystallization and preliminary X-ray diffraction analysis of two N-terminal fragments of the DNA-cleavage domain of topoisomerase IV from Staphylococcus aureus

    SciTech Connect

    Carr, Stephen B.; Makris, George; Phillips, Simon E. V.; Thomas, Christopher D.

    2006-11-01

    The crystallization and data collection of topoisomerase IV from S. aureus is described. Phasing by molecular replacement proved difficult owing to the presence of translational NCS and strategies used to overcome this are discussed. DNA topoisomerase IV removes undesirable topological features from DNA molecules in order to help maintain chromosome stability. Two constructs of 56 and 59 kDa spanning the DNA-cleavage domain of the A subunit of topoisomerase IV from Staphylococcus aureus (termed GrlA56 and GrlA59) have been crystallized. Crystals were grown at 291 K using the sitting-drop vapour-diffusion technique with PEG 3350 as a precipitant. Preliminary X-ray analysis revealed that GrlA56 crystals belong to space group P2{sub 1}, diffract to a resolution of 2.9 Å and possess unit-cell parameters a = 83.6, b = 171.5, c = 87.8 Å, β = 90.1°, while crystals of GrlA59 belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 41.5, b = 171.89, c = 87.9 Å. These crystals diffract to a resolution of 2.8 Å. This is the first report of the crystallization and preliminary X-ray analysis of the DNA-cleavage domain of a topoisomerase IV from a Gram-positive organism.

  8. Metal-based biologically active compounds: synthesis, characterization, DNA interaction, antibacterial, cytotoxic and SOD mimic activities.

    PubMed

    Patel, Mohan N; Patel, Chintan R; Joshi, Hardik N

    2013-02-01

    The square pyramidal copper(II) complexes of N, O- donor ligand and ciprofloxacin have been synthesized. Synthesized complexes were characterized by physicochemical parameters like elemental analysis, electronic, FT-IR and LC-MS spectra. The complexes were screened for their antimicrobial activity against Gram(+Ve), i.e. Staphylococcus aureus, Bacillus subtilis, and Gram(-Ve), i.e. Serratia marcescens, Pseudomonas aeruginosa and Escherichia coli, microorganisms in terms of minimum inhibitory concentration and colony-forming unit. To determine the binding mode of complexes with Herring Sperm DNA, absorption titration and viscosity measurement were employed. DNA cleavage activity was carried out by gel electrophoresis experiment using supercoiled form of pUC19 DNA. The complexes were tested for their superoxide dismutase mimic activity in terms of IC(50) value. Synthesized complexes were also screened for their cytotoxicity using brine shrimp lethality assay method. PMID:23306896

  9. Self-cleavage of Human CLCA1 Protein by a Novel Internal Metalloprotease Domain Controls Calcium-activated Chloride Channel Activation*♦

    PubMed Central

    Yurtsever, Zeynep; Sala-Rabanal, Monica; Randolph, David T.; Scheaffer, Suzanne M.; Roswit, William T.; Alevy, Yael G.; Patel, Anand C.; Heier, Richard F.; Romero, Arthur G.; Nichols, Colin G.; Holtzman, Michael J.; Brett, Tom J.

    2012-01-01

    The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface. PMID:23112050

  10. Simple Bond Cleavage

    SciTech Connect

    Gary S. Groenewold

    2005-08-01

    Simple bond cleavage is a class of fragmentation reactions in which a single bond is broken, without formation of new bonds between previously unconnected atoms. Because no bond making is involved, simple bond cleavages are endothermic, and activation energies are generally higher than for rearrangement eliminations. The rate of simple bond cleavage reactions is a strong function of the internal energy of the molecular ion, which reflects a loose transition state that resembles reaction products, and has a high density of accessible states. For this reason, simple bond cleavages tend to dominate fragmentation reactions for highly energized molecular ions. Simple bond cleavages have negligible reverse activation energy, and hence they are used as valuable probes of ion thermochemistry, since the energy dependence of the reactions can be related to the bond energy. In organic mass spectrometry, simple bond cleavages of odd electron ions can be either homolytic or heterolytic, depending on whether the fragmentation is driven by the radical site or the charge site. Simple bond cleavages of even electron ions tend to be heterolytic, producing even electron product ions and neutrals.

  11. New unsymmetric dinuclear Cu(II)Cu(II) complexes and their relevance to copper(II) containing metalloenzymes and DNA cleavage.

    PubMed

    Peralta, Rosely A; Neves, Ademir; Bortoluzzi, Adailton J; Dos Anjos, Ademir; Xavier, Fernando R; Szpoganicz, Bruno; Terenzi, Hernán; de Oliveira, Mauricio C B; Castellano, Eduardo; Friedermann, Geraldo R; Mangrich, Antonio S; Novak, Miguel A

    2006-05-01

    . Since these complexes are redox active species, we analyzed their activity toward the nucleic acid DNA, a macromolecule prone to oxidative damage. Interestingly these complexes promoted DNA cleavage following an oxygen dependent pathway. PMID:16563512

  12. Chamomile flower extract-directed CuO nanoparticle formation for its antioxidant and DNA cleavage properties.

    PubMed

    Duman, Fatih; Ocsoy, Ismail; Kup, Fatma Ozturk

    2016-03-01

    In this study, we report the synthesis of copper oxide nanoparticles (CuO NPs) using a medicinal plant (Matricaria chamomilla) flower extract as both reducing and capping agent and investigate their antioxidant activity and interaction with plasmid DNA (pBR322).The CuO NPs were characterized using Uv-Vis spectroscopy, FT-IR (Fourier transform infrared spectroscopy), DLS (dynamic light scattering), XRD (X-ray diffraction), EDX (energy-dispersive X-ray) spectroscopy and SEM (scanning electron microscopy). The CuO NPs exhibited nearly mono-distributed and spherical shapes with diameters of 140 nm size. UV-Vis absorption spectrum of CuO NPs gave a broad peak around 285 and 320 nm. The existence of functional groups on the surface of CuO NPs was characterized with FT-IR analysis. XRD pattern showed that the NPs are in the form of a face-centered cubic crystal. Zeta potential value was measured as -20 mV due to the presence of negatively charged functional groups in plant extract. Additionally, we demonstrated concentration-dependent antioxidant activity of CuO NPs and their interaction with plasmid DNA. We assumed that the CuO NPs both cleave and break DNA double helix structure. PMID:26706538

  13. Phylogenomics of caspase-activated DNA fragmentation factor

    SciTech Connect

    Eckhart, Leopold . E-mail: leopold.eckhart@meduniwien.ac.at; Fischer, Heinz; Tschachler, Erwin

    2007-04-27

    The degradation of nuclear DNA by DNA fragmentation factor (DFF) is a key step in apoptosis of mammalian cells. Using comparative genomics, we have here determined the evolutionary history of the genes encoding the two DFF subunits, DFFA (also known as ICAD) and DFFB (CAD). Orthologs of DFFA and DFFB were identified in Nematostella vectensis, a representative of the primitive metazoan clade cnidarians, and in various vertebrates and insects, but not in representatives of urochordates, echinoderms, and nematodes. The domains mediating the interaction of DFFA and DFFB, a caspase cleavage site in DFFA, and the amino acid residues critical for endonuclease activity of DFFB were conserved in Nematostella. These findings suggest that DFF has been a part of the primordial apoptosis system of the eumetazoan common ancestor and that the ancient cell death machinery has degenerated in several evolutionary lineages, including the one leading to the prototypical apoptosis model, Caenorhabditis elegans.

  14. Cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor L.; Brow, Mary Ann D.; Dahlberg, James E.

    2007-12-11

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

  15. Cleavage of nucleic acids

    SciTech Connect

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow; Mary Ann D.; Dahlberg, James E.

    2010-11-09

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

  16. Cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

    2000-01-01

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

  17. Probing the Run-On Oligomer of Activated SgrAI Bound to DNA

    PubMed Central

    Shah, Santosh; Sanchez, Jonathan; Stewart, Andrew; Piperakis, Michael M.; Cosstick, Richard; Nichols, Claire; Park, Chad K.; Ma, Xin; Wysocki, Vicki; Bitinaite, Jurate; Horton, Nancy C.

    2015-01-01

    SgrAI is a type II restriction endonuclease with an unusual mechanism of activation involving run-on oligomerization. The run-on oligomer is formed from complexes of SgrAI bound to DNA containing its 8 bp primary recognition sequence (uncleaved or cleaved), and also binds (and thereby activates for DNA cleavage) complexes of SgrAI bound to secondary site DNA sequences which contain a single base substitution in either the 1st/8th or the 2nd/7th position of the primary recognition sequence. This modulation of enzyme activity via run-on oligomerization is a newly appreciated phenomenon that has been shown for a small but increasing number of enzymes. One outstanding question regarding the mechanistic model for SgrAI is whether or not the activating primary site DNA must be cleaved by SgrAI prior to inducing activation. Herein we show that an uncleavable primary site DNA containing a 3’-S-phosphorothiolate is in fact able to induce activation. In addition, we now show that cleavage of secondary site DNA can be activated to nearly the same degree as primary, provided a sufficient number of flanking base pairs are present. We also show differences in activation and cleavage of the two types of secondary site, and that effects of selected single site substitutions in SgrAI, as well as measured collisional cross-sections from previous work, are consistent with the cryo-electron microscopy model for the run-on activated oligomer of SgrAI bound to DNA. PMID:25880668

  18. Cleavage by Caspase 8 and Mitochondrial Membrane Association Activate the BH3-only Protein Bid during TRAIL-induced Apoptosis.

    PubMed

    Huang, Kai; Zhang, Jingjing; O'Neill, Katelyn L; Gurumurthy, Channabasavaiah B; Quadros, Rolen M; Tu, Yaping; Luo, Xu

    2016-05-27

    The BH3-only protein Bid is known as a critical mediator of the mitochondrial pathway of apoptosis following death receptor activation. However, since full-length Bid possesses potent apoptotic activity, the role of a caspase-mediated Bid cleavage is not established in vivo In addition, due to the fact that multiple caspases cleave Bid at the same site in vitro, the identity of the Bid-cleaving caspase during death receptor signaling remains uncertain. Moreover, as Bid maintains its overall structure following its cleavage by caspase 8, it remains unclear how Bid is activated upon cleavage. Here, Bid-deficient (Bid KO) colon cancer cells were generated by gene editing, and were reconstituted with wild-type or mutants of Bid. While the loss of Bid blocked apoptosis following treatment by TNF-related apoptosis inducing ligand (TRAIL), this blockade was relieved by re-introduction of the wild-type Bid. In contrast, the caspase-resistant mutant Bid(D60E) and a BH3 defective mutant Bid(G94E) failed to restore TRAIL-induced apoptosis. By generating Bid/Bax/Bak-deficient (TKO) cells, we demonstrated that Bid is primarily cleaved by caspase 8, not by effector caspases, to give rise to truncated Bid (tBid) upon TRAIL treatment. Importantly, despite the presence of an intact BH3 domain, a tBid mutant lacking the mitochondrial targeting helices (α6 and α7) showed diminished apoptotic activity. Together, these results for the first time establish that cleavage by caspase 8 and the subsequent association with the outer mitochondrial membrane are two critical events that activate Bid during death receptor-mediated apoptosis. PMID:27053107

  19. DNA binding, nuclease activity, DNA photocleavage and cytotoxic properties of Cu(II) complexes of N-substituted sulfonamides.

    PubMed

    García-Giménez, José Luis; Hernández-Gil, Javier; Martínez-Ruíz, Aloma; Castiñeiras, Alfonso; Liu-González, Malva; Pallardó, Federico V; Borrás, Joaquín; Alzuet Piña, Gloria

    2013-04-01

    Ternary copper(II) complexes [Cu(NST)2(phen)] (1) and [Cu(NST)2(NH3)2]·H2O (2) [HNST=N-(4,5-dimethylthiazol-2-yl)naphthalene-1-sulfonamide] were prepared and characterized by physico-chemical techniques. Both 1 and 2 were structurally characterized by X-ray crystallography. The crystal structures show the presence of a distorted square planar CuN4 geometry in which the deprotonated sulfonamide, acting as monodentate ligand, binds to the metal ion through the thiazole N atom. Both complexes present intermolecular π-π stacking interactions between phenanthroline rings (compound 1) and between naphthalene rings (compound 2). The interaction of the complexes with CT DNA was studied by means of thermal denaturation, viscosity measurements and fluorescence spectroscopy. The complexes display good binding propensity to the calf thymus DNA giving the order: 1>2. Complex 1, which has a higher capability for binding to DNA, showed better nuclease activity than 2 in the presence of ascorbate/H2O2. Both the kinetics and the mechanism of the DNA cleavage reaction were investigated. Furthermore, complex 1 showed efficient photo-induced DNA cleavage activity on irradiation with UV light in the absence of any external reagent. The UV light induced DNA cleavage follows a photo-redox pathway with generation of hydroxyl radicals as reactive species. In addition, the cytotoxic properties of both complexes (1 and 2) were evaluated in human cancer cells (HeLa, Caco-2 and MDA-468). The low IC50 values, in particular those against Caco-2, have indicated that the compounds can be considered as promising chemotherapeutic agents. PMID:23384854

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

    PubMed

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

    2015-12-01

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

  1. Analysis of the recognition mechanism involved in the EcoRV catalyzed cleavage of DNA using modified oligodeoxynucleotides.

    PubMed Central

    Fliess, A; Wolfes, H; Seela, F; Pingoud, A

    1988-01-01

    We have prepared a series of undecadeoxynucleotides that contain changes in the functional group pattern present within the EcoRV recognition site - GATATC-. Oligonucleotides were synthesized on solid phase using normal and modified beta-cyanoethylphosphoramidites and analyzed in steady state cleavage experiments with the EcoRV restriction endonuclease. The following groups appear to interact strongly with the enzyme, since their modification or substitution renders the oligonucleotides refractory to cleavage: the exocyclic NH2-groups of both A residues, the N7 of the first A residue, the exocyclic NH2-group of the C residue and the CH3-groups of both T residues. The exocyclic NH-group of the G residue supports effective recognition, since its absence lowers the kcat of the cleavage reaction. The N7 of the second A residue and the C5 position of the C residue apparently are not recognized by EcoRV; their substitution by -CH- or modification with -Br or -CH3, resp., does not considerably change the rate of cleavage. All oligonucleotides investigated compete with the unmodified substrate for binding to the enzyme. We conclude that EcoRV recognizes its substrate presumably through hydrogen bonds to the exocyclic NH2-group and the N7 of the first A residue, the exocyclic NH2-groups of the second A and the C residue, as well as through hydrophobic interactions with both T residues. PMID:3062581

  2. Mononuclear dioxomolybdenum(VI) thiosemicarbazonato complexes: Synthesis, characterization, structural illustration, in vitro DNA binding, cleavage, and antitumor properties.

    PubMed

    Hussein, Mouayed A; Guan, Teoh S; Haque, Rosenani A; Khadeer Ahamed, Mohamed B; Abdul Majid, Amin M S

    2015-02-01

    Four dioxomolybdenum(VI) complexes were synthesized by reacting [MoO2(acac)2] with N-ethyl-2-(5-bromo-2-hydroxybenzylidene) hydrazinecarbothioamide (1), N-ethyl-2-(5-allyl-3-methoxy-2-hydroxybenzylidene) hydrazinecarbothioamide (2), N-methyl-2-(3-tert-butyl-2-hydroxybenzylidene) hydrazinecarbothioamide (3), and N-ethyl-2-(3-methyl-2-hydroxybenzylidene) hydrazinecarbothioamide (4). The molecular structures of 1, 2, and all the synthesized complexes were determined using single crystal X-ray crystallography. The binding properties of the ligand and complexes with calf thymus DNA (CT-DNA) were investigated via UV, fluorescence titrations, and viscosity measurement. Gel electrophoresis revealed that all the complexes cleave pBR 322 plasmid DNA. The cytotoxicity of the complexes were studied against the HCT 116 human colorectal cell line. All the complexes exhibited more pronounced activity than the standard reference drug 5-fluorouracil (IC50 7.3μM). These studies show that dioxomolybdenum(VI) complexes could be potentially useful in chemotherapy. PMID:25456676

  3. Mononuclear dioxomolybdenum(VI) thiosemicarbazonato complexes: Synthesis, characterization, structural illustration, in vitro DNA binding, cleavage, and antitumor properties

    NASA Astrophysics Data System (ADS)

    Hussein, Mouayed A.; Guan, Teoh S.; Haque, Rosenani A.; Khadeer Ahamed, Mohamed B.; Abdul Majid, Amin M. S.

    2015-02-01

    Four dioxomolybdenum(VI) complexes were synthesized by reacting [MoO2(acac)2] with N-ethyl-2-(5-bromo-2-hydroxybenzylidene) hydrazinecarbothioamide (1), N-ethyl-2-(5-allyl-3-methoxy-2-hydroxybenzylidene) hydrazinecarbothioamide (2), N-methyl-2-(3-tert-butyl-2-hydroxybenzylidene) hydrazinecarbothioamide (3), and N-ethyl-2-(3-methyl-2-hydroxybenzylidene) hydrazinecarbothioamide (4). The molecular structures of 1, 2, and all the synthesized complexes were determined using single crystal X-ray crystallography. The binding properties of the ligand and complexes with calf thymus DNA (CT-DNA) were investigated via UV, fluorescence titrations, and viscosity measurement. Gel electrophoresis revealed that all the complexes cleave pBR 322 plasmid DNA. The cytotoxicity of the complexes were studied against the HCT 116 human colorectal cell line. All the complexes exhibited more pronounced activity than the standard reference drug 5-fluorouracil (IC50 7.3 μM). These studies show that dioxomolybdenum(VI) complexes could be potentially useful in chemotherapy.

  4. DNA repair by the cryptic endonuclease activity of Mu transposase.

    PubMed

    Choi, Wonyoung; Harshey, Rasika M

    2010-06-01

    Phage Mu transposes by two distinct pathways depending on the specific stage of its life cycle. A common strand transfer intermediate is resolved differentially in the two pathways. During lytic growth, the intermediate is resolved by replication of Mu initiated within the flanking target DNA; during integration of infecting Mu, it is resolved without replication, by removal and repair of DNA from a previous host that is still attached to the ends of the incoming Mu genome. We have discovered that the cryptic endonuclease activity reported for the isolated C-terminal domain of the transposase MuA [Wu Z, Chaconas G (1995) A novel DNA binding and nuclease activity in domain III of Mu transposase: Evidence for a catalytic region involved in donor cleavage. EMBO J 14:3835-3843], which is not observed in the full-length protein or in the assembled transpososome in vitro, is required in vivo for removal of the attached host DNA or "5'flap" after the infecting Mu genome has integrated into the E. coli chromosome. Efficient flap removal also requires the host protein ClpX, which is known to interact with the C-terminus of MuA to remodel the transpososome for replication. We hypothesize that ClpX constitutes part of a highly regulated mechanism that unmasks the cryptic nuclease activity of MuA specifically in the repair pathway. PMID:20167799

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

    PubMed Central

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

    2009-01-01

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

  6. Processing of the yellow fever virus nonstructural polyprotein: a catalytically active NS3 proteinase domain and NS2B are required for cleavages at dibasic sites.

    PubMed Central

    Chambers, T J; Grakoui, A; Rice, C M

    1991-01-01

    The vaccinia virus-T7 transient expression system was used to further examine the role of the NS3 proteinase in processing of the yellow fever (YF) virus nonstructural polyprotein in BHK cells. YF virus-specific polyproteins and cleavage products were identified by immunoprecipitation with region-specific antisera, by size, and by comparison with authentic YF virus polypeptides. A YF virus polyprotein initiating with a signal sequence derived from the E protein fused to the N terminus of NS2A and extending through the N-terminal 356 amino acids of NS5 exhibited processing at the 2A-2B, 2B-3, 3-4A, 4A-4B, and 4B-5 cleavage sites. Similar results were obtained with polyproteins whose N termini began within NS2A (position 110) or with NS2B. When the NS3 proteinase domain was inactivated by replacing the proposed catalytic Ser-138 with Ala, processing at all sites was abolished. The results suggest that an active NS3 proteinase domain is necessary for cleavage at the diabasic nonstructural cleavage sites and that cleavage at the proposed 4A-4B signalase site requires prior cleavage at the 4B-5 site. Cleavages were not observed with a polyprotein whose N terminus began with NS3, but cleavage at the 4B-5 site could be restored by supplying the the NS2B protein in trans. Several experimental results suggested that trans cleavage at the 4B-5 site requires association of NS2B and the NS3 proteinase domain. Coexpression of different proteinases and catalytically inactive polyprotein substrates revealed that trans cleavage at the 2B-3 and 4B-5 sites was relatively efficient when compared with trans cleavage at the 2A-2B and 3-4A sites. Images PMID:1833562

  7. Detection of endopeptidase activity and analysis of cleavage specificity using a radiometric solid-phase enzymatic assay

    SciTech Connect

    Jean, F.; Basak, A.; Chretien, M.; Lazure, C. , Quebec )

    1991-05-01

    A radiometric procedure to detect the presence of proteolytic enzymes and analyze their substrate specificity is described. The enzymatic activity is first measured by the release into solution of a radiolabeled reporter group from an immobilized peptidyl substrate. Two peptidyl substrates encompassing multiple cleavage sites, a derivative of Leu-enkephalin and a peptide related to the bait region of human {alpha} 2-macroglobulin, are prepared and linked via a spacer molecule to an insoluble support. The labeled peptides released are then separated by high-performance liquid chromatography. The position of the released peptides upon chromatography allows direct identification of the sites of cleavage. The assay, using a radioactive iodinated tyrosine residue as reporter group, is extremely sensitive (less than 0.02 pg/ml of trypsin), reproducible, and easy to perform while yielding unambiguous identification of the sites of cleavage. This assay can be used to detect the presence of enzymatic activities and/or of enzyme inhibitors. Furthermore, it can be easily adapted to detect from a variety of sources all four classes of enzymes known by using appropriate peptidyl substrate sequences, buffer, pH, and incubation conditions.

  8. Two Spin-State Reactivity in the Activation and Cleavage of CO2 by [ReO2](.).

    PubMed

    Canale, Valentino; Robinson, Robert; Zavras, Athanasios; Khairallah, George N; d'Alessandro, Nicola; Yates, Brian F; O'Hair, Richard A J

    2016-05-19

    The rhenium dioxide anion [ReO2](-) reacts with carbon dioxide in a linear ion trap mass spectrometer to produce [ReO3](-) corresponding to activation and cleavage of a C-O bond. Isotope labeling experiments using [Re(18)O2](-) reveal that (18)O/(16)O scrambling does not occur prior to cleavage of the C-O bond. Density functional theory calculations were performed to examine the mechanism for this oxygen atom abstraction reaction. Because the spins of the ground states are different for the reactant and product ions ((3)[ReO2](-) versus (1)[ReO3](-)), both reaction surfaces were examined in detail and multiple [O2Re-CO2](-) intermediates and transition structures were located and minimum energy crossing points were calculated. The computational results show that the intermediate [O2Re(η(2)-C,O-CO2)](-) species most likely initiates C-O bond activation and cleavage. The stronger binding affinity of CO2 within this species and the greater instabilities of other [O2Re-CO2)](-) intermediates are significant enough that oxygen atom exchange is avoided. PMID:27193088

  9. Rottlerin potentiates camptothecin-induced cytotoxicity in human hormone refractory prostate cancers through increased formation and stabilization of topoisomerase I-DNA cleavage complexes in a PKCδ-independent pathway

    PubMed Central

    Hsu, Jui-Ling; Ho, Yunn-Fang; Li, Tsai-Kun; Chen, Ching-Shih; Hsu, Lih-Ching; Guh, Jih-Hwa

    2014-01-01

    Combination therapy, which can optimize killing activity to cancers and minimize drug resistance, is a mainstream therapy against hormone-refractory prostate cancers (HRPCs). Rottlerin, a natural polyphenolic component, synergistically increased PC-3 (a HRPC cell line) apoptosis induced by camptothecin (a topoisomerase I inhibitor). Using siRNA technique to knockdown protein kinase C-δ (PKCδ), the data showed that rottlerin-mediated synergistic effect was PKCδ-independent, although rottlerin has been used as a PKCδ inhibitor. Rottlerin potentiated camptothecin-induced DNA fragmentation at S phase and ATM phosphorylation at Ser1981. The effect was correlated to apoptosis (r2 = 0.9). To detect upstream signals, the data showed that camptothecin acted on and stabilized topoisomerase I-DNA complex, leading to the formation of camptothecin-trapped cleavage complexes (TOP1cc). The effect was potentiated by rottlerin. To determine DNA repair capability, the time-related γH2A.X formation was examined after camptothecin removal. Consequently, rottlerin significantly inhibited camptothecin removal-mediated decline of γH2A.X formation at S phase, indicating the impairment of DNA repair activity in the presence of rottlerin. The combinatory treatment of camptothecin and rottlerin induced conformational change and activation of Bax and formation of truncated Bad, suggesting the contribution of mitochondria stress to apoptosis. In summary, the data suggest that rottlerin-mediated camptothecin sensitization is through the augmented stabilization of TOP1cc, leading to an increase of DNA damage stress and, possibly, an impairment of DNA repair capability. Subsequently, mitochondria-involved apoptosis is triggered through Bax activation and truncated Bad formation. The novel discovery may provide an anticancer approach of combinatory use between rottlerin and camptothecin for the treatment of HRPCs. PMID:22490701

  10. Tautomerization lowers the activation barriers for N-glycosidic bond cleavage of protonated uridine and 2'-deoxyuridine.

    PubMed

    Wu, R R; Rodgers, M T

    2016-09-21

    The gas-phase conformations of protonated uridine, [Urd+H](+), and its 2'-deoxy form, protonated 2'-deoxyuridine, [dUrd+H](+), have been examined in detail previously by infrared multiple photon dissociation action spectroscopy techniques. Both 2,4-dihydroxy tautomers and O4 protonated conformers of [Urd+H](+) and [dUrd+H](+) were found to coexist in the experiments with the 2,4-dihydroxy tautomers dominating the population. In the present study, the kinetic energy dependence of the collision-induced dissociation behavior of [Urd+H](+) and [dUrd+H](+) are examined using a guided ion beam tandem mass spectrometer to probe the mechanisms and energetics for activated dissociation of these protonated nucleosides. The primary dissociation pathways observed involve N-glycosidic bond cleavage leading to competitive elimination of protonated or neutral uracil. The potential energy surfaces (PESs) for these N-glycosidic bond cleavage pathways are mapped out via electronic structure calculations for the mixture of 2,4-dihydroxy tautomers and O4 protonated conformers of [Urd+H](+) and [dUrd+H](+) populated in the experiments. The calculated activation energies (AEs) and heats of reaction (ΔHrxns) for N-glycosidic bond cleavage at both the B3LYP and MP2(full) levels of theory are compared to the measured values. The agreement between experiment and theory indicates that B3LYP provides better estimates of the energetics of the species along the PESs for N-glycosidic bond cleavage than MP2, and that the 2,4-dihydroxy tautomers, which are stabilized by strong hydrogen-bonding interactions, predominantly influence the observed threshold dissociation behavior of [Urd+H](+) and [dUrd+H](+). PMID:27536972

  11. Extracellular granzyme K mediates endothelial activation through the cleavage of protease-activated receptor-1.

    PubMed

    Sharma, Mehul; Merkulova, Yulia; Raithatha, Sheetal; Parkinson, Leigh G; Shen, Yue; Cooper, Dawn; Granville, David J

    2016-05-01

    Granzymes are a family of serine proteases that were once thought to function exclusively as mediators of cytotoxic lymphocyte-induced target cell death. However, non-apoptotic roles for granzymes, including granzyme K (GzK), have been proposed. As recent studies have observed elevated levels of GzK in the plasma of patients diagnosed with clinical sepsis, we hypothesized that extracellular GzK induces a proinflammatory response in endothelial cells. In the present study, extracellular GzK proteolytically activated protease-activated receptor-1 leading to increased interleukin 6 and monocyte chemotactic protein 1 production in endothelial cells. Enhanced expression of intercellular adhesion molecule 1 along with an increased capacity for adherence of THP-1 cells was also observed. Characterization of downstream pathways implicated the mitogen-activated protein kinase p38 pathway for intercellular adhesion molecule 1 expression, and both the p38 and the extracellular signal-regulated protein kinases 1 and 2 pathways in cytokine production. GzK also increased tumour necrosis factor α-induced inflammatory adhesion molecule expression. Furthermore, the physiological inhibitor of GzK, inter-α-inhibitor protein, significantly inhibited GzK activity in vitro. In summary, extracellular GzK promotes a proinflammatory response in endothelial cells. PMID:26936634

  12. Toward the design of a catalytic metallodrug: selective cleavage of G-quadruplex telomeric DNA by an anticancer copper-acridine-ATCUN complex.

    PubMed

    Yu, Zhen; Han, Menglu; Cowan, James A

    2015-02-01

    Telomeric DNA represents a novel target for the development of anticancer drugs. By application of a catalytic metallodrug strategy, a copper-acridine-ATCUN complex (CuGGHK-Acr) has been designed that targets G-quadruplex telomeric DNA. Both fluorescence solution assays and gel sequencing demonstrate the CuGGHK-Acr catalyst to selectively bind and cleave the G-quadruplex telomere sequence. The cleavage pathway has been mapped by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) experiments. CuGGHK-Acr promotes significant inhibition of cancer cell proliferation and shortening of telomere length. Both senescence and apoptosis are induced in the breast cancer cell line MCF7. PMID:25504651

  13. Toward the Design of a Catalytic Metallodrug: Selective Cleavage of G-Quadruplex Telomeric DNA by an Anticancer Copper–Acridine–ATCUN Complex**

    PubMed Central

    Yu, Zhen; Han, Menglu

    2015-01-01

    Telomeric DNA represents a novel target for the development of anticancer drugs. By application of a catalytic metallodrug strategy, a copper–acridine–ATCUN complex (CuGGHK-Acr) has been designed that targets G-quadruplex telomeric DNA. Both fluorescence solution assays and gel sequencing demonstrate the CuGGHK-Acr catalyst to selectively bind and cleave the G-quadruplex telomere sequence. The cleavage pathway has been mapped by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) experiments. CuGGHK-Acr promotes significant inhibition of cancer cell proliferation and shortening of telomere length. Both senescence and apoptosis are induced in the breast cancer cell line MCF7. PMID:25504651

  14. Cleavage at the 586aa caspase-6 site in mutant huntingtin influences caspase-6 activation in vivo

    PubMed Central

    Graham, Rona K.; Deng, Yu; Carroll, Jeffery; Vaid, Kuljeet; Cowan, Catherine; Pouladi, Mahmoud A.; Metzler, Martina; Bissada, Nagat; Wang, Lili; Faull, Richard L. M.; Gray, Michelle; Yang, X. William; Raymond, Lynn A.; Hayden, Michael R.

    2010-01-01

    Caspase cleavage of huntingtin (htt) and nuclear htt accumulation represent early neuropathological changes in brains of patients with Huntington disease (HD). However the relationship between caspase cleavage of htt and caspase activation patterns in the pathogenesis of HD remains poorly understood. The lack of a phenotype in YAC mice expressing caspase-6-resistant (C6R) mutant htt (mhtt) highlights proteolysis of htt at the 586aa caspase-6 (casp6) site as a key mechanism in the pathology of HD. The goal of this study was to investigate how proteolysis of htt at residue 586 plays a role in the pathogenesis of HD and determine whether inhibiting casp6 cleavage of mhtt alters cell death pathways in vivo. Here we demonstrate that activation of casp6, and not caspase-3, is observed before onset of motor abnormalities in human and murine HD brain. Active casp6 levels correlate directly with CAG size and inversely with age of onset. In contrast, in vivo expression of C6R mhtt attenuates caspase activation. Increased casp6 activity and apoptotic cell death is evident in primary striatal neurons expressing caspase-cleavable, but not C6R, mhtt following NMDA application. Pretreatment with a casp6 inhibitor rescues the apoptotic cell death observed in this paradigm. These data demonstrate that activation of casp6 is an early marker of disease in HD. Furthermore, these data provide a clear link between excitotoxic pathways and proteolysis and suggest that C6R mhtt protects against neurodegeneration by influencing the activation of neuronal cell death and excitotoxic pathways operative in HD. PMID:21068307

  15. Inhibition of peroxynitrite-mediated DNA strand cleavage and hydroxyl radical formation by aspirin at pharmacologically relevant concentrations: Implications for cancer intervention

    SciTech Connect

    Chen, Wei; Zhu, Hong; Jia, Zhenquan; Li, Jianrong; Misra, Hara P.; Zhou, Kequan; Li, Yunbo

    2009-12-04

    Epidemiological studies have suggested that the long-term use of aspirin is associated with a decreased incidence of human malignancies, especially colorectal cancer. Since accumulating evidence indicates that peroxynitrite is critically involved in multistage carcinogenesis, this study was undertaken to investigate the ability of aspirin to inhibit peroxynitrite-mediated DNA damage. Peroxynitrite and its generator 3-morpholinosydnonimine (SIN-1) were used to cause DNA strand breaks in {phi}X-174 plasmid DNA. We demonstrated that the presence of aspirin at concentrations (0.25-2 mM) compatible with amounts in plasma during chronic anti-inflammatory therapy resulted in a significant inhibition of DNA cleavage induced by both peroxynitrite and SIN-1. Moreover, the consumption of oxygen caused by 250 {mu}M SIN-1 was found to be decreased in the presence of aspirin, indicating that aspirin might affect the auto-oxidation of SIN-1. Furthermore, EPR spectroscopy using 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap demonstrated the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from authentic peroxynitrite, and that aspirin at 0.25-2 mM potently diminished the radical adduct formation in a concentration-dependent manner. Taken together, these results demonstrate for the first time that aspirin at pharmacologically relevant concentrations can inhibit peroxynitrite-mediated DNA strand breakage and hydroxyl radical formation. These results may have implications for cancer intervention by aspirin.

  16. Half-sandwich RuCl2(η(6)-p-cymene) core complexes containing sulfur donor aroylthiourea ligands: DNA and protein binding, DNA cleavage and cytotoxic studies.

    PubMed

    Jeyalakshmi, Kumaramangalam; Haribabu, Jebiti; Bhuvanesh, Nattamai S P; Karvembu, Ramasamy

    2016-08-01

    A series of Ru(ii)(η(6)-p-cymene) complexes (1-4) bearing the general formula [RuCl2(η(6)-p-cymene)L] (L = monodentate aroylthiourea ligand) has been synthesized and characterized by analytical and various spectroscopic techniques. The neutral monodentate coordination of aroylthiourea with Ru via an S atom was confirmed by single crystal X-ray diffraction study. The complexes were tested for their ability to interact with DNA and protein. The complexes bound with calf thymus DNA (CT DNA) with the intrinsic binding constant value in the order of 10(4) M(-1). The intercalative mode of binding was confirmed by the ethidium bromide (EB) displacement study. The interaction of the complexes with CT DNA was further supported by viscosity measurements and circular dichroic (CD) spectra. The Ru(ii) complexes cleaved the supercoiled DNA without the need of any external agent. The spectroscopic evidence showed good binding efficacy of the complexes with BSA (Bovine Serum Albumin). The alterations in the secondary structure of BSA by the Ru(ii) complexes were confirmed by synchronous fluorescence spectra. Cytotoxicity examination by MTT assay was carried out in two cancer cell lines (MCF7 and A549) and one non-cancerous cell line (L929). Complex 4 showed significant activity [IC50 = 52.3 (MCF7) and 54.6 (A549) μM] which was comparable with that of similar known complexes. The morphological changes assessed by Hoechst staining revealed that the cell death occurred by apoptosis. PMID:27435011

  17. DNA Methyltransferase Activity Assays: Advances and Challenges

    PubMed Central

    Poh, Wan Jun; Wee, Cayden Pang Pee; Gao, Zhiqiang

    2016-01-01

    DNA methyltransferases (MTases), a family of enzymes that catalyse the methylation of DNA, have a profound effect on gene regulation. A large body of evidence has indicated that DNA MTase is potentially a predictive biomarker closely associated with genetic disorders and genetic diseases like cancer. Given the attention bestowed onto DNA MTases in molecular biology and medicine, highly sensitive detection of DNA MTase activity is essential in determining gene regulation, epigenetic modification, clinical diagnosis and therapeutics. Conventional techniques such as isotope labelling are effective, but they often require laborious sample preparation, isotope labelling, sophisticated equipment and large amounts of DNA, rendering them unsuitable for uses at point-of-care. Simple, portable, highly sensitive and low-cost assays are urgently needed for DNA MTase activity screening. In most recent technological advances, many alternative DNA MTase activity assays such as fluorescent, electrochemical, colorimetric and chemiluminescent assays have been proposed. In addition, many of them are coupled with nanomaterials and/or enzymes to significantly enhance their sensitivity. Herein we review the progress in the development of DNA MTase activity assays with an emphasis on assay mechanism and performance with some discussion on challenges and perspectives. It is hoped that this article will provide a broad coverage of DNA MTase activity assays and their latest developments and open new perspectives toward the development of DNA MTase activity assays with much improved performance for uses in molecular biology and clinical practice. PMID:26909112

  18. Specialization of the DNA-Cleaving Activity of a Group I Ribozyme Through In Vitro Evolution

    NASA Technical Reports Server (NTRS)

    Tsang, Joyce; Joyce, Gerald F.

    1996-01-01

    In an earlier study, an in vitro evolution procedure was applied to a large population of variants of the Tetrahymena group 1 ribozyme to obtain individuals with a 10(exp 5)-fold improved ability to cleave a target single-stranded DNA substrate under simulated physiological conditions. The evolved ribozymes also showed a twofold improvement, compared to the wild-type, in their ability to cleave a single-stranded RNA substrate. Here, we report continuation of the in vitro evolution process using a new selection strategy to achieve both enhanced DNA and diminished RNA-cleavage activity. Our strategy combines a positive selection for DNA cleavage with a negative selection against RNA binding. After 36 "generations" of in vitro evolution, the evolved population showed an approx. 100-fold increase in the ratio of DNA to RNA-cleavage activity. Site-directed mutagenesis experiment confirmed the selective advantage of two covarying mutations within the catalytic core of ribozyme that are largely responsible for this modified behavior. The population of ribozymes has now undergone a total of 63 successive generations of evolution, resulting in an average 28 mutations relative to the wild-type that are responsible for the altered phenotype.

  19. Cleavage of extracellular matrix in periodontitis: gingipains differentially affect cell adhesion activities of fibronectin and tenascin-C

    PubMed Central

    Ruggiero, Sabrina; Cosgarea, Raluca; Potempa, Jan; Potempa, Barbara; Eick, Sigrun; Chiquet, Matthias

    2014-01-01

    Gingipains are cysteine proteases that represent major virulence factors of the periodontopathogenic bacterium Porphyromonas gingivalis. Gingipains are reported to degrade extracellular matrix (ECM) of periodontal tissues, leading to tissue destruction and apoptosis. The exact mechanism is not known, however. Fibronectin and tenascin-C are pericellular ECM glycoproteins present in periodontal tissues. Whereas fibronectin mediates fibroblast adhesion, tenascin-C binds to fibronectin and inhibits its cell-spreading activity. Using purified proteins in vitro, we asked whether fibronectin and tenascin-C are cleaved by gingipains at clinically relevant concentrations, and how fragmentation by the bacterial proteases affects their biological activity in cell adhesion. Fibronectin was cleaved into distinct fragments by all three gingipains; however, only arginine-specific HRgpA and RgpB but not lysine-specific Kgp destroyed its cell-spreading activity. This result was confirmed with recombinant cell-binding domain of fibronectin. Of the two major tenascin-C splice variants, the large but not the small was a substrate for gingipains, indicating that cleavage occurred primarily in the alternatively spliced domain. Surprisingly, cleavage of large tenascin-C variant by all three gingipains generated fragments with increased anti-adhesive activity towards intact fibronectin. Fibronectin and tenascin-C fragments were detected in gingival crevicular fluid of a subset of periodontitis patients. We conclude that cleavage by gingipains directly affects the biological activity of both fibronectin and tenascin-C in a manner that might lead to increased cell detachment and loss during periodontal disease. PMID:23313574

  20. Cleavage of extracellular matrix in periodontitis: gingipains differentially affect cell adhesion activities of fibronectin and tenascin-C.

    PubMed

    Ruggiero, Sabrina; Cosgarea, Raluca; Potempa, Jan; Potempa, Barbara; Eick, Sigrun; Chiquet, Matthias

    2013-04-01

    Gingipains are cysteine proteases that represent major virulence factors of the periodontopathogenic bacterium Porphyromonas gingivalis. Gingipains are reported to degrade extracellular matrix (ECM) of periodontal tissues, leading to tissue destruction and apoptosis. The exact mechanism is not known, however. Fibronectin and tenascin-C are pericellular ECM glycoproteins present in periodontal tissues. Whereas fibronectin mediates fibroblast adhesion, tenascin-C binds to fibronectin and inhibits its cell-spreading activity. Using purified proteins in vitro, we asked whether fibronectin and tenascin-C are cleaved by gingipains at clinically relevant concentrations, and how fragmentation by the bacterial proteases affects their biological activity in cell adhesion. Fibronectin was cleaved into distinct fragments by all three gingipains; however, only arginine-specific HRgpA and RgpB but not lysine-specific Kgp destroyed its cell-spreading activity. This result was confirmed with recombinant cell-binding domain of fibronectin. Of the two major tenascin-C splice variants, the large but not the small was a substrate for gingipains, indicating that cleavage occurred primarily in the alternatively spliced domain. Surprisingly, cleavage of large tenascin-C variant by all three gingipains generated fragments with increased anti-adhesive activity towards intact fibronectin. Fibronectin and tenascin-C fragments were detected in gingival crevicular fluid of a subset of periodontitis patients. We conclude that cleavage by gingipains directly affects the biological activity of both fibronectin and tenascin-C in a manner that might lead to increased cell detachment and loss during periodontal disease. PMID:23313574

  1. Microarrays Made Simple: "DNA Chips" Paper Activity

    ERIC Educational Resources Information Center

    Barnard, Betsy

    2006-01-01

    DNA microarray technology is revolutionizing biological science. DNA microarrays (also called DNA chips) allow simultaneous screening of many genes for changes in expression between different cells. Now researchers can obtain information about genes in days or weeks that used to take months or years. The paper activity described in this article…

  2. Initial Stages of V(D)J Recombination: the Organization of RAG1/2 and RSS DNA in the Post-cleavage Complex

    PubMed Central

    Grundy, Gabrielle J.; Ramón-Maiques, Santiago; Dimitriadis, Emilios K.; Kotova, Svetlana; Biertümpfel, Christian; Heymann, J. Bernard; Steven, Alasdair C.; Gellert, Martin; Yang, Wei

    2009-01-01

    SUMMARY To obtain structural information on the early stages of V(D)J recombination, we isolated a complex of the core RAG1 and RAG2 proteins with DNA containing a pair of cleaved recombination signal sequences (RSS). Stoichiometric and molecular mass analysis established that this signal end complex (SEC) contains two protomers each of RAG1 and RAG2. Visualization of the SEC by negative staining electron microscopy revealed an anchor-shaped particle with approximate two-fold symmetry. Consistent with a parallel arrangement of DNA and protein subunits, the N-termini of RAG1 and RAG2 are positioned at opposing ends of the complex, and the DNA chains beyond the RSS nonamer emerge from the same face of the complex, near to the RAG1 N-termini. These first images of the V(D)J recombinase in its post-cleavage state provide a framework for modeling RAG domains and their interactions with DNA. PMID:19647518

  3. Internal guide RNA interactions interfere with Cas9-mediated cleavage.

    PubMed

    Thyme, Summer B; Akhmetova, Laila; Montague, Tessa G; Valen, Eivind; Schier, Alexander F

    2016-01-01

    The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9-gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9-gRNA complex formation. PMID:27282953

  4. Internal guide RNA interactions interfere with Cas9-mediated cleavage

    PubMed Central

    Thyme, Summer B.; Akhmetova, Laila; Montague, Tessa G.; Valen, Eivind; Schier, Alexander F.

    2016-01-01

    The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9–gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9–gRNA complex formation. PMID:27282953

  5. Synthesis, spectroscopic characterization and structural investigation of a new charge transfer complex of 2,6-diaminopyridine with 4-nitrophenylacetic acid: Antimicrobial, DNA binding/cleavage and antioxidant studies

    NASA Astrophysics Data System (ADS)

    Murugesan, Venkatesan; Saravanabhavan, Munusamy; Sekar, Marimuthu

    2015-08-01

    A new hydrogen-bonded charge-transfer complex (CT) formed by the reaction between donor, 2,6-diaminopyridine and acceptor, 4-nitrophenylacetic acid in methanol at room temperature. The crystal was characterized by elemental analysis, IR, NMR spectroscopic studies and thermal studies. The elemental analysis of CT complex, obtained data revealed that the formation of 1:1 ratio CT complex was proposed. Infrared and NMR studies confirm the chemical constituents and molecular structure of the synthesized complex crystal. The high thermal stability is due to the molecular frame work through H-bonding interactions. Structural investigation indicates that cation and anion are linked through strong N+-H⋯O- type of hydrogen bond. The hydrogen bonded charge transfer crystal was screened for its pharmacology, such as antimicrobial, DNA binding/cleavage and antioxidant studies. The CT complex was screened for its antibacterial and antifungal activity against various bacterial and fungal species, which shows good antimicrobial activity. The DNA binding results indicated that the compound could interact with DNA through intercalation. It should have weak to moderate capacity of scavenging with DPPH.

  6. Sin Nombre hantavirus nucleocapsid protein exhibits a metal-dependent DNA-specific endonucleolytic activity.

    PubMed

    Möncke-Buchner, Elisabeth; Szczepek, Michal; Bokelmann, Marcel; Heinemann, Patrick; Raftery, Martin J; Krüger, Detlev H; Reuter, Monika

    2016-09-01

    We demonstrate that the nucleocapsid protein of Sin Nombre hantavirus (SNV-N) has a DNA-specific endonuclease activity. Upon incubation of SNV-N with DNA in the presence of magnesium or manganese, we observed DNA digestion in sequence-unspecific manner. In contrast, RNA was not affected under the same conditions. Moreover, pre-treatment of SNV-N with RNase before DNA cleavage increased the endonucleolytic activity. Structure-based protein fold prediction using known structures from the PDB database revealed that Asp residues in positions 88 and 103 of SNV-N show sequence similarity with the active site of the restriction endonuclease HindIII. Crystal structure of HindIII predicts that residues Asp93 and Asp108 are essential for coordination of the metal ions required for HindIII DNA cleavage. Therefore, we hypothesized that homologous residues in SNV-N, Asp88 and Asp103, may have a similar function. Replacing Asp88 and Asp103 by alanine led to an SNV-N protein almost completely abrogated for endonuclease activity. PMID:27261891

  7. Cytosines, but not purines, determine recombination activating gene (RAG)-induced breaks on heteroduplex DNA structures: implications for genomic instability.

    PubMed

    Naik, Abani Kanta; Lieber, Michael R; Raghavan, Sathees C

    2010-03-01

    The sequence specificity of the recombination activating gene (RAG) complex during V(D)J recombination has been well studied. RAGs can also act as structure-specific nuclease; however, little is known about the mechanism of its action. Here, we show that in addition to DNA structure, sequence dictates the pattern and efficiency of RAG cleavage on altered DNA structures. Cytosine nucleotides are preferentially nicked by RAGs when present at single-stranded regions of heteroduplex DNA. Although unpaired thymine nucleotides are also nicked, the efficiency is many fold weaker. Induction of single- or double-strand breaks by RAGs depends on the position of cytosines and whether it is present on one or both of the strands. Interestingly, RAGs are unable to induce breaks when adenine or guanine nucleotides are present at single-strand regions. The nucleotide present immediately next to the bubble sequence could also affect RAG cleavage. Hence, we propose "C((d))C((S))C((S))" (d, double-stranded; s, single-stranded) as a consensus sequence for RAG-induced breaks at single-/double-strand DNA transitions. Such a consensus sequence motif is useful for explaining RAG cleavage on other types of DNA structures described in the literature. Therefore, the mechanism of RAG cleavage described here could explain facets of chromosomal rearrangements specific to lymphoid tissues leading to genomic instability. PMID:20051517

  8. New Oxidovanadium Complexes Incorporating Thiosemicarbazones and 1, 10-Phenanthroline Derivatives as DNA Cleavage, Potential Anticancer Agents, and Hydroxyl Radical Scavenger.

    PubMed

    Ying, Peng; Zeng, Pengfei; Lu, Jiazheng; Chen, Hongyuan; Liao, Xiangwen; Yang, Ning

    2015-10-01

    Four novel oxidovanadium(IV) complexes, [VO(hntdtsc)(PHIP)] (1) (hntdtsc = 2-hydroxy-1-naphthaldehyde thiosemicarbazone, PHIP= 2-phenyl-imidazo[4,5-f]1,10-phenanthroline), [VO(hntdtsc)(DPPZ)](2)(DPPZ= dipyrido[3,2-a:2',3'-c]phenazine), [VO(satsc)(PHIP)](3) (satsc=salicylaldehyde thiosemicarbazone), and [VO(satsc)(DPPZ)](4), have been prepared and characterized. The chemical nuclease activities and photocleavage reactions of the complexes were tested. All four complexes can efficiently cleave pBR322 DNA, and complex 1 has the best cleaving ability. The antitumor properties of these complexes were examined with three different tumor cell lines using MTT assay. Their antitumor mechanism has been analyzed using cell cycle analysis, fluorescence microscopy of apoptosis, and Annexin V-FITC/PI assay. The results showed that the growth of human neuroblastoma (SH-SY5Y, SK-N-SH) and human breast adenocarcinoma (MCF-7) cells were inhibited significantly with very low IC50 values. Complex 1 was found to be the most potent antitumor agent among the four complexes. It can cause G0/G1 phase arrest of the cell cycle and exhibited significant induced apoptosis in SK-N-SH cells and displayed typical morphological apoptotic characteristics. In addition, they all displayed reasonable abilities to scavenge hydroxyl radical, and complex 1 was the best inhibitor. PMID:25659415

  9. Conventional and microwave-assisted synthesis, characterization, DFT calculations, in vitro DNA binding and cleavage studies of potential chemotherapeutic diorganotin(IV) mandelates.

    PubMed

    Mridula; Nath, Mala

    2016-09-01

    Diorganotin(IV) complexes of the general formulae {[R2Sn(L)]2O}(R=Me (1), n-Bu (2), and n-Oct (3); L=anion of mandelic acid) and {[R2Sn(L)]2Cl2}(R=Ph (4)) have been synthesized by conventional thermal method (1a-3a), except 4a and by microwave-assisted reactions (1b-4b). The elemental analysis, IR, NMR ((1)H, (13)C and (119)Sn) and ESI-MS/DART-mass spectral studies revealed that dimeric 1:1 complexes with SnOSn bridges (1-3) are formed possessing distorted trigonal bipyramidal geometry around the Sn atoms, except 4b which exhibits octahedral geometry with SnClSn bridges. The proposed geometries have been validated by density functional theory calculations. Thermal behavior of 1b-4b, studied by using thermogravimetry (TG), differential thermal analysis (DTA) and derivative thermogravimetric (DTG) techniques, indicated that all except 4b are stable up to 200°C. In vitro interaction studies of 1b-4b with CT-DNA were performed by UV-Vis, fluorescence titrations and results suggest that the complexes are binding to DNA via an intercalative mode. The binding affinity and quenching ability were quantified in terms of intrinsic binding constant (Kb) (3.74×10(4)M(-1), 2b; >3.67×10(4)M(-1), 4b; >3.03×10(4)M(-1), 3b; >0.72×10(4)M(-1), 1b) and Stern-Volmer quenching constant (Ksv) (2.16×10(5), 2b; >1.73×10(5), 4b; >1.66×10(5)3b; >1.51×10(5), 1b) which showed high binding affinity of 2b with CT-DNA. The cleavage studies of 1b-4b with pBR322 plasmid DNA was ascertained by agarose gel electrophoresis. They exhibited effective cleavage of supercoiled plasmid DNA into its nicked form (1b, 3b, 4b) and even into its linear form in presence of 2b. PMID:27423117

  10. In vitro DNA binding, pBR322 plasmid cleavage and molecular modeling study of chiral benzothiazole Schiff-base-valine Cu(II) and Zn(II) complexes to evaluate their enantiomeric biological disposition for molecular target DNA

    NASA Astrophysics Data System (ADS)

    Alizadeh, Rahman; Afzal, Mohd; Arjmand, Farukh

    2014-10-01

    Bicyclic heterocyclic compounds viz. benzothiazoles are key components of deoxyribonucleic acid (DNA) molecules and participate directly in the encoding of genetic information. Benzothiazoles, therefore, represent a potent and selective class of antitumor compounds. The design and synthesis of chiral antitumor chemotherapeutic agents of Cu(II) and Zn(II), L- and -D benzothiazole Schiff base-valine complexes 1a &b and 2a &b, respectively were carried out and thoroughly characterized by spectroscopic and analytical techniques. Interaction of 1a and b and 2a and b with CT DNA by employing UV-vis, florescence, circular dichroic methods and cleavage studies of 1a with pBR322 plasmid, molecular docking were done in order to demonstrate their enantiomeric disposition toward the molecular drug target DNA. Interestingly, these studies unambiguously demonstrated the greater potency of L-enantiomer in comparison to D-enantiomer.

  11. DNA intercalation, topoisomerase II inhibition and cytotoxic activity of the plant alkaloid neocryptolepine.

    PubMed

    Bailly, C; Laine, W; Baldeyrou, B; De Pauw-Gillet, M C; Colson, P; Houssier, C; Cimanga, K; Van Miert, S; Vlietinck, A J; Pieters, L

    2000-06-01

    Cryptolepine and neocryptolepine are two indoloquinoline alkaloids isolated from the roots of the African plant Cryptolepis sanguinolenta. Both drugs have revealed antibacterial and antiparasitic activities and are strongly cytotoxic to tumour cells. We have recently shown that cryptolepine can intercalate into DNA and stimulates DNA cleavage by human topoisomerase II. In this study, we have investigated the mechanism of action and cytotoxicity of neocryptolepine, which differs from the parent isomer only by the orientation of the indole unit with respect to the quinoline moiety. The biochemical and physicochemical results presented here indicate that neocryptolepine also intercalates into DNA, preferentially at GC-rich sequences, but exhibits a reduced affinity for DNA compared with cryptolepine. The two alkaloids interfere with the catalytic activity of human topoisomerase II but the poisoning activity is slightly more pronounced with cryptolepine than with its isomer. The data provide a molecular basis to account for the reduced cytotoxicity of neocryptolepine compared with the parent drug. PMID:11049087

  12. DNA-Binding, Photocleavage, and Photodynamic Anti-cancer Activities of Pyridyl Corroles.

    PubMed

    Liang, Zhen-Hua; Liu, Hai-Yang; Zhou, Rong; Zhang, Zao; Ali, Atif; Han, Bing-Jie; Liu, Yun-Jun; Xiao, Xin-Yan

    2016-08-01

    The DNA-binding, photocleavage, and antitumor activity of three free base pyridyl corroles 1, 2, and 3 have been investigated. The binding affinity toward CT-DNA decreases with increasing number of pentafluorophenyl, whereas the photocleavage activity toward pBR322 DNA becomes more efficient. Singlet oxygen was demonstrated as active species responsible for DNA cleavage. These corroles exhibited high cytotoxicity against three tested cancer cells (Hela, HapG2, and A549) and the cytotoxicity could be further enhanced under irradiation. Intracellular reactive oxygen species level was also monitored using HeLa Cells upon the combined treatment of corroles and light. These corroles could be absorbed by HeLa cells at low concentration. They can induce the decrease of mitochondrial membrane potential and apoptosis of tumor cells under irradiation. PMID:26895317

  13. Mixed-ligand copper(ii) Schiff base complexes: the role of the co-ligand in DNA binding, DNA cleavage, protein binding and cytotoxicity.

    PubMed

    Lian, Wen-Jing; Wang, Xin-Tian; Xie, Cheng-Zhi; Tian, He; Song, Xue-Qing; Pan, He-Ting; Qiao, Xin; Xu, Jing-Yuan

    2016-05-31

    Four novel mononuclear Schiff base copper(ii) complexes, namely, [Cu(L)(OAc)]·H2O (), [Cu(HL)(C2O4)(EtOH)]·EtOH (), [Cu(L)(Bza)] () and [Cu(L)(Sal)] () (HL = 1-(((2-((2-hydroxypropyl)amino)ethyl)imino)methyl)naphthalene-2-ol), Bza = benzoic acid, Sal = salicylic acid), were synthesized and characterized by X-ray crystallography, elemental analysis and infrared spectroscopy. Single-crystal diffraction analysis revealed that all the complexes were mononuclear molecules, in which the Schiff base ligand exhibited different coordination modes and conformations. The N-HO and O-HO inter- and intramolecular hydrogen bonding interactions linked these molecules into multidimensional networks. Their interactions with calf thymus DNA (CT-DNA) were investigated by UV-visible and fluorescence spectrometry, as well as by viscosity measurements. The magnitude of the Kapp values of the four complexes was 10(5), indicating a moderate intercalative binding mode between the complexes and DNA. Electrophoresis results showed that all these complexes induced double strand breaks of pUC19 plasmid DNA in the presence of H2O2 through an oxidative pathway. In addition, the fluorescence spectrum of human serum albumin (HSA) with the complexes suggested that the quenching mechanism of HSA by the complexes was a static process. Moreover, the antiproliferative activity of the four complexes against HeLa (human cervical carcinoma) and HepG-2 (human liver hepatocellular carcinoma) cells evaluated by colorimetric cell proliferation assay and clonogenic assay revealed that all four complexes had improved cytotoxicity against cancer cells. Inspiringly, complex , with salicylic acid as the auxiliary ligand, displayed a stronger anticancer activity, suggesting that a synergistic effect of the Schiff base complex and the nonsteroidal anti-inflammatory drug may be involved in the cell killing process. The biological features of mixed-ligand copper(ii) Schiff base complexes and how acetic auxiliary

  14. A New Signaling Pathway for HCV Inhibition by Estrogen: GPR30 Activation Leads to Cleavage of Occludin by MMP-9.

    PubMed

    Ulitzky, Laura; Lafer, Manuel M; KuKuruga, Mark A; Silberstein, Erica; Cehan, Nicoleta; Taylor, Deborah R

    2016-01-01

    Poor outcome in response to hepatitis C virus, including higher viral load, hepatocellular carcinoma and cirrhosis, is more associated with men and postmenopausal women than with premenopausal women and women receiving hormone replacement therapy, suggesting that β-estradiol plays an innate role in preventing viral infection and liver disease. Consequently, most research in the field has concluded that estrogen affects HCV replication through viral interactions with estrogen receptor-α. Previously, estrogen-like antagonists, including Tamoxifen, were shown to reduce HCV RNA production and prevent viral entry, although the authors did not identify host factors involved. Estrogen can act alternatively through the membrane-bound G-protein-coupled estrogen receptor, GPR30. Here, human hepatoma Huh7.5 cells were infected with HCV J6/JFH-1 and treated with estrogen or Tamoxifen, resulting in a marked decrease in detectable virus. The effect was mimicked by G1, a GPR30-specific agonist, and was reversed by the GPR30-specific antagonist, G15. While previous studies have demonstrated that estrogen down-regulated occludin in cervical cancer cells, its action on liver cells was unknown. Occludin is a tight junction protein and HCV receptor and here we report that activation and cellular export of MMP-9 led to the cleavage of occludin upon estrogen treatment of liver cells. This is the first report of the cleavage of an HCV receptor in response to estrogen. We also identify the occludin cleavage site in extracellular Domain D; the motif required for HCV entry and spread. This pathway gives new insight into a novel innate antiviral pathway and the suboptimal environment that estrogen provides for the proliferation of the virus. It may also explain the disparate host-virus responses to HCV demonstrated by the two sexes. Moreover, these data suggest that hormone replacement therapy may have beneficial antiviral enhancement properties for HCV-infected postmenopausal women and

  15. A New Signaling Pathway for HCV Inhibition by Estrogen: GPR30 Activation Leads to Cleavage of Occludin by MMP-9

    PubMed Central

    Ulitzky, Laura; Lafer, Manuel M.; KuKuruga, Mark A.; Silberstein, Erica; Cehan, Nicoleta; Taylor, Deborah R.

    2016-01-01

    Poor outcome in response to hepatitis C virus, including higher viral load, hepatocellular carcinoma and cirrhosis, is more associated with men and postmenopausal women than with premenopausal women and women receiving hormone replacement therapy, suggesting that β-estradiol plays an innate role in preventing viral infection and liver disease. Consequently, most research in the field has concluded that estrogen affects HCV replication through viral interactions with estrogen receptor-α. Previously, estrogen-like antagonists, including Tamoxifen, were shown to reduce HCV RNA production and prevent viral entry, although the authors did not identify host factors involved. Estrogen can act alternatively through the membrane-bound G-protein-coupled estrogen receptor, GPR30. Here, human hepatoma Huh7.5 cells were infected with HCV J6/JFH-1 and treated with estrogen or Tamoxifen, resulting in a marked decrease in detectable virus. The effect was mimicked by G1, a GPR30-specific agonist, and was reversed by the GPR30-specific antagonist, G15. While previous studies have demonstrated that estrogen down-regulated occludin in cervical cancer cells, its action on liver cells was unknown. Occludin is a tight junction protein and HCV receptor and here we report that activation and cellular export of MMP-9 led to the cleavage of occludin upon estrogen treatment of liver cells. This is the first report of the cleavage of an HCV receptor in response to estrogen. We also identify the occludin cleavage site in extracellular Domain D; the motif required for HCV entry and spread. This pathway gives new insight into a novel innate antiviral pathway and the suboptimal environment that estrogen provides for the proliferation of the virus. It may also explain the disparate host-virus responses to HCV demonstrated by the two sexes. Moreover, these data suggest that hormone replacement therapy may have beneficial antiviral enhancement properties for HCV-infected postmenopausal women and

  16. Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

    SciTech Connect

    Spannaus, Ralf; Bodem, Jochen

    2014-04-15

    In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies. The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity.

  17. Site specific cleavage of thetaX-174 replicative form DNA after modification by N-acetoxy-N-2-acetylaminofluorene

    SciTech Connect

    Bases, R.; Mendez, F.; Mendez, L.

    1983-01-01

    Three kinds of structural disturbances were found in an 88 base pair (bp) fragment of thetaX-174 DNA after exposure to N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF). Frequent strand scissions at two specific guanine sites on the 5' /sup 32/P-end-labeled fragment were identified by base sequence analysis. Scissions at these two sites were induced at neutral pH and they were not increased by treatment with apurinic endonuclease. They are an immediate consequence of N-Aco-AAF action and are not primarily apurinic sites. Alkali treatment with 1 M piperidine at 90/sup 0/C induced strand scissions at every guanine, demonstrating adduct slices, depurination and strand scissions. Adducted DNA was sensitive to single-strand specific nuclease digestion, suggesting unwound DNA. These studies indicate the prediliction of N-Aco-AAF for certain DNA sites and they suggest three kinds of DNA modifications which can be expected after adduction by this carcinogen. Some of the sites may be premutational carcinogen-induced DNA structural modifications.

  18. Determining the Architecture of a Protein-DNA Complex by Combining FeBABE Cleavage Analyses, 3-D Printed Structures, and the ICM Molsoft Program.

    PubMed

    James, Tamara; Hsieh, Meng-Lun; Knipling, Leslie; Hinton, Deborah

    2015-01-01

    Determining the structure of a protein-DNA complex can be difficult, particularly if the protein does not bind tightly to the DNA, if there are no homologous proteins from which the DNA binding can be inferred, and/or if only portions of the protein can be crystallized. If the protein comprises just a part of a large multi-subunit complex, other complications can arise such as the complex being too large for NMR studies, or it is not possible to obtain the amounts of protein and nucleic acids needed for crystallographic analyses. Here, we describe a technique we used to map the position of an activator protein relative to the DNA within a large transcription complex. We determined the position of the activator on the DNA from data generated using activator proteins that had been conjugated at specific residues with the chemical cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). These analyses were combined with 3-D models of the available structures of portions of the activator protein and B-form DNA to obtain a 3-D picture of the protein relative to the DNA. Finally, the Molsoft program was used to refine the position, revealing the architecture of the protein-DNA within the transcription complex. PMID:26404142

  19. The RNA cleavage activity of RNA polymerase III is mediated by an essential TFIIS-like subunit and is important for transcription termination

    PubMed Central

    Chédin, Stéphane; Riva, Michel; Schultz, Patrick; Sentenac, André; Carles, Christophe

    1998-01-01

    Budding yeast RNA polymerase III (Pol III) contains a small, essential subunit, named C11, that is conserved in humans and shows a strong homology to TFIIS. A mutant Pol III, heterocomplemented with Schizosaccharomyces pombe C11, was affected in transcription termination in vivo. A purified form of the enzyme (Pol III Δ), deprived of C11 subunit, initiated properly but ignored pause sites and was defective in termination. Remarkably, Pol III Δ lacked the intrinsic RNA cleavage activity of complete Pol III. In vitro reconstitution experiments demonstrated that Pol III RNA cleavage activity is mediated by C11. Mutagenesis in C11 of two conserved residues, which are critical for the TFIIS-dependent cleavage activity of Pol II, is lethal. Immunoelectron microscopy data suggested that C11 is localized on the mobile thumb-like stalk of the polymerase. We propose that C11 allows the enzyme to switch between an RNA elongation and RNA cleavage mode and that the essential role of the Pol III RNA cleavage activity is to remove the kinetic barriers to the termination process. The integration of TFIIS function into a specific Pol III subunit may stem from the opposite requirements of Pol III and Pol II in terms of transcript length and termination efficiency. PMID:9869639

  20. Pannexin 1, an ATP Release Channel, Is Activated by Caspase Cleavage of Its Pore-associated C-terminal Autoinhibitory Region*♦

    PubMed Central

    Sandilos, Joanna K.; Chiu, Yu-Hsin; Chekeni, Faraaz B.; Armstrong, Allison J.; Walk, Scott F.; Ravichandran, Kodi S.; Bayliss, Douglas A.

    2012-01-01

    Pannexin 1 (PANX1) channels mediate release of ATP, a “find-me” signal that recruits macrophages to apoptotic cells; PANX1 activation during apoptosis requires caspase-mediated cleavage of PANX1 at its C terminus, but how the C terminus inhibits basal channel activity is not understood. Here, we provide evidence suggesting that the C terminus interacts with the human PANX1 (hPANX1) pore and that cleavage-mediated channel activation requires disruption of this inhibitory interaction. Basally silent hPANX1 channels localized on the cell membrane could be activated directly by protease-mediated C-terminal cleavage, without additional apoptotic effectors. By serial deletion, we identified a C-terminal region just distal to the caspase cleavage site that is required for inhibition of hPANX1; point mutations within this small region resulted in partial activation of full-length hPANX1. Consistent with the C-terminal tail functioning as a pore blocker, we found that truncated and constitutively active hPANX1 channels could be inhibited, in trans, by the isolated hPANX1 C terminus either in cells or when applied directly as a purified peptide in inside-out patch recordings. Furthermore, using a cysteine cross-linking approach, we showed that relief of inhibition following cleavage requires dissociation of the C terminus from the channel pore. Collectively, these data suggest a mechanism of hPANX1 channel regulation whereby the intact, pore-associated C terminus inhibits the full-length hPANX1 channel and a remarkably well placed caspase cleavage site allows effective removal of key inhibitory C-terminal determinants to activate hPANX1. PMID:22311983

  1. Mutation of Gly721 Alters DNA Topoisomerase I Active Site Architecture and Sensitivity to Camptothecin*

    PubMed Central

    van der Merwe, Marie; Bjornsti, Mary-Ann

    2015-01-01

    DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA via a concerted mechanism of DNA strand cleavage and religation. Top1p is the cellular target of the anticancer drug camptothecin (CPT), which reversibly stabilizes a covalent enzyme-DNA intermediate. Top1p clamps around duplex DNA, wherein the core and C-terminal domains are connected by extended α-helices (linker domain), which position the active site Tyr of the C-terminal domain within the catalytic pocket. The physical connection of the linker with the Top1p clamp as well as linker flexibility affect enzyme sensitivity to CPT. Crystallographic data reveal that a conserved Gly residue (located at the juncture between the linker and C-terminal domains) is at one end of a short α-helix, which extends to the active site Tyr covalently linked to the DNA. In the presence of drug, the linker is rigid and this α-helix extends to include Gly and the preceding Leu. We report that mutation of this conserved Gly in yeast Top1p alters enzyme sensitivity to CPT. Mutating Gly to Asp, Glu, Asn, Gln, Leu, or Ala enhanced enzyme CPT sensitivity, with the acidic residues inducing the greatest increase in drug sensitivity in vivo and in vitro. By contrast, Val or Phe substituents rendered the enzyme CPT-resistant. Mutation-induced alterations in enzyme architecture preceding the active site Tyr suggest these structural transitions modulate enzyme sensitivity to CPT, while enhancing the rate of DNA cleavage. We postulate that this conserved Gly residue provides a flexible hinge within the Top1p catalytic pocket to facilitate linker dynamics and the structural alterations that accompany drug binding of the covalent enzyme-DNA intermediate. PMID:18056711

  2. Dimeric Fe (II, III) complex of quinoneoxime as functional model of PAP enzyme: Mössbauer, magneto-structural and DNA cleavage studies

    NASA Astrophysics Data System (ADS)

    Salunke-Gawali, Sunita; Ahmed, Khursheed; Varret, François; Linares, Jorge; Zaware, Santosh; Date, Sadgopal; Rane, Sandhya

    2008-07-01

    value of antiferromagnetic exchange leads to Fe+3μ-(OH) Fe + 2 bridging in Fe-1 dimer instead of μ-oxo bridge. The intermolecular association through H-bonds may lead to weakly coupled antiferromagnetic interaction between two Fe-2 molecules having Fe + 3(h.s.) centers. Using S = 5/2, 5/2 spin pair model we obtained best-fitted parameters such as J = -12.4 cm - 1, g = 2.3 with R = 3.58 × 10 - 5. Synthetic strategy results in non-equivalent iron sites in Fe-1 dimer analogues to PAP enzyme hence its reconstitution results in pUC-19 DNA cleavage activity, as physiological functionality of APase. It is compared with nuclease activity of Fe-2 RAPase.

  3. The Role of an Active Site Mg2+ in HDV Ribozyme Self-Cleavage: Insights from QM/MM Calculations

    PubMed Central

    Mlýnský, Vojtěch; Šponer, Jiří

    2014-01-01

    The hepatitis delta virus (HDV) ribozyme is a catalytic RNA motif embedded in the human pathogenic HDV RNA. It catalyzes self-cleavage of its sugar-phosphate backbone with direct participation of the active site cytosine C75. Biochemical and structural data support a general acid role of C75. Here, we used hybrid quantum mechanical/molecular mechanical (QM/MM) calculations to probe the reaction mechanism and changes in Gibbs energy along the ribozyme's reaction pathway with an N3-protonated C75H+ in the active site, which acts as the general acid, and a partially hydrated Mg2+ ion with one deprotonated, inner-shell coordinated water molecule that acts as the general base. We followed eight reaction paths with distinct position and coordination of the catalytically important active site Mg2+ ion. For six of them, we observed feasible activation barriers ranging from 14.2 to 21.9 kcal/mol, indicating that the specific position of the Mg2+ ion in the active site is predicted to strongly affect the kinetics of self-cleavage. The deprotonation of the U-1(2′-OH) nucleophile and the nucleophilic attack of the resulting U-1(2′-O−) on the scissile phosphodiester are found to be separate steps, as deprotonation precedes the nucleophilic attack. This sequential mechanism of the HDV ribozyme differs from the concerted nucleophilic activation and attack suggested for the hairpin ribozyme. We estimated the pKa of the U-1(2′-OH) group to range from 8.8 to 11.2, suggesting that the pKa is lowered by several units from that of a free ribose, comparable to and most likely smaller than the pKa of the solvated active site Mg2+ ion. Our results thus support the notion that the structure of the HDV ribozyme, and particularly the positioning of the active site Mg2+ ion, facilitates deprotonation and activation of the 2′-OH nucleophile. PMID:25412464

  4. Regulation of zygotic genome activation and DNA damage checkpoint acquisition at the mid-blastula transition

    PubMed Central

    Zhang, Maomao; Kothari, Priyanka; Mullins, Mary; Lampson, Michael A.

    2014-01-01

    Following fertilization, oviparous embryos undergo rapid, mostly transcriptionally silent cleavage divisions until the mid-blastula transition (MBT), when large-scale developmental changes occur, including zygotic genome activation (ZGA) and cell cycle remodeling, via lengthening and checkpoint acquisition. Despite their concomitant appearance, whether these changes are co-regulated is unclear. Three models have been proposed to account for the timing of (ZGA). One model implicates a threshold nuclear to cytoplasmic (N:C) ratio, another stresses the importance cell cycle elongation, while the third model invokes a timer mechanism. We show that precocious Chk1 activity in pre-MBT zebrafish embryos elongates cleavage cycles, thereby slowing the increase in the N:C ratio. We find that cell cycle elongation does not lead to transcriptional activation. Rather, ZGA slows in parallel with the N:C ratio. We show further that the DNA damage checkpoint program is maternally supplied and independent of ZGA. Although pre-MBT embryos detect damage and activate Chk2 after induction of DNA double-strand breaks, the Chk1 arm of the DNA damage response is not activated, and the checkpoint is nonfunctional. Our results are consistent with the N:C ratio model for ZGA. Moreover, the ability of precocious Chk1 activity to delay pre-MBT cell cycles indicate that lack of Chk1 activity limits checkpoint function during cleavage cycles. We propose that Chk1 gain-of-function at the MBT underlies cell cycle remodeling, whereas ZGA is regulated independently by the N:C ratio. PMID:25558827

  5. Nuclease activity of Saccharomyces cerevisiae Dna2 inhibits its potent DNA helicase activity

    PubMed Central

    Levikova, Maryna; Klaue, Daniel; Seidel, Ralf; Cejka, Petr

    2013-01-01

    Dna2 is a nuclease-helicase involved in several key pathways of eukaryotic DNA metabolism. The potent nuclease activity of Saccharomyces cerevisiae Dna2 was reported to be required for all its in vivo functions tested to date. In contrast, its helicase activity was shown to be weak, and its inactivation affected only a subset of Dna2 functions. We describe here a complex interplay of the two enzymatic activities. We show that the nuclease of Dna2 inhibits its helicase by cleaving 5′ flaps that are required by the helicase domain for loading onto its substrate. Mutational inactivation of Dna2 nuclease unleashes unexpectedly vigorous DNA unwinding activity, comparable with that of the most potent eukaryotic helicases. Thus, the ssDNA-specific nuclease activity of Dna2 limits and controls the enzyme's capacity to unwind dsDNA. We postulate that regulation of this interplay could modulate the biochemical properties of Dna2 and thus license it to carry out its distinct cellular functions. PMID:23671118

  6. Acetylation of Werner syndrome protein (WRN): relationships with DNA damage, DNA replication and DNA metabolic activities

    PubMed Central

    Lozada, Enerlyn; Yi, Jingjie; Luo, Jianyuan; Orren, David K.

    2014-01-01

    Loss of WRN function causes Werner Syndrome, characterized by increased genomic instability, elevated cancer susceptibility and premature aging. Although WRN is subject to acetylation, phosphorylation and sumoylation, the impact of these modifications on WRN’s DNA metabolic function remains unclear. Here, we examined in further depth the relationship between WRN acetylation and its role in DNA metabolism, particularly in response to induced DNA damage. Our results demonstrate that endogenous WRN is acetylated somewhat under unperturbed conditions. However, levels of acetylated WRN significantly increase after treatment with certain DNA damaging agents or the replication inhibitor hydroxyurea. Use of DNA repair-deficient cells or repair pathway inhibitors further increase levels of acetylated WRN, indicating that induced DNA lesions and their persistence are at least partly responsible for increased acetylation. Notably, acetylation of WRN correlates with inhibition of DNA synthesis, suggesting that replication blockage might underlie this effect. Moreover, WRN acetylation modulates its affinity for and activity on certain DNA structures, in a manner that may enhance its relative specificity for physiological substrates. Our results also show that acetylation and deacetylation of endogenous WRN is a dynamic process, with sirtuins and other histone deacetylases contributing to WRN deacetylation. These findings advance our understanding of the dynamics of WRN acetylation under unperturbed conditions and following DNA damage induction, linking this modification not only to DNA damage persistence but also potentially to replication stalling caused by specific DNA lesions. Our results are consistent with proposed metabolic roles for WRN and genomic instability phenotypes associated with WRN deficiency. PMID:24965941

  7. Architecture of the bacteriophage T4 activator MotA/promoter DNA interaction during sigma appropriation.

    PubMed

    Hsieh, Meng-Lun; James, Tamara D; Knipling, Leslie; Waddell, M Brett; White, Stephen; Hinton, Deborah M

    2013-09-20

    Gene expression can be regulated through factors that direct RNA polymerase to the correct promoter sequence at the correct time. Bacteriophage T4 controls its development in this way using phage proteins that interact with host RNA polymerase. Using a process called σ appropriation, the T4 co-activator AsiA structurally remodels the σ(70) subunit of host RNA polymerase, while a T4 activator, MotA, engages the C terminus of σ(70) and binds to a DNA promoter element, the MotA box. Structures for the N-terminal (NTD) and C-terminal (CTD) domains of MotA are available, but no structure exists for MotA with or without DNA. We report the first molecular map of the MotA/DNA interaction within the σ-appropriated complex, which we obtained by using the cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). We conjugated surface-exposed, single cysteines in MotA with FeBABE and performed cleavage reactions in the context of stable transcription complexes. The DNA cleavage sites were analyzed using ICM Molsoft software and three-dimensional physical models of MotA(NTD), MotA(CTD), and the DNA to investigate shape complementarity between the protein and the DNA and to position MotA on the DNA. We found that the unusual "double wing" motif present within MotA(CTD) resides in the major groove of the MotA box. In addition, we have used surface plasmon resonance to show that MotA alone is in a very dynamic equilibrium with the MotA element. Our results demonstrate the utility of fine resolution FeBABE mapping to determine the architecture of protein-DNA complexes that have been recalcitrant to traditional structure analyses. PMID:23902794

  8. Cleavage of Type I Collagen by Fibroblast Activation Protein-α Enhances Class A Scavenger Receptor Mediated Macrophage Adhesion

    PubMed Central

    Mazur, Anna; Holthoff, Emily; Vadali, Shanthi; Kelly, Thomas; Post, Steven R.

    2016-01-01

    Pathophysiological conditions such as fibrosis, inflammation, and tumor progression are associated with modification of the extracellular matrix (ECM). These modifications create ligands that differentially interact with cells to promote responses that drive pathological processes. Within the tumor stroma, fibroblasts are activated and increase the expression of type I collagen. In addition, activated fibroblasts specifically express fibroblast activation protein-α (FAP), a post-prolyl peptidase. Although FAP reportedly cleaves type I collagen and contributes to tumor progression, the specific pathophysiologic role of FAP is not clear. In this study, the possibility that FAP-mediated cleavage of type I collagen modulates macrophage interaction with collagen was examined using macrophage adhesion assays. Our results demonstrate that FAP selectively cleaves type I collagen resulting in increased macrophage adhesion. Increased macrophage adhesion to FAP-cleaved collagen was not affected by inhibiting integrin-mediated interactions, but was abolished in macrophages lacking the class A scavenger receptor (SR-A/CD204). Further, SR-A expressing macrophages localize with activated fibroblasts in breast tumors of MMTV-PyMT mice. Together, these results demonstrate that FAP-cleaved collagen is a substrate for SR-A-dependent macrophage adhesion, and suggest that by modifying the ECM, FAP plays a novel role in mediating communication between activated fibroblasts and macrophages. PMID:26934296

  9. Fuzzy logic sensing of G-quadruplex DNA and its cleavage reagents based on reduced graphene oxide.

    PubMed

    Huang, Wei Tao; Zhang, Jian Rong; Xie, Wan Yi; Shi, Yan; Luo, Hong Qun; Li, Nian Bing

    2014-07-15

    Herein, by combining the merits of nanotechnology and fuzzy logic theory, we develop a simple, label-free, and general strategy based on an organic dye-graphene hybrid system for fluorescence intelligent sensing of G-quadruplexes (G4) formation, hydroxyl radical (HO∙), and Fe(2+) in vitro. By exploiting acridine orange (AO) dyes-graphene as a nanofilter and nanoswitch and the ability of graphene to interact with DNA with different structures, our approach can efficiently distinguish, quantitatively detect target analytes. In vitro assays with G4DNA demonstrated increases in fluorescence intensity of the AO-rGO system with a linear range of 16-338 nM and a detection limit as low as 2.0 nM. The requenched fluorescence of the G4TBA-AO-rGO system has a non-linear response to Fenton reagent. But this requenching reduces the fluorescence intensity in a manner proportional to the logarithm to the base 10 of the concentration of Fenton reagent in the range of 0.1-100 μM and 100-2000 μM, respectively. Furthermore, we develop a novel and intelligent sensing method based on fuzzy logic which mimics human reasoning, solves complex and non-linear problems, and transforms the numerical output into the language description output for potential application in biochemical systems, environmental monitoring systems, and molecular-level fuzzy logic computing system. PMID:24561526

  10. A dual amplification fluorescent strategy for sensitive detection of DNA methyltransferase activity based on strand displacement amplification and DNAzyme amplification.

    PubMed

    Cui, Wanling; Wang, Lei; Jiang, Wei

    2016-03-15

    DNA methyltransferase (MTase) plays a critical role in many biological processes and has been regarded as a predictive cancer biomarker and a therapeutic target in cancer treatment. Sensitive detection of DNA MTase activity is essential for early cancer diagnosis and therapeutics. Here, we developed a dual amplification fluorescent strategy for sensitive detection of DNA MTase activity based on strand displacement amplification (SDA) and DNAzyme amplification. A trifunctional double-stranded DNA (dsDNA) probe was designed including a methylation site for DNA MTase recognition, a complementary sequence of 8-17 DNAzyme for synthesizing DNAzyme, and a nicking site for nicking enzyme cleavage. Firstly, the trifunctional dsDNA probe was methylated by DNA MTase to form the methylated dsDNA. Subsequently, HpaII restriction endonuclease specifically cleaved the residue of unmethylated dsDNA. Next, under the action of polymerase and nicking enzyme, the methylared dsDNA initiated SDA, releasing numbers of 8-17 DNAzymes. Finally, the released 8-17 DNAzymes triggered DNAzyme amplification reaction to induce a significant fluorescence enhancement. This strategy could detect DNA MTase activity as low as 0.0082U/mL. Additionally, the strategy was successfully applied for evaluating the inhibitions of DNA MTase using two anticancer drugs, 5-azacytidine and 5-aza-2'-deoxycytidine. The results indicate the proposed strategy has a potential application in early cancer diagnosis and therapeutics. PMID:26492469

  11. Synthesis, characterization, DNA interaction, antioxidant and anticancer activity of new ruthenium(II) complexes of thiosemicarbazone/semicarbazone bearing 9,10-phenanthrenequinone.

    PubMed

    Anitha, Panneerselvam; Chitrapriya, Nataraj; Jang, Yoon Jung; Viswanathamurthi, Periasamy

    2013-12-01

    A new series of octahedral ruthenium(II) complexes supported by tridentate ligands derived from phenanthrenequinone and derivatives of thiosemicarbazide/semicarbazide and other co-ligands have been synthesized and characterized. DNA binding experiments indicated that ruthenium(II) complexes can interact with DNA through non-intercalation and the apparent binding constant value (Kb) of [RuCl(CO)(PPh₃)(L₃)] (3) at room temperature was calculated to be 2.27 × 10(3)M(-1). The DNA cleavage studies showed that the complexes have better cleavage of pBR 322 DNA. Antioxidative activity proved that the complexes have significant radical scavenging activity against free radicals. Cytotoxic activities showed that the ruthenium(II) complexes exhibited more effective cytotoxic activity against selected cancer cells. PMID:24144689

  12. Modulation of the DNA scanning activity of the Micrococcus luteus UV endonuclease

    SciTech Connect

    Hamilton, R.W.; Lloyd, R.S. )

    1989-10-15

    Micrococcus luteus UV endonuclease incises DNA at the sites of ultraviolet (UV) light-induced pyrimidine dimers. The mechanism of incision has been previously shown to be a glycosylic bond cleavage at the 5'-pyrimidine of the dimer followed by an apyrimidine endonuclease activity which cleaves the phosphodiester backbone between the pyrimidines. The process by which M. luteus UV endonuclease locates pyrimidine dimers within a population of UV-irradiated plasmids was shown to occur, in vitro, by a processive or sliding mechanism on non-target DNA as opposed to a distributive or random hit mechanism. Form I plasmid DNA containing 25 dimers per molecule was incubated with M. luteus UV endonuclease in time course reactions. The three topological forms of plasmid DNA generated were analyzed by agarose gel electrophoresis. When the enzyme encounters a pyrimidine dimer, it is significantly more likely to make only the glycosylase cleavage as opposed to making both the glycosylic and phosphodiester bond cleavages. Thus, plasmids are accumulated with many alkaline-labile sites relative to single-stranded breaks. In addition, reactions were performed at both pH 8.0 and pH 6.0, in the absence of NaCl, as well as 25,100, and 250 mM NaCl. The efficiency of the DNA scanning reaction was shown to be dependent on both the ionic strength and pH of the reaction. At low ionic strengths, the reaction was shown to proceed by a processive mechanism and shifted to a distributive mechanism as the ionic strength of the reaction increased. Processivity at pH 8.0 is shown to be more sensitive to increases in ionic strength than reactions performed at pH 6.0.

  13. Rh(III)-Catalyzed Synthesis of N-Unprotected Indoles from Imidamides and Diazo Ketoesters via C-H Activation and C-C/C-N Bond Cleavage.

    PubMed

    Qi, Zisong; Yu, Songjie; Li, Xingwei

    2016-02-19

    The synthesis of N-unprotected indoles has been realized via Rh(III)-catalyzed C-H activation/annulation of imidamides with α-diazo β-ketoesters. The reaction occurs with the release of an amide coproduct, which originates from both the imidamide and the diazo as a result of C═N cleavage of the imidamide and C-C(acyl) cleavage of the diazo. A rhodacyclic intermediate has been isolated and a plausible mechanism has been proposed. PMID:26824751

  14. A novel method for detecting apoptosis shows that hepatocytes undergo a time dependent increase in DNA cleavage and chromatin condensation which is augmented after TGF-beta 1 treatment.

    PubMed

    Cain, K; Inayat-Hussain, S H; Couet, C; Qin, H M; Oberhammer, F A

    1996-04-01

    This study describes a new method for quantitating apoptosis in hepatocyte monolayers in which nuclei were isolated from the cells and DNA strand breaks detected by in situ end-labeling and flow cytometry. Most (97%) nuclei from untreated hepatocytes had low end-labelling and were derived from non-apoptotic cells. Approximately 2-3% of the nuclei had high end-labelling and originated from apoptotic hepatocytes. The numbers of these nuclei increased linearly from 3 to 85% between 0 and 48 h after treatment with transforming growth factor-beta 1 (TGF-beta 1). However, a morphological assessment of apoptosis with Hoechst H33258 showed that the proportion of apoptotic nuclei plateaued at 18-19% between 24 and 48 h after TGF-beta 1 treatment. Thus, the in situ end-labeling technique also detected DNA cleavage in nuclei which did not have an obvious apoptotic morphology. Confocal microscopy of low and high end-labelled nuclei which had been separated by fluorescent cell sorting showed that nuclei with high levels of end-labeling exhibited a wide diversity of morphologies. These included nuclei with little or no chromatin condensation and nuclei with characteristic apoptotic morphology. In addition, nuclei from untreated hepatocytes contained low levels of DNA cleavage, which were localized in areas of condensed chromatin and increased according to the time in culture. Thus, hepatocytes undergo a progressive and cumulative process of DNA cleavage/chromatin condensation which is markedly enhanced by TGF-beta 1. PMID:8900474

  15. Development of Novel DNA Cleavage Systems Based on Copper Complexes. Synthesis and Characterisation of Cu(II) Complexes of Hydroxyflavones

    PubMed Central

    el Amrani, F. Ben-Allal; Perelló, L.; Torres, L.

    2000-01-01

    Copper(II) complexes of several hydroxyflavones were prepared and characterised through their physico-chemical properties. The nuclease activity of three synthesised complexes is reported. These copper(II) complexes present more nuclease activity than the ligands and the copper(II) ion. PMID:18475969

  16. Proteolytic regulation of epithelial sodium channels by urokinase plasminogen activator: cutting edge and cleavage sites.

    PubMed

    Ji, Hong-Long; Zhao, Runzhen; Komissarov, Andrey A; Chang, Yongchang; Liu, Yongfeng; Matthay, Michael A

    2015-02-27

    Plasminogen activator inhibitor 1 (PAI-1) level is extremely elevated in the edematous fluid of acutely injured lungs and pleurae. Elevated PAI-1 specifically inactivates pulmonary urokinase-type (uPA) and tissue-type plasminogen activators (tPA). We hypothesized that plasminogen activation and fibrinolysis may alter epithelial sodium channel (ENaC) activity, a key player in clearing edematous fluid. Two-chain urokinase (tcuPA) has been found to strongly stimulate heterologous human αβγ ENaC activity in a dose- and time-dependent manner. This activity of tcuPA was completely ablated by PAI-1. Furthermore, a mutation (S195A) of the active site of the enzyme also prevented ENaC activation. By comparison, three truncation mutants of the amino-terminal fragment of tcuPA still activated ENaC. uPA enzymatic activity was positively correlated with ENaC current amplitude prior to reaching the maximal level. In sharp contrast to uPA, neither single-chain tPA nor derivatives, including two-chain tPA and tenecteplase, affected ENaC activity. Furthermore, γ but not α subunit of ENaC was proteolytically cleaved at ((177)GR↓KR(180)) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of γ ENaC, incremental increase in opening rate, and activation of closed (electrically "silent") channels. This study for the first time demonstrates multifaceted mechanisms for uPA-mediated up-regulation of ENaC, which form the cellular and molecular rationale for the beneficial effects of urokinase in mitigating mortal pulmonary edema and pleural effusions. PMID:25555911

  17. Mechanistic insight into conjugated N-N bond cleavage by Rh(III)-catalyzed redox-neutral C-H activation of pyrazolones.

    PubMed

    Wu, Weirong; Liu, Yuxia; Bi, Siwei

    2015-08-14

    Density functional theory (DFT) calculations have been performed to investigate the detailed mechanism of Rh(III)-catalyzed redox-neutral C-H activation of pyrazolones with PhC≡CPh. It is found that (1) the methylene C-H activation is prior to the phenyl C-H activation, (2) the N-N bond cleavage is realized via Rh(III) → Rh(I) → Rh(III) rather than via Rh(III) → Rh(V) → Rh(III). The zwitterionic Rh(I) complex is identified to be a key intermediate in promoting the N-N bond cleavage. (3) Different from the Rh(III)-catalyzed hydrazine-directed C-H activation for indole synthesis, the rate-determining step of the reaction studied in this work is the Rh(III) → Rh(I) → Rh(III) process resulting in the N-N bond cleavage rather than the alkyne insertion step. The present theoretical study provides new insight into the mechanism of the conjugated N-N bond cleavage. PMID:26138233

  18. Asymmetric Cleavage of B-Carotene Yields a Transcriptional Repressor of Retinoid X Receptor and Peroxisome Proliferator-Activated Receptor Responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    B-Carotene and its metabolites exert a broad range of effects, in part by regulating transcriptional responses through specific nuclear receptor activation. Symmetric cleavage of B-carotene can yield 9-cis retinoic acid (9-cisRA), the natural ligand for the nuclear receptor RXR, the obligate heterod...

  19. Invasive cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

    1999-01-01

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

  20. Invasive cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

    2002-01-01

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

  1. Activity and Regulation of Archaeal DNA Alkyltransferase

    PubMed Central

    Perugino, Giuseppe; Vettone, Antonella; Illiano, Giuseppina; Valenti, Anna; Ferrara, Maria C.; Rossi, Mosè; Ciaramella, Maria

    2012-01-01

    Agents that form methylation adducts in DNA are highly mutagenic and carcinogenic, and organisms have evolved specialized cellular pathways devoted to their repair, including DNA alkyltransferases. These are proteins conserved in eucarya, bacteria and archaea, acting by a unique reaction mechanism, which leads to direct repair of DNA alkylation damage and irreversible protein alkylation. The alkylated form of DNA alkyltransferases is inactive, and in eukaryotes, it is rapidly directed to degradation. We report here in vitro and in vivo studies on the DNA alkyltransferase from the thermophilic archaeon Sulfolobus solfataricus (SsOGT). The development of a novel, simple, and sensitive fluorescence-based assay allowed a careful characterization of the SsOGT biochemical and DNA binding activities. In addition, transcriptional and post-translational regulation of SsOGT by DNA damage was studied. We show that although the gene transcription is induced by alkylating agent treatment, the protein is degraded in vivo by an alkylation-dependent mechanism. These experiments suggest a striking conservation, from archaea to humans, of this important pathway safeguarding genome stability. PMID:22167184

  2. Synthesis, crystal structure, DNA interaction and anticancer activity of tridentate copper(II) complexes.

    PubMed

    Li, Guan-Ying; Du, Ke-Jie; Wang, Jin-Quan; Liang, Jie-Wen; Kou, Jun-Feng; Hou, Xiao-Juan; Ji, Liang-Nian; Chao, Hui

    2013-02-01

    Three new tridentate copper(II) complexes [Cu(dthp)Cl(2)] (1) (dthp=2,6-di(thiazol-2-yl)pyridine), [Cu(dmtp)Cl(2)] (2) (dmtp=2,6-di(5-methyl-4H-1,2,4-triazol-3-yl)pyridine) and [Cu(dtp)Cl(2)] (3) (dtp=2,6-di(4H-1,2,4-triazol-3-yl)pyridine) have been synthesized and characterized. Crystal structure of complex 1 shows that the complex existed as distorted square pyramid with five co-ordination sites occupied by the tridentate ligand and the two chlorine anions. Ethidium bromide displacement assay, viscosity measurements, circular dichroism studies and cyclic voltammetric experiments suggested that these complexes bound to DNA via an intercalative mode. Three Cu(II) complexes were found to efficiently cleave DNA in the presence of sodium ascorbate, and singlet oxygen ((1)O(2)) and hydrogen peroxide were proved to contribute to the DNA cleavage process. They exhibited anticancer activity against HeLa, Hep-G2 and BEL-7402 cell lines. Nuclear chromatin cleavage has also been observed with AO/EB staining assay and the alkaline single-cell gel electrophoresis (comet assay). The results demonstrated that three Cu(II) complexes cause DNA damage that can induce the apoptosis of BEL-7402 cells. PMID:23186647

  3. Predictions of Cleavability of Calpain Proteolysis by Quantitative Structure-Activity Relationship Analysis Using Newly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array*

    PubMed Central

    Shinkai-Ouchi, Fumiko; Koyama, Suguru; Ono, Yasuko; Hata, Shoji; Ojima, Koichi; Shindo, Mayumi; duVerle, David; Ueno, Mika; Kitamura, Fujiko; Doi, Naoko; Takigawa, Ichigaku; Mamitsuka, Hiroshi; Sorimachi, Hiroyuki

    2016-01-01

    Calpains are intracellular Ca2+-regulated cysteine proteases that are essential for various cellular functions. Mammalian conventional calpains (calpain-1 and calpain-2) modulate the structure and function of their substrates by limited proteolysis. Thus, it is critically important to determine the site(s) in proteins at which calpains cleave. However, the calpains' substrate specificity remains unclear, because the amino acid (aa) sequences around their cleavage sites are very diverse. To clarify calpains' substrate specificities, 84 20-mer oligopeptides, corresponding to P10-P10′ of reported cleavage site sequences, were proteolyzed by calpains, and the catalytic efficiencies (kcat/Km) were globally determined by LC/MS. This analysis revealed 483 cleavage site sequences, including 360 novel ones. The kcat/Kms for 119 sites ranged from 12.5–1,710 M−1s−1. Although most sites were cleaved by both calpain-1 and −2 with a similar kcat/Km, sequence comparisons revealed distinct aa preferences at P9-P7/P2/P5′. The aa compositions of the novel sites were not statistically different from those of previously reported sites as a whole, suggesting calpains have a strict implicit rule for sequence specificity, and that the limited proteolysis of intact substrates is because of substrates' higher-order structures. Cleavage position frequencies indicated that longer sequences N-terminal to the cleavage site (P-sites) were preferred for proteolysis over C-terminal (P′-sites). Quantitative structure-activity relationship (QSAR) analyses using partial least-squares regression and >1,300 aa descriptors achieved kcat/Km prediction with r = 0.834, and binary-QSAR modeling attained an 87.5% positive prediction value for 132 reported calpain cleavage sites independent of our model construction. These results outperformed previous calpain cleavage predictors, and revealed the importance of the P2, P3′, and P4′ sites, and P1-P2 cooperativity. Furthermore, using our

  4. Predictions of Cleavability of Calpain Proteolysis by Quantitative Structure-Activity Relationship Analysis Using Newly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array.

    PubMed

    Shinkai-Ouchi, Fumiko; Koyama, Suguru; Ono, Yasuko; Hata, Shoji; Ojima, Koichi; Shindo, Mayumi; duVerle, David; Ueno, Mika; Kitamura, Fujiko; Doi, Naoko; Takigawa, Ichigaku; Mamitsuka, Hiroshi; Sorimachi, Hiroyuki

    2016-04-01

    Calpains are intracellular Ca(2+)-regulated cysteine proteases that are essential for various cellular functions. Mammalian conventional calpains (calpain-1 and calpain-2) modulate the structure and function of their substrates by limited proteolysis. Thus, it is critically important to determine the site(s) in proteins at which calpains cleave. However, the calpains' substrate specificity remains unclear, because the amino acid (aa) sequences around their cleavage sites are very diverse. To clarify calpains' substrate specificities, 84 20-mer oligopeptides, corresponding to P10-P10' of reported cleavage site sequences, were proteolyzed by calpains, and the catalytic efficiencies (kcat/Km) were globally determined by LC/MS. This analysis revealed 483 cleavage site sequences, including 360 novel ones. Thekcat/Kms for 119 sites ranged from 12.5-1,710 M(-1)s(-1) Although most sites were cleaved by both calpain-1 and -2 with a similarkcat/Km, sequence comparisons revealed distinct aa preferences at P9-P7/P2/P5'. The aa compositions of the novel sites were not statistically different from those of previously reported sites as a whole, suggesting calpains have a strict implicit rule for sequence specificity, and that the limited proteolysis of intact substrates is because of substrates' higher-order structures. Cleavage position frequencies indicated that longer sequences N-terminal to the cleavage site (P-sites) were preferred for proteolysis over C-terminal (P'-sites). Quantitative structure-activity relationship (QSAR) analyses using partial least-squares regression and >1,300 aa descriptors achievedkcat/Kmprediction withr= 0.834, and binary-QSAR modeling attained an 87.5% positive prediction value for 132 reported calpain cleavage sites independent of our model construction. These results outperformed previous calpain cleavage predictors, and revealed the importance of the P2, P3', and P4' sites, and P1-P2 cooperativity. Furthermore, using our binary-QSAR model

  5. Next-Generation Sequencing of Apoptotic DNA Breakpoints Reveals Association with Actively Transcribed Genes and Gene Translocations

    PubMed Central

    Fullwood, Melissa J.; Lee, Joanne; Lin, Lifang; Li, Guoliang; Huss, Mikael; Ng, Patrick; Sung, Wing-Kin; Shenolikar, Shirish

    2011-01-01

    DNA fragmentation is a well-recognized hallmark of apoptosis. However, the precise DNA sequences cleaved during apoptosis triggered by distinct mechanisms remain unclear. We used next-generation sequencing of DNA fragments generated in Actinomycin D-treated human HL-60 leukemic cells to generate a high-throughput, global map of apoptotic DNA breakpoints. These data highlighted that DNA breaks are non-random and show a significant association with active genes and open chromatin regions. We noted that transcription factor binding sites were also enriched within a fraction of the apoptotic breakpoints. Interestingly, extensive apoptotic cleavage was noted within genes that are frequently translocated in human cancers. We speculate that the non-random fragmentation of DNA during apoptosis may contribute to gene translocations and the development of human cancers. PMID:22087219

  6. DNA interaction and cytotoxic activities of square planar platinum(II) complexes with N, S-donor ligands

    NASA Astrophysics Data System (ADS)

    Patel, Mohan N.; Patel, Chintan R.; Joshi, Hardik N.; Thakor, Khyati P.

    2014-06-01

    The platinum(II) complexes with N, S-donor ligands have been synthesized and characterized by physicochemical methods viz. elemental, electronic, FT-IR, 1H NMR and LC-MS spectra. The binding mode and potency of the complexes with HS DNA (Herring Sperm) have been examined by absorption titration and viscosity measurement studies. The results revealed that complexes bind to HS DNA via covalent mode with the intrinsic binding constant (Kb) in the range 1.37-7.76 × 105 M-1. Decrease in the relative viscosity of HS DNA also supports the covalent mode of binding. The DNA cleavage activity of synthesized complexes has been carried out by gel electrophoresis experiment using supercoiled form of pUC19 DNA; showing the unwinding of the negatively charged supercoiled DNA. Brine shrimp (Artemia Cysts) lethality bioassay technique has been applied for the determination of toxic property of synthesized complexes in terms of μM.

  7. CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage

    PubMed Central

    Yakhnin, Alexander V.; Baker, Carol S.; Vakulskas, Christopher A.; Yakhnin, Helen; Berezin, Igor; Romeo, Tony; Babitzke, Paul

    2013-01-01

    Summary Csr is a conserved global regulatory system that controls expression of several hundred Escherichia coli genes. CsrA protein represses translation of numerous genes by binding to mRNA and inhibiting ribosome access. CsrA also activates gene expression, although an activation mechanism has not been reported. CsrA activates flhDC expression, encoding the master regulator of flagellum biosynthesis and chemotaxis, by stabilizing the mRNA. Computer modeling, gel mobility shift, and footprint analyses identified two CsrA binding sites extending from positions 1–12 (BS1) and 44–55 (BS2) of the 198-nt flhDC leader transcript. flhD'-'lacZ expression was reduced by mutations in csrA and/or the CsrA binding sites. The position of BS1 suggested that bound CsrA might inhibit 5' end-dependent RNase E cleavage of flhDC mRNA. Consistent with this hypothesis, CsrA protected flhDC leader RNA from RNase E cleavage in vitro and protection depended on BS1 and BS2. Primer extension studies identified flhDC decay intermediates in vivo that correspond to in vitro RNase E cleavage sites. Deletion of these RNase E cleavage sites resulted in increased flhD'-'lacZ expression. Data from mRNA decay studies and quantitative primer extension assays support a model in which bound CsrA activates flhDC expression by inhibiting the 5' end-dependent RNase E cleavage pathway. PMID:23305111

  8. CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage.

    PubMed

    Yakhnin, Alexander V; Baker, Carol S; Vakulskas, Christopher A; Yakhnin, Helen; Berezin, Igor; Romeo, Tony; Babitzke, Paul

    2013-02-01

    Csr is a conserved global regulatory system that controls expression of several hundred Escherichia coli genes. CsrA protein represses translation of numerous genes by binding to mRNA and inhibiting ribosome access. CsrA also activates gene expression, although an activation mechanism has not been reported. CsrA activates flhDC expression, encoding the master regulator of flagellum biosynthesis and chemotaxis, by stabilizing the mRNA. Computer modelling, gel mobility shift and footprint analyses identified two CsrA binding sites extending from positions 1-12 (BS1) and 44-55 (BS2) of the 198 nt flhDC leader transcript. flhD'-'lacZ expression was reduced by mutations in csrA and/or the CsrA binding sites. The position of BS1 suggested that bound CsrA might inhibit 5' end-dependent RNase E cleavage of flhDC mRNA. Consistent with this hypothesis, CsrA protected flhDC leader RNA from RNase E cleavage in vitro and protection depended on BS1 and BS2. Primer extension studies identified flhDC decay intermediates in vivo that correspond to in vitro RNase E cleavage sites. Deletion of these RNase E cleavage sites resulted in increased flhD'-'lacZ expression. Data from mRNA decay studies and quantitative primer extension assays support a model in which bound CsrA activates flhDC expression by inhibiting the 5' end-dependent RNase E cleavage pathway. PMID:23305111

  9. In vitro peptide cleavage assay for detection of Botulinum Neurotoxin-A activity in food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The gold standard assay for measuring the activity and typing of Clostridium botulinum neurotoxins is the mouse bioassay. The mouse bioassay is sensitive, robust and does not require specialized equipment. However, the mouse bioassay is slow, not practical for many settings and results in the death ...

  10. Water-soluble DNA minor groove binders as potential chemotherapeutic agents: synthesis, characterization, DNA binding and cleavage, antioxidation, cytotoxicity and HSA interactions.

    PubMed

    Fu, Xia-Bing; Liu, Dan-Dan; Lin, Yuan; Hu, Wei; Mao, Zong-Wan; Le, Xue-Yi

    2014-06-21

    Two new water-soluble copper(ii)-dipeptide complexes: [Cu(glygly)(PyTA)]ClO4·1.5H2O (1) and [Cu(glygly)(PzTA)]ClO4·1.5H2O (2) (glygly = glycylglycine anion, PyTA = 2,4-diamino-6-(2'-pyridyl)-1,3,5-triazine and PzTA = 2,4-diamino-6-(2'-pyrazino)-1,3,5-triazine), utilizing two interrelated DNA base-like ligands (PyTA and PzTA), have been synthesized and characterized. The structure elucidation for 1 performed by single crystal X-ray diffraction showed a one dimensional chain conformation in which the central copper ions arrange in a five-coordinate distorted square-pyramidal geometry. Spectroscopic titration, viscosity and electrophoresis measurements revealed that the complexes bound to DNA via an outside groove binding mode, and cleaved pBR322 DNA efficiently in the presence of ascorbate, probably via an oxidative mechanism with the involvement of ˙OH and ˙O2(-). Notably, the complexes exhibited considerable in vitro cytotoxicity against four human carcinoma cell lines (HepG2, HeLa, A549 and U87) with IC50 values ranging from 41.68 to 159.17 μM, in addition to their excellent SOD mimics (IC50 ~ 0.091 and 0.114 μM). Besides, multispectroscopic evidence suggested their HSA-binding at the cavity containing Trp-214 in subdomain IIA with moderate affinity, mainly via hydrophobic interaction. Further, the molecular docking technique utilized for ascertaining the mechanism and mode of action towards DNA and HSA theoretically verified the experimental results. PMID:24770345

  11. The 'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring '2A-like' sequences.

    PubMed

    Donnelly, M L; Hughes, L E; Luke, G; Mendoza, H; ten Dam, E; Gani, D; Ryan, M D

    2001-05-01

    The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated by their 2A proteins 'cleaving' at their own C termini. We have analysed this activity using artificial reporter polyprotein systems comprising green fluorescent protein (GFP) linked via foot-and-mouth disease virus (FMDV) 2A to beta-glucuronidase (GUS) -- forming a single, long, open reading frame. Analysis of the distribution of radiolabel showed a high proportion of the in vitro translation products (approximately 90%) were in the form of the 'cleavage' products GUS and [GFP2A]. Alternative models have been proposed to account for the 'cleavage' activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect. To investigate the mechanism of this cleavage event constructs encoding site-directed mutant and naturally occurring '2A-like' sequences were used to program in vitro translation systems and the gel profiles analysed. Analysis of site-directed mutant 2A sequences showed that 'cleavage' occurred in constructs in which all the candidate nucleophilic residues were substituted -- with the exception of aspartate-12. This residue is not, however, conserved amongst all functional '2A-like' sequences. '2A-like' sequences were identified within insect virus polyproteins, the NS34 protein of type C rotaviruses, repeated sequences in Trypanosoma spp. and a eubacterial alpha-glucosiduronasesequence(Thermatoga maritima aguA). All of the 2A-like sequences analysed were active (to various extents), other than the eubacterial alpha-glucosiduronase 2A-like sequence. This method of control of protein biogenesis may well not, therefore, be confined to members of the PICORNAVIRIDAE: Taken together, these data provide additional evidence that neither FMDV 2A nor '2A-like' sequences are autoproteolytic elements. PMID:11297677

  12. TspanC8 tetraspanins differentially regulate the cleavage of ADAM10 substrates, Notch activation and ADAM10 membrane compartmentalization.

    PubMed

    Jouannet, Stéphanie; Saint-Pol, Julien; Fernandez, Laurent; Nguyen, Viet; Charrin, Stéphanie; Boucheix, Claude; Brou, Christel; Milhiet, Pierre-Emmanuel; Rubinstein, Eric

    2016-05-01

    The metalloprotease ADAM10 mediates the shedding of the ectodomain of various cell membrane proteins, including APP, the precursor of the amyloid peptide Aβ, and Notch receptors following ligand binding. ADAM10 associates with the members of an evolutionary conserved subgroup of tetraspanins, referred to as TspanC8, which regulate its exit from the endoplasmic reticulum. Here we show that 4 of these TspanC8 (Tspan5, Tspan14, Tspan15 and Tspan33) which positively regulate ADAM10 surface expression levels differentially impact ADAM10-dependent Notch activation and the cleavage of several ADAM10 substrates, including APP, N-cadherin and CD44. Sucrose gradient fractionation, single molecule tracking and quantitative mass-spectrometry analysis of the repertoire of molecules co-immunoprecipitated with Tspan5, Tspan15 and ADAM10 show that these two tetraspanins differentially regulate ADAM10 membrane compartmentalization. These data represent a unique example where several tetraspanins differentially regulate the function of a common partner protein through a distinct membrane compartmentalization. PMID:26686862

  13. Duality of polynucleotide substrates for Phi29 DNA polymerase: 3′→5′ RNase activity of the enzyme

    PubMed Central

    Lagunavicius, Arunas; Kiveryte, Zivile; Zimbaite-Ruskuliene, Vilma; Radzvilavicius, Tomas; Janulaitis, Arvydas

    2008-01-01

    Phi29 DNA polymerase is a small DNA-dependent DNA polymerase that belongs to eukaryotic B-type DNA polymerases. Despite the small size, the polymerase is a multifunctional proofreading-proficient enzyme. It catalyzes two synthetic reactions (polymerization and deoxynucleotidylation of Phi29 terminal protein) and possesses two degradative activities (pyrophosphorolytic and 3′→5′ DNA exonucleolytic activities). Here we report that Phi29 DNA polymerase exonucleolyticaly degrades ssRNA. The RNase activity acts in a 3′ to 5′ polarity. Alanine replacements in conserved exonucleolytic site (D12A/D66A) inactivated RNase activity of the enzyme, suggesting that a single active site is responsible for cleavage of both substrates: DNA and RNA. However, the efficiency of RNA hydrolysis is ∼10-fold lower than for DNA. Phi29 DNA polymerase is widely used in rolling circle amplification (RCA) experiments. We demonstrate that exoribonuclease activity of the enzyme can be used for the target RNA conversion into a primer for RCA, thus expanding application potential of this multifunctional enzyme and opening new opportunities for RNA detection. PMID:18230765

  14. Loss of TINCR expression promotes proliferation, metastasis through activating EpCAM cleavage in colorectal cancer

    PubMed Central

    Zhang, Zhe-ying; Chang, Ya-ya; Zheng, Lin; Yuan, Li; Zhang, Fan; Hu, Yu-han; Zhang, Wen-juan; Li, Xue-nong

    2016-01-01

    Long non-coding RNAs (lncRNAs) are involved in kinds of human diseases, including colorectal cancer (CRC). TINCR, a 3.7 kb long non coding RNA, was associated with cell differentiation in keratinocyte and gastric cancer cells. However, little is known about the role of TINCR in regulation CRC progression. Here, we showed that lncRNA TINCR was associated with CRC proliferation and metastasis. TINCR was statistically downregulated in CRC tissues and metastatic CRC cell lines compared with their counterparts. TINCR was reversely correlated with CRC progression and promoted tumor cells growth, metastasis in vivo and in vitro. While overexpression of TINCR had opposite effect. In addition, we also found that TINCR specifically bound to EpCAM through RNA IP and RNA pull down assays. Loss of TINCR promoted hydrolysis of EpCAM and then released EpICD, subsequently, activated the Wnt/β-catenin pathway. Further studies shown that c-Myc repressed the expression of TINCR through repressing sp1 transcriptive activity, which established a positive feedback loop controlling c-Myc and TINCR expression. These findings elucidate that loss of TINCR expression promotes proliferation and metastasis in CRC and it could be considered as a potential cancer suppressor gene. PMID:27009809

  15. Synthesis, spectroscopic, antimicrobial and DNA cleavage studies of new Co(II), Ni(II), Cu(II), Cd(II), Zn(II) and Hg(II) complexes with naphthofuran-2-carbohydrazide Schiff base

    NASA Astrophysics Data System (ADS)

    Halli, Madappa B.; Sumathi, R. B.

    2012-08-01

    A series of Co(II), Ni(II), Cu(II), Cd(II), Zn(II) and Hg(II) complexes have been synthesized with newly synthesized Schiff base derived from naphthofuran-2-carbohydrazide and cinnamaldehyde. The elemental analyses of the complexes are confined to the stoichiometry of the type MLCl2 [M = Co(II) and Cu(II)], ML2Cl2 [M = Ni(II), Cd(II), Zn(II) and Hg(II)] respectively, where L is Schiff base ligand. Structures have been proposed from elemental analyses, IR, electronic, mass, 1H NMR, ESR spectral data, magnetic, and thermal studies. The measured low molar conductance values in DMF indicate that the complexes are non-electrolytes. Spectroscopic studies suggest coordination occurs through azomethine nitrogen and carbonyl oxygen of the ligand with the metal ions. The Schiff base and its complexes have been screened for their antibacterial (Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Salmonella typhi) and antifungal (Aspergillus niger, Aspergillus flavus, Cladosporium and Candida albicans) activities by minimum inhibitory concentration (MIC) method. The DNA cleavage studies by agarose gel electrophoresis method was studied for all the complexes.

  16. Fluorescence "turn-on" determination of H2O2 using multilayer porous SiO2/NGQDs and PdAu mimetics enzymatic/oxidative cleavage of single-stranded DNA.

    PubMed

    Liang, Linlin; Lan, Feifei; Li, Li; Su, Min; Ge, Shenguang; Yu, Jinghua; Liu, Haiyun; Yan, Mei

    2016-08-15

    A 3D microfluidic paper-based fluorescence analytical device with hollow channels based on the turn-on switching of a resonance energy transfer triggered by the •OH induced cleavage of a DNA strand was successfully constructed. And this fluorescent nanoplatform was first designed to achieve in situ and real-time determination of H2O2 released from cancer cells to obtain an accurate determination. With optimal conditions, the proposed method displayed excellent analytical performance for the detection of H2O2 ranging from 0.3 to 1.0mM with a detection limit of 0.1nM. The favorable performances of this sensor were due to the peroxidase-like activity of nitrogen-doped graphene quantum dots (multilayer porous SiO2 act as stabilizer to load more nitrogen-doped graphene quantum dots for signal amplification) and folic acid-pPdAu/GO (which also could act as an efficient fluorescence quencher and a recognition element of cancer cells by folic acid). It was worth noting that it could be used for visually determined the flux of H2O2 from the cells. Therefore, the developed biosensor holds potential for ultrasensitive quantitative analysis of H2O2 and supplies valuable information for diabetes mellitus research and clinical diagnosis. PMID:27085952

  17. Cathepsin S Cleavage of Protease-Activated Receptor-2 on Endothelial Cells Promotes Microvascular Diabetes Complications.

    PubMed

    Kumar Vr, Santhosh; Darisipudi, Murthy N; Steiger, Stefanie; Devarapu, Satish Kumar; Tato, Maia; Kukarni, Onkar P; Mulay, Shrikant R; Thomasova, Dana; Popper, Bastian; Demleitner, Jana; Zuchtriegel, Gabriele; Reichel, Christoph; Cohen, Clemens D; Lindenmeyer, Maja T; Liapis, Helen; Moll, Solange; Reid, Emma; Stitt, Alan W; Schott, Brigitte; Gruner, Sabine; Haap, Wolfgang; Ebeling, Martin; Hartmann, Guido; Anders, Hans-Joachim

    2016-06-01

    Endothelial dysfunction is a central pathomechanism in diabetes-associated complications. We hypothesized a pathogenic role in this dysfunction of cathepsin S (Cat-S), a cysteine protease that degrades elastic fibers and activates the protease-activated receptor-2 (PAR2) on endothelial cells. We found that injection of mice with recombinant Cat-S induced albuminuria and glomerular endothelial cell injury in a PAR2-dependent manner. In vivo microscopy confirmed a role for intrinsic Cat-S/PAR2 in ischemia-induced microvascular permeability. In vitro transcriptome analysis and experiments using siRNA or specific Cat-S and PAR2 antagonists revealed that Cat-S specifically impaired the integrity and barrier function of glomerular endothelial cells selectively through PAR2. In human and mouse type 2 diabetic nephropathy, only CD68(+) intrarenal monocytes expressed Cat-S mRNA, whereas Cat-S protein was present along endothelial cells and inside proximal tubular epithelial cells also. In contrast, the cysteine protease inhibitor cystatin C was expressed only in tubules. Delayed treatment of type 2 diabetic db/db mice with Cat-S or PAR2 inhibitors attenuated albuminuria and glomerulosclerosis (indicators of diabetic nephropathy) and attenuated albumin leakage into the retina and other structural markers of diabetic retinopathy. These data identify Cat-S as a monocyte/macrophage-derived circulating PAR2 agonist and mediator of endothelial dysfunction-related microvascular diabetes complications. Thus, Cat-S or PAR2 inhibition might be a novel strategy to prevent microvascular disease in diabetes and other diseases. PMID:26567242

  18. Mimicking Cdk2 phosphorylation of Bcl-xL at Ser73 results in caspase activation and Bcl-xL cleavage

    PubMed Central

    Seng, NS; Megyesi, J; Tarcsafalvi, A; Price, PM

    2016-01-01

    Cisplatin is a widely used chemotherapeutic agent, yet its efficacy is limited by nephrotoxicity. The severity of nephrotoxicity is associated with the extent of kidney cell death. Previously, we found that cisplatin-induced kidney cell death was dependent on Cdk2 activation, and inhibition of Cdk2 protected cells from cisplatin-induced apoptosis. Using an in vitro kination assay, we showed that Cdk2 phosphorylated Bcl-xL, an anti-apoptotic member of Bcl-2 family proteins, at serine 73. We also found that this phosphorylated Bcl-xL participated in cell death, as a phosphomimetic mutant of Bcl-xL at the serine 73 site (S73D-Bcl-xL) activated caspases. We now find that S73D-Bcl-xL was cleaved at D61 and D76, which are putative caspase cleavage sites, to generate 15-kDa and 12-kDa fragments. Unlike full-length Bcl-xL, these cleavage products of Bcl-xL were previously reported to be pro-apoptotic. We sought to determine whether these Bcl-xL fragments were necessary for the induction of cell death by S73D-Bcl-xL. Mutation of these caspase cleavage sites prevented the formation of the 15-kDa and 12-kDa Bcl-xL cleavage products, but apoptosis still persisted in a S73D modified Bcl-xL. Our findings show that Cdk2 phosphorylation of Bcl-xL at Ser73, but not the Bcl-xL cleavage products, is necessary and sufficient to induce cell death.

  19. DNA-based control of protein activity

    PubMed Central

    Engelen, W.; Janssen, B. M. G.

    2016-01-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  20. DNA-based control of protein activity.

    PubMed

    Engelen, W; Janssen, B M G; Merkx, M

    2016-03-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  1. Inhibition of human DNA topoisomerase IB by nonmutagenic ruthenium(II)-based compounds with antitumoral activity.

    PubMed

    de Camargo, Mariana S; da Silva, Monize M; Correa, Rodrigo S; Vieira, Sara D; Castelli, Silvia; D'Anessa, Ilda; De Grandis, Rone; Varanda, Eliana; Deflon, Victor M; Desideri, Alessandro; Batista, Alzir A

    2016-02-01

    Herein we synthesized two new ruthenium(II) compounds [Ru(pySH)(bipy)(dppb)]PF6 (1) and [Ru(HSpym)(bipy)(dppb)]PF6 (2) that are analogs to an antitumor agent recently described, [Ru(SpymMe2)(bipy)(dppb)]PF6 (3), where [(Spy) = 2-mercaptopyridine anion; (Spym) = 2-mercaptopyrimidine anion and (SpymMe2) = 4,6-dimethyl-2-mercaptopyrimidine anion]. In vitro cell culture experiments revealed significant anti-proliferative activity for 1-3 against HepG2 and MDA-MB-231 tumor cells, higher than the standard anti-cancer drugs doxorubicin and cisplatin. No mutagenicity is detected when compounds are evaluated by cytokinesis-blocked micronucleus cytome and Ames test in the presence and absence of S9 metabolic activation from rat liver. Interaction studies show that compounds 1-3 can bind to DNA through electrostatic interactions and to albumin through hydrophobic interactions. The three compounds are able to inhibit the DNA supercoiled relaxation mediated by human topoisomerase IB (Top1). Compound 3 is the most efficient Top1 inhibitor and the inhibitory effect is enhanced upon pre-incubation with the enzyme. Analysis of different steps of Top1 catalytic cycle indicates that 3 inhibits the cleavage reaction impeding the binding of the enzyme to DNA and slows down the religation reaction. Molecular docking shows that 3 preferentially binds closer to the residues of the active site when Top1 is free and lies on the DNA groove downstream of the cleavage site in the Top1-DNA complex. Thus, 3 can be considered in further studies for a possible use as an anticancer agent. PMID:26758075

  2. A label-free DNA-templated silver nanocluster probe for fluorescence on-off detection of endonuclease activity and inhibition.

    PubMed

    Qian, Yunxia; Zhang, Yaodong; Lu, Lu; Cai, Yanan

    2014-01-15

    Endonuclease cleavage of DNA plays an important role in biological and medicinal chemistry. This study aimed to develop a reliable and sensitive method for nuclease activity assay by combining the high specificity of DNA cleavage reactions with ultrahigh fluorescence turn-on abilities of guanine-rich (G-rich) DNA sequences in proximity to silver nanoclusters (Ag NCs). The DNA-templated Ag NC (DNA-Ag NC) probe with endonuclease recognition sequence consists of NC and a G-rich probe. The NC probe was designed by adding Ag NC nucleation sequence at the 5'-end. The G-rich probe is the complementary DNA sequence modified by adding a G-rich overhang sequence at the 3'-end. Thus, the fluorescence of DNA-Ag NC probe was activated because of DNA hybridization. When these DNA-Ag NC probes were exposed to the targeted endonucleases, specific DNA cleavages occurred, and pieces of G-rich DNA fragments separated from Ag NCs, resulting in fluorescence turn-off. The endonuclease activity was quantified by monitoring the change in the fluorescence intensity. Detection was demonstrated by assaying EcoRI activity. Under optimized conditions, the fluorescence reduction efficiency was linear with the EcoRI concentration in the range of 5.0×10(-4) U μL(-1) to 3.0×10(-3) U μL(-1), with a detection limit of 3.5×10(-4) U μL(-1), which is much better than or at least comparable with that in previous reports. The potential application of the proposed method for screening endonuclease inhibitors was also demonstrated. The presented assay protocol proved to be convenient, effective, sensitive, and easy in preparing the fluorescent probe. PMID:24001584

  3. Structure-specific nuclease activity of RAGs is modulated by sequence, length and phase position of flanking double-stranded DNA.

    PubMed

    Kumari, Rupa; Raghavan, Sathees C

    2015-01-01

    RAGs (recombination activating genes) are responsible for the generation of antigen receptor diversity through the process of combinatorial joining of different V (variable), D (diversity) and J (joining) gene segments. In addition to its physiological property, wherein RAG functions as a sequence-specific nuclease, it can also act as a structure-specific nuclease leading to genomic instability and cancer. In the present study, we investigate the factors that regulate RAG cleavage on non-B DNA structures. We find that RAG binding and cleavage on heteroduplex DNA is dependent on the length of the double-stranded flanking region. Besides, the immediate flanking double-stranded region regulates RAG activity in a sequence-dependent manner. Interestingly, the cleavage efficiency of RAGs at the heteroduplex region is influenced by the phasing of DNA. Thus, our results suggest that sequence, length and phase positions of the DNA can affect the efficiency of RAG cleavage when it acts as a structure-specific nuclease. These findings provide novel insights on the regulation of the pathological functions of RAGs. PMID:25327637

  4. The anti-apoptotic effect of leukotriene D4 involves the prevention of caspase 8 activation and Bid cleavage.

    PubMed Central

    Wikström, Katarina; Juhas, Maria; Sjölander, Anita

    2003-01-01

    We have shown in a previous study that leukotriene D(4) (LTD(4)) signalling increases cell survival and proliferation in intestinal epithelial cells [Ohd, Wikström and Sjölander (2000) Gastroenterology 119, 1007-1018]. This is highly interesting since inflammatory conditions of the bowel are associated with an increased risk of developing colon cancer. The enzyme cyclo-oxygenase 2 (COX-2) is important in this context since it is up-regulated in colon cancer tissues and in tumour cell lines. Treatment with the COX-2-specific inhibitor N -(2-cyclohexyloxy-4-nitrophenyl)methane sulphonamide has been shown previously to cause apoptosis in intestinal epithelial cells. In the present study, we attempted to elucidate the underlying mechanisms and we can now show that a mitochondrial pathway is employed. Inhibition of COX-2 causes release of cytochrome c, as shown by both Western-blot and microscopy studies, and as with apoptosis, this is significantly decreased by LTD(4). Since previous studies showed increased Bcl-2 levels on LTD(4) stimulation, we further studied apoptotic regulation at the mitochondrial level. From this we could exclude the involvement of the anti-apoptotic protein Bcl-X(L) as well as its pro-apoptotic counterpart Bax, since they are not expressed. Furthermore, the activity of the pro-apoptotic protein Bad (Bcl-2/Bcl-X(L)-antagonist, causing cell death) was completely unaffected. However, inhibition of COX-2 caused cleavage of caspase 8 into a 41 kDa fragment associated with activation and caused the appearance of an activated 15 kDa fragment of Bid. This indicates that N -(2-cyclohexyloxy-4-nitrophenyl)methane sulphonamide-induced apoptosis is mediated by the activation of caspase 8, via generation of truncated Bid, and thereafter release of cytochrome c. Interestingly, LTD(4) not only reverses the effects induced by inhibition of COX-2 but also reduces the apoptotic potential by lowering the basal level of caspase 8 activation and truncated Bid

  5. Design, RNA cleavage and antiviral activity of new artificial ribonucleases derived from mono-, di- and tripeptides connected by linkers of different hydrophobicity.

    PubMed

    Tamkovich, Nikolay; Koroleva, Lyudmila; Kovpak, Mikhail; Goncharova, Elena; Silnikov, Vladimir; Vlassov, Valentin; Zenkova, Marina

    2016-03-15

    A novel series of metal-free artificial ribonucleases (aRNases) was designed, synthesized and assessed in terms of ribonuclease activity and ability to inactivate influenza virus WSN/A33/H1N1 in vitro. The compounds were built of two short peptide fragments, which include Lys, Ser, Arg, Glu and imidazole residues in various combinations, connected by linkers of different hydrophobicity (1,12-diaminododecane or 4,9-dioxa-1,12-diaminododecane). These compounds efficiently cleaved different RNA substrates under physiological conditions at rates three to five times higher than that of artificial ribonucleases described earlier and displayed RNase A-like cleavage specificity. aRNases with the hydrophobic 1,12-diaminododecane linker displayed ribonuclease activity 3-40 times higher than aRNases with the 4,9-dioxa-1,12-diaminododecane linker. The assumed mechanism of RNA cleavage was typical for natural ribonucleases, that is, general acid-base catalysis via the formation of acid/base pairs by functional groups of amino acids present in the aRNases; the pH profile of cleavage confirmed this mechanism. The most active aRNases under study exhibited high antiviral activity and entirely inactivated influenza virus A/WSN/33/(H1N1) after a short incubation period of viral suspension under physiological conditions. PMID:26899594

  6. Passive kimberlite intrusion into actively dilating dyke-fracture arrays: evidence from fibrous calcite veins and extensional fracture cleavage

    NASA Astrophysics Data System (ADS)

    Basson, I. J.; Viola, G.

    2004-09-01

    Calcite veins are invariably associated with en-echelon kimberlite dyke-fracture arrays. A detailed microstructural study of veining indicates four vein types. Type I stretched or ataxial veins are defined by high aspect ratio calcite fibers that are crystallographically continuous with calcite of the kimberlite matrix wall rock, by elongated phenocrystic phlogopite with sharp crystal terminations centered on contacts between adjacent calcite fibers and by phenocrystic phlogopite that grows or extends across these veins. Type I vein mineralogy indicates syn-dilational crystallization of vein minerals in local tensional areas within the kimberlite. Vein Types II (stretched to syntaxial elongate-blocky) and III (antitaxial) indicate late crystallization vein mineral growth during subsequent or repeated dilation. Calcite fibers in Type I to Type III veins are orthogonal to the contacts of their host dykes regardless of the orientation of vein margins. Type IV calcite veins, with blocky or mosaic/polycrystalline textures, are attributed to minor post-intrusion extension, which was potentially accompanied by repeated kimberlite intrusion within a given dyke array. Syn-crystallization/syn-intrusion Type I veins and an ubiquitous dyke-parallel fracture cleavage, in a zone up to 4 m on either side of dyke contacts, suggest that en-echelon kimberlite dyke-fracture arrays occupied the approximate center of zones of active dilation within the brittle carapace of the upper crust. Type II and III veins indicate that extension or dilation continued, independently of an occupying kimberlite fluid phase, after initial intrusion. Arrested mobile hydrofracturing, under low differential stress within the upper brittle or seismic carapace of the continental crust, followed by repeated dilation of the dyke-fracture system, is proposed as a mechanism for producing the features observed in this study. The conditions constrained in this study indicate passive dyke intrusion into dilating

  7. Assessment of preferential cleavage of an actively transcribed retroviral hybrid gene in murine cells by deoxyribonuclease I, bleomycin, neocarzinostatin, or ionizing radiation

    SciTech Connect

    Beckmann, R.P.; Agostino, M.J.; McHugh, M.M.; Sigmund, R.D.; Beerman, T.A.

    1987-08-25

    Preferential cleavage induced by bleomycin, neocarzinostatin, or ionizing radiation in a transcribed cellular gene was evaluated through comparisons with deoxyribonuclease I. The glucocorticoid-inducible LTL gene previously described served as the specific DNA target. A Southern blot analysis was used to specifically assess cleavage of the LTL gene in nuclei isolated from cells either treated or untreated with the synthetic glucocorticoid dexamethasone. Hypersensitivity of the gene to bleomycin or neocarzinostatin, which paralleled deoxyribonuclease I hypersensitivity, was evident only in nuclei isolated from dexamethasone-treated cells. Like deoxyribonuclease I, sites of dexamethasone-inducible drug hypersensitivity were coincident with the binding region for the glucocorticoid receptor found within the regulatory sequences of the LTL gene. In contrast, no hypersensitivity to ionizing radiation was evident. Although bleomycin and neocarzinostatin showed qualitatively similar preferences for the threshold LTL gene, quantitative evaluations of damage to total cellular DNA by filter elution showed that the relative specificity of bleomycin for the hypersensitive region was much less than that of either deoxyribonuclease I or neocarzinostatin.

  8. Evolution of the R2 Retrotransposon Ribozyme and Its Self-Cleavage Site

    PubMed Central

    Eickbush, Danna G.; Burke, William D.; Eickbush, Thomas H.

    2013-01-01

    R2 is a non-long terminal repeat retrotransposon that inserts site-specifically in the tandem 28S rRNA genes of many animals. Previously, R2 RNA from various species of Drosophila was shown to self-cleave from the 28S rRNA/R2 co-transcript by a hepatitis D virus (HDV)-like ribozyme encoded at its 5' end. RNA cleavage was at the precise 5' junction of the element with the 28S gene. Here we report that RNAs encompassing the 5' ends of R2 elements from throughout its species range fold into HDV-like ribozymes. In vitro assays of RNA self-cleavage conducted in many R2 lineages confirmed activity. For many R2s, RNA self-cleavage was not at the 5' end of the element but at 28S rRNA sequences up to 36 nucleotides upstream of the junction. The location of cleavage correlated well with the types of endogenous R2 5' junctions from different species. R2 5' junctions were uniform for most R2s in which RNA cleavage was upstream in the rRNA sequences. The 28S sequences remaining on the first DNA strand synthesized during retrotransposition are postulated to anneal to the target site and uniformly prime second strand DNA synthesis. In species where RNA cleavage occurred at the R2 5' end, the 5' junctions were variable. This junction variation is postulated to result from the priming of second strand DNA synthesis by chance microhomologies between the target site and the first DNA strand. Finally, features of R2 ribozyme evolution, especially changes in cleavage site and convergence on the same active site sequences, are discussed. PMID:24066021

  9. A new ternary copper(II) complex derived from 2-(2'-pyridyl)benzimidazole and glycylglycine: synthesis, characterization, DNA binding and cleavage, antioxidation and HSA interaction.

    PubMed

    Fu, Xia-Bing; Lin, Zi-Hua; Liu, Hai-Feng; Le, Xue-Yi

    2014-03-25

    A new ternary copper(II)-dipeptide complex [Cu(glygly)(HPB)(Cl)]⋅2H2O (glygly=glycylglycine anion, HPB=2-(2'-pyridyl)benzimidazole) has been synthesized and characterized. The DNA interaction of the complex was studied by spectroscopic methods, viscosity, and electrophoresis measurements. The antioxidant activity was also investigated using the pyrogallol autoxidation assay. Besides, the interaction of the complex with human serum albumin (HSA) in vitro was examined by multispectroscopic techniques. The complex partially intercalated to CT-DNA with a high binding constant (Kb=7.28×10(5) M(-1)), and cleaved pBR322 DNA efficiently via an oxidative mechanism in the presence of Vc, with the HO· and O2(-) as the active species, and the SOD as a promoter. Furthermore, the complex shows a considerable SOD-like activity with the IC50 value of 3.8386 μM. The complex exhibits desired binding affinity to HSA, in which hydrogen bond or vander Waals force played a major role. The alterations of HSA secondary structure induced by the complex were confirmed by UV-visible, CD, synchronous fluorescence and 3D fluorescence spectroscopy. PMID:24291450

  10. A new ternary copper(II) complex derived from 2-(2";-pyridyl)benzimidazole and glycylglycine: Synthesis, characterization, DNA binding and cleavage, antioxidation and HSA interaction

    NASA Astrophysics Data System (ADS)

    Fu, Xia-Bing; Lin, Zi-Hua; Liu, Hai-Feng; Le, Xue-Yi

    2014-03-01

    A new ternary copper(II)-dipeptide complex [Cu(glygly)(HPB)(Cl)]ṡ2H2O (glygly = glycylglycine anion, HPB = 2-(2";-pyridyl)benzimidazole) has been synthesized and characterized. The DNA interaction of the complex was studied by spectroscopic methods, viscosity, and electrophoresis measurements. The antioxidant activity was also investigated using the pyrogallol autoxidation assay. Besides, the interaction of the complex with human serum albumin (HSA) in vitro was examined by multispectroscopic techniques. The complex partially intercalated to CT-DNA with a high binding constant (Kb = 7.28 × 105 M-1), and cleaved pBR322 DNA efficiently via an oxidative mechanism in the presence of Vc, with the HO· and O2-rad as the active species, and the SOD as a promoter. Furthermore, the complex shows a considerable SOD-like activity with the IC50 value of 3.8386 μM. The complex exhibits desired binding affinity to HSA, in which hydrogen bond or vander Waals force played a major role. The alterations of HSA secondary structure induced by the complex were confirmed by UV-visible, CD, synchronous fluorescence and 3D fluorescence spectroscopy.

  11. The down regulation of target genes by photo activated DNA nanoscissors.

    PubMed

    Tsai, Tsung-Lin; Shieh, Dar-Bin; Yeh, Chen-Sheng; Tzeng, Yonhua; Htet, Khant; Chuang, Kao-Shu; Hwu, Jih Ru; Su, Wu-Chou

    2010-09-01

    An artificial, targeted, light-activated nanoscissor (ATLANS) was developed for precision photonic cleavage of DNA at selectable target sequences. The ATLANS is comprised of nanoparticle core and a monolayer of hydrazone-modified triplex-forming oligonucleotides (TFOs), which recognize and capture the targeted DNA duplex. Upon photo-illumination (lambda = 460 nm), the attached hydrazone scissor specifically cleaves the targeted DNA at a pre-designed nucleotide pair. Electrophoretic mobility shift and co-precipitation assays revealed sequence-specific binding with the short-fragment and long-form plasmid DNA of both TFO and TFO-nanoparticle probes. Upon photo-illumination, ATLANS introduced a precise double-stranded break 12bp downstream the TFO binding sequence and down-regulated the target gene in HeLa cell system. Gold nanoparticles multiplexed the cutting efficiency and potential for simultaneous manipulation of multiple targets, as well as protected DNA from non-specific photo-damage. This photon-mediated DNA manipulation technology will facilitate high spatial and temporal precision in simultaneous silencing at the genome level, and advanced simultaneous manipulation of multiple targeted genes. PMID:20605206

  12. Differential, dominant activation and inhibition of Notch signalling and APP cleavage by truncations of PSEN1 in human disease.

    PubMed

    Newman, Morgan; Wilson, Lachlan; Verdile, Giuseppe; Lim, Anne; Khan, Imran; Moussavi Nik, Seyyed Hani; Pursglove, Sharon; Chapman, Gavin; Martins, Ralph N; Lardelli, Michael

    2014-02-01

    PRESENILIN1 (PSEN1) is the major locus for mutations causing familial Alzheimer's disease (FAD) and is also mutated in Pick disease of brain, familial acne inversa and dilated cardiomyopathy. It is a critical facilitator of Notch signalling and many other signalling pathways and protein cleavage events including production of the Amyloidβ (Aβ) peptide from the AMYLOID BETA A4 PRECURSOR PROTEIN (APP). We previously reported that interference with splicing of transcripts of the zebrafish orthologue of PSEN1 creates dominant negative effects on Notch signalling. Here, we extend this work to show that various truncations of human PSEN1 (or zebrafish Psen1) protein have starkly differential effects on Notch signalling and cleavage of zebrafish Appa (a paralogue of human APP). Different truncations can suppress or stimulate Notch signalling but not Appa cleavage and vice versa. The G183V mutation possibly causing Pick disease causes production of aberrant transcripts truncating the open reading frame after exon 5 sequence. We show that the truncated protein potentially translated from these transcripts avidly incorporates into very stable Psen1-dependent higher molecular weight complexes and suppresses cleavage of Appa but not Notch signalling. In contrast, the truncated protein potentially produced by the P242LfsX11 acne inversa mutation has no effect on Appa cleavage but, unexpectedly, enhances Notch signalling. Our results suggest novel hypotheses for the pathological mechanisms underlying these diseases and illustrate the importance of investigating the function of dominant mutations at physiologically relevant expression levels and in the normally heterozygous state in which they cause human disease rather than in isolation from healthy alleles. PMID:24101600

  13. Exploring the DNA binding mode of transition metal based biologically active compounds

    NASA Astrophysics Data System (ADS)

    Raman, N.; Sobha, S.

    2012-01-01

    Few novel 4-aminoantipyrine derived Schiff bases and their metal complexes were synthesized and characterized. Their structural features and other properties were deduced from the elemental analysis, magnetic susceptibility and molar conductivity as well as from mass, IR, UV-vis, 1H NMR and EPR spectral studies. The binding of the complexes with CT-DNA was analyzed by electronic absorption spectroscopy, viscosity measurement, and cyclic voltammetry. The interaction of the metal complexes with DNA was also studied by molecular modeling with special reference to docking. The experimental and docking results revealed that the complexes have the ability of interaction with DNA of minor groove binding mode. The intrinsic binding constants ( Kb) of the complexes with CT-DNA were found out which show that they are minor groove binders. Gel electrophoresis assay demonstrated the ability of the complexes to cleave the pUC19 DNA in the presence of AH 2 (ascorbic acid). Moreover, the oxidative cleavage studies using distamycin revealed the minor groove binding for the newly synthesized 4-aminoantipyrine derived Schiff bases and their metal complexes. Evaluation of antibacterial activity of the complexes against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, and Klebsiella pneumoniae exhibited that the complexes have potent biocidal activity than the free ligands.

  14. Role of trypsin-like cleavage at arginine 192 in the enzymatic and cytotonic activities of Escherichia coli heat-labile enterotoxin.

    PubMed Central

    Grant, C C; Messer, R J; Cieplak, W

    1994-01-01

    Previous studies of cholera toxin and Escherichia coli heat-labile enterotoxin have suggested that proteolytic cleavage plays an important role in the expression of ADP-ribosyltransferase activity and toxicity. Specifically, several studies have implicated a trypsin-like cleavage at arginine 192, which lies within an exposed region subtended by a disulfide bond in the intact A subunit, in toxicity. To investigate the role of this modification in the enzymatic and cytotonic properties of heat-labile enterotoxin, the response of purified, recombinant A subunit to tryptic activation and the effect of substituting arginine 192 with glycine on the activities of the holotoxin were examined. The recombinant A subunit of heat-labile enterotoxin exhibited significant levels of ADP-ribosyltransferase activity that were only nominally increased (approximately twofold) by prior limited trypsinolysis. The enzymatic activity also did not appear to be affected by auto-ADP-ribosylation that occurs during the high-level synthesis of the recombinant A subunit in E. coli. A mutant form of the holotoxin containing the arginine 192-to-glycine substitution exhibited levels of cytotonic activity for CHO cells that were similar to that of the untreated, wild-type holotoxin but exhibited a marked delay in the ability to increase intracellular levels of cyclic AMP in Caco-2 cells. The results indicate that trypsin-like cleavage of the A subunit of E. coli heat-labile enterotoxin at arginine 192 is not requisite to the expression of enzymatic activity by the A subunit and further reveal that this modification, although it enhances the biological and enzymatic activities of the toxin, is not absolutely required for the enterotoxin to elicit cytotonic effects. Images PMID:7927684

  15. HIV-1 Vpu accessory protein induces caspase-mediated cleavage of IRF3 transcription factor.

    PubMed

    Park, Sang Yoon; Waheed, Abdul A; Zhang, Zai-Rong; Freed, Eric O; Bonifacino, Juan S

    2014-12-19

    Vpu is an accessory protein encoded by HIV-1 that interferes with multiple host-cell functions. Herein we report that expression of Vpu by transfection into 293T cells causes partial proteolytic cleavage of interferon regulatory factor 3 (IRF3), a key transcription factor in the innate anti-viral response. Vpu-induced IRF3 cleavage is mediated by caspases and occurs mainly at Asp-121. Cleavage produces a C-terminal fragment of ∼37 kDa that comprises the IRF dimerization and transactivation domains but lacks the DNA-binding domain. A similar cleavage is observed upon infection of the Jurkat T-cell line with vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-1. Two other HIV-1 accessory proteins, Vif and Vpr, also contribute to the induction of IRF3 cleavage in both the transfection and the infection systems. The C-terminal IRF3 fragment interferes with the transcriptional activity of full-length IRF3. Cleavage of IRF3 under all of these conditions correlates with cleavage of poly(ADP-ribose) polymerase, an indicator of apoptosis. We conclude that Vpu contributes to the attenuation of the anti-viral response by partial inactivation of IRF3 while host cells undergo apoptosis. PMID:25352594

  16. Comparison of the cleavage of pyrimidine dimers by the bacteriophage T4 and Micrococcus luteus uv-specific endonucleases

    SciTech Connect

    Gordon, L.K.; Haseltine, W.A.

    1980-12-25

    A comparison was made of the activity of the uv-specific endonucleases of bacteriophage T4 (T4 endonuclease V) and of Micrococcus luteus on ultraviolet light-irradiated DNA substrates of defined sequence. The two enzyms cleave DNA at the site of pyrimidine dimers with the same frequency. The products of the cleavage reaction are the same. The pyrimidine dimer DNA-glycosylase activity of both enzymes is more active on double-stranded DNA than it is on single-stranded DNA.

  17. Metal-catalyzed activation of ethers via C-O bond cleavage: a new strategy for molecular diversity.

    PubMed

    Cornella, Josep; Zarate, Cayetana; Martin, Ruben

    2014-12-01

    In 1979, the seminal work of Wenkert set the standards for the utilization of aryl and vinyl ethers as coupling partners via C-O bond-cleavage. Although the topic remained dormant for almost three decades, the last few years have witnessed a renaissance in this area of expertise, experiencing an exponential growth and becoming a significant discipline within the cross-coupling arena. The means to utilize readily accessible aryl or vinyl ethers as counterparts does not only represent a practical, powerful and straightforward alternative to organic halides, but also constitutes an excellent opportunity to improve our chemical knowledge about a relatively unexplored area of expertise. This review summarizes the most significant developments in the area of C-O bond-cleavage when employing aryl or vinyl ethers, providing a detailed overview of the current state of the art and including future aspects, when applicable. PMID:25157613

  18. DNA topoisomerase II structures and anthracycline activity: insights into ternary complex formation.

    PubMed

    Dal Ben, D; Palumbo, M; Zagotto, G; Capranico, G; Moro, S

    2007-01-01

    DNA Topoisomerase II (Top2) is an essential nuclear enzyme that regulates the topological state of the DNA, and a target of very effective anticancer drugs including anthracycline antibiotics. Even though several aspects of drug activity against Top2 are understood, the drug receptor site is not yet known. Several Top2 mutants have altered drug sensitivity and have provided information of structural features determining drug action. Here, we have revised the published crystal structures of eukaryotic and prokaryotic Top2s and relevant biochemical investigations of enzyme activity and anthracycline action. In particular, we have considered Top2 mutations conferring resistance to anthracyclines and related agents. Following a previous study (Moro et al, Biochemistry, 2004; 43: 7503-13), we have then re-built a molecular model of the entire enzyme in complex with DNA after the cleavage reaction, and used it to define the receptor site of anthracyclines. The results suggest a model wherein the drug specifically contacts the cleaved DNA as well as amino acid residues of the enzyme CAP-like domain. The findings can explain several established structure-activity relationships of antitumour anthracyclines, and provide a framework for further developments of effective Top2 poison. PMID:17897022

  19. Thrombin Cleavage of Osteopontin Modulates Its Activities in Human Cells In Vitro and Mouse Experimental Autoimmune Encephalomyelitis In Vivo

    PubMed Central

    Boggio, Elena; Gigliotti, Casimiro Luca; Soluri, Maria Felicia; Clemente, Nausicaa; Toth, Erika; Raineri, Davide; Ferrara, Benedetta; Chiocchetti, Annalisa

    2016-01-01

    Osteopontin is a proinflammatory cytokine and plays a pathogenetic role in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), by recruiting autoreactive T cells into the central nervous system. Osteopontin functions are modulated by thrombin cleavage generating N- and C-terminal fragment, whose individual roles are only partly known. Published data are difficult to compare since they have been obtained with heterogeneous approaches. Interestingly, thrombin cleavage of osteopontin unmasks a cryptic domain of interaction with α4β1 integrin that is the main adhesion molecule involved in lymphocyte transmigration to the brain and is the target for natalizumab, the most potent drug preventing relapses. We produced recombinant osteopontin and its N- and C-terminal fragments in an eukaryotic system in order to allow their posttranslational modifications. We investigated, in vitro, their effect on human cells and in vivo in EAE. We found that the osteopontin cleavage plays a key role in the function of this cytokine and that the two fragments exert distinct effects both in vitro and in vivo. These findings suggest that drugs targeting each fragment may be used to fine-tune the pathological effects of osteopontin in several diseases. PMID:27478856

  20. Thrombin Cleavage of Osteopontin Modulates Its Activities in Human Cells In Vitro and Mouse Experimental Autoimmune Encephalomyelitis In Vivo.

    PubMed

    Boggio, Elena; Dianzani, Chiara; Gigliotti, Casimiro Luca; Soluri, Maria Felicia; Clemente, Nausicaa; Cappellano, Giuseppe; Toth, Erika; Raineri, Davide; Ferrara, Benedetta; Comi, Cristoforo; Dianzani, Umberto; Chiocchetti, Annalisa

    2016-01-01

    Osteopontin is a proinflammatory cytokine and plays a pathogenetic role in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), by recruiting autoreactive T cells into the central nervous system. Osteopontin functions are modulated by thrombin cleavage generating N- and C-terminal fragment, whose individual roles are only partly known. Published data are difficult to compare since they have been obtained with heterogeneous approaches. Interestingly, thrombin cleavage of osteopontin unmasks a cryptic domain of interaction with α 4 β 1 integrin that is the main adhesion molecule involved in lymphocyte transmigration to the brain and is the target for natalizumab, the most potent drug preventing relapses. We produced recombinant osteopontin and its N- and C-terminal fragments in an eukaryotic system in order to allow their posttranslational modifications. We investigated, in vitro, their effect on human cells and in vivo in EAE. We found that the osteopontin cleavage plays a key role in the function of this cytokine and that the two fragments exert distinct effects both in vitro and in vivo. These findings suggest that drugs targeting each fragment may be used to fine-tune the pathological effects of osteopontin in several diseases. PMID:27478856

  1. Reactive oxygen species-dependent HSP90 protein cleavage participates in arsenical As{sup +3}- and MMA{sup +3}-induced apoptosis through inhibition of telomerase activity via JNK activation

    SciTech Connect

    Shen, S.-C.; Yang, L.-Y.; Lin, H.-Y.; Wu, C.-Y.; Su, T.-H.; Chen, Y.-C.

    2008-06-01

    The effects of six arsenic compounds including As{sup +3}, MMA{sup +3}, DMA{sup +3}, As{sup +5}, MMA{sup +5}, and DMA{sup +5} on the viability of NIH3T3 cells were examined. As{sup +3} and MMA{sup +3}, but not the others, exhibited significant cytotoxic effects in NIH3T3 cells through apoptosis induction. The apoptotic events such as DNA fragmentation and chromosome condensation induced by As{sup +3} and MMA{sup +3} were prevented by the addition of NAC and CAT, and induction of HO-1 gene expression in accordance with cleavage of the HSP90 protein, and suppression of telomerase activity were observed in NIH3T3 cells under As{sup +3} and MMA{sup +3} treatments. An increase in the intracellular peroxide level was examined in As{sup +3}- and MMA{sup +3}-treated NIH3T3 cells, and As{sup +3}- and MMA{sup +3}-induced apoptotic events were blocked by NAC, CAT, and DPI addition. HSP90 inhibitors, GA and RD, significantly attenuated the telomerase activity in NIH3T3 cells with an enhancement of As{sup +3}- and MMA{sup +3}-induced cytotoxicity. Suppression of JNKs significantly inhibited As{sup +3}- and MMA{sup +3}-induced apoptosis by blocking HSP90 protein cleavage and telomerase reduction in NIH3T3 cells. Furthermore, Hb, SnPP, and dexferosamine showed no effect against As{sup +3}- and MMA{sup +3}-induced apoptosis, and overexpression of HO-1 protein or inhibition of HO-1 protein expression did not affect the apoptosis induced by As{sup +3} or MMA{sup +3}. These data provide the first evidence to indicate that apoptosis induced by As{sup +3} and MMA{sup +3} is mediated by an ROS-dependent degradation of HSP90 protein and reduction of telomerase via JNK activation, and HO-1 induction might not be involved.

  2. Cleavage at both Arg306 and Arg506 is required and sufficient for timely and efficient inactivation of factor Va by activated protein C*

    PubMed Central

    Barhoover, Melissa A.; Kalafatis, Michael

    2011-01-01

    Background Activated protein C (APC) inactivates membrane-bound factor Va following cleavages of the heavy chain at Arg306, Arg506, and Arg679. Objectives To examine which cleavage is most important for inactivation, recombinant factor V molecules were constructed as follows: factor V306Q (R306→Q), factor V506Q (R506→Q), and factor V306Q/506Q (R306→Q and R506→Q). Methods The recombinant molecules were expressed in mammalian cells, purified, and assayed prior and after incubation with APC and lipids for 30 min (factor Vai) in clotting assays and in an assay using purified reagents and saturating concentrations of factor Va. Results Clotting assays demonstrated that factor VaiWT, factor Vai306Q and factor Vai506Q were devoid of activity, while factor Vai306Q/506Q maintained ~70% activity following a 30 minute incubation with APC. Prothrombinase assembled with all mutant cofactor molecules before and after treatment with APC had Km values similar to values found with prothrombinase assembled with factor VaWT. Prothrombinase assembled with factor VaiWT demonstrated 20-fold reduction in kcat, while prothrombinase assembled with factor Vai506Q had a 2-fold reduction in kcat as compared to prothrombinase assembled with factor VaWT. In contrast, factor Vai306Q and factor Vai306Q/506Q didn’t show any loss in kcat under similar experimental conditions. Conclusion Our data demonstrate that: 1) the activity of an APC-treated factor Va molecule bearing a single mutation at Arg306 or Arg506 depends on the assay used; and 2) regardless of the assay employed, in the absence of the APC-cleavage sites at Arg306 and Arg506, the active cofactor is unable to be significantly inactivated by APC in the presence of a membrane surface. PMID:21467919

  3. Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation

    PubMed Central

    Huo, Yanwu; Nam, Ki Hyun; Ding, Fang; Lee, Heejin; Wu, Lijie; Xiao, Yibei; Farchione, F. Daniel; Zhou, Sharleen; Rajashankar, Raj; Kurinov, Igor; Zhang, Rongguang; Ke, Ailong

    2014-01-01

    CRISPR drives prokaryotic adaptation to invasive nucleic acids such as phages and plasmids using an RNA-mediated interference mechanism. Interference in Type I CRISPR-Cas systems requires a targeting Cascade complex and a degradation machine Cas3, which contains both nuclease and helicase activities. Here we report the crystal structures of Cas3 bound to ss-DNA substrate and show that it is an obligated 3′-to-5′ ss-DNase preferentially accepting substrate directly from the helicase moiety. Conserved residues in the HD-type nuclease coordinate two irons for ss-DNA cleavage. ATP coordination and conformational flexibility are revealed for the SF2-type helicase moiety. Cas3 is specifically guided towards Cascade-bound target DNA with a correct PAM sequence, through physical interactions to both the non-target substrate strand and the CasA protein. The cascade of recognition events ensures a well-controlled DNA targeting and degradation of alien DNA by Cascade and Cas3. PMID:25132177

  4. A DNA polymerase activity is associated with Cauliflower Mosaic Virus.

    PubMed Central

    Menissier, J; Laquel, P; Lebeurier, G; Hirth, L

    1984-01-01

    A DNA polymerase activity is found within the Cauliflower Mosaic Virus (CaMV) particle. Analysis of the reaction product reveals that the linear form of the virion DNA is preferentially labelled. The molecular weight of the DNA polymerase as determined on an "activity gel" is 76 kDa. Images PMID:6514573

  5. Double-stranded DNA-templated cleavage of oligonucleotides containing a P3'->N5' linkage triggered by triplex formation: the effects of chemical modifications and remarkable enhancement in reactivity.

    PubMed

    Ito, Kosuke Ramon; Kodama, Tetsuya; Tomizu, Masaharu; Negoro, Yoshinori; Orita, Ayako; Osaki, Tomohisa; Hosoki, Noritsugu; Tanaka, Takaya; Imanishi, Takeshi; Obika, Satoshi

    2010-11-01

    We recently reported double-stranded DNA-templated cleavage of oligonucleotides as a sequence-specific DNA-detecting method. In this method, triplex-forming oligonucleotides (TFOs) modified with 5'-amino-2',4'-BNA were used as a DNA-detecting probe. This modification introduced a P3'→N5' linkage (P-N linkage) in the backbone of the TFO, which was quickly cleaved under acidic conditions when it formed a triplex. The prompt fission of the P-N linkage was assumed to be driven by a conformational strain placed on the linkage upon triplex formation. Therefore, chemical modifications around the P-N linkage should change the reactivity by altering the microenvironment. We synthesized 5'-aminomethyl type nucleic acids, and incorporated them into TFOs instead of 5'-amino-2',4'-BNA to investigate the effect of 5'-elongation. In addition, 2',4'-BNA/LNA or 2',5'-linked DNA were introduced at the 3'- and/or 5'-neighboring residues of 5'-amino-2',4'-BNA to reveal neighboring residual effects. We evaluated the triplex stability and reaction properties of these TFOs, and found out that chemical modifications around the P-N linkage greatly affected their reaction properties. Notably, 2',5'-linked DNA at the 3' position flanking 5'-amino-2',4'-BNA brought significantly higher reactivity, and we succeeded in indicating that a TFO with this modification is promising as a DNA analysis tool. PMID:20615902

  6. Activation of 12/23-RSS-Dependent RAG Cleavage by hSWI/SNF Complex in the Absence of Transcription

    PubMed Central

    Du, Hansen; Ishii, Haruhiko; Pazin, Michael J.; Sen, Ranjan

    2015-01-01

    SUMMARY Maintenance of genomic integrity during antigen receptor gene rearrangements requires (1) regulated access of the V(D)J recombinase to specific loci and (2) generation of double-strand DNA breaks only after recognition of a pair of matched recombination signal sequences (RSSs). Here we recapitulate both key aspects of regulated recombinase accessibility in a cell-free system using plasmid substrates assembled into chromatin. We show that recruitment of the SWI/SNF chromatin-remodeling complex to both RSSs increases coupled cleavage by RAG1 and RAG2 proteins. SWI/SNF functions by altering local chromatin structure in the absence of RNA polymerase II-dependent transcription or histone modifications. These observations demonstrate a direct role for cis-sequence-regulated local chromatin remodeling in RAG1/2-dependent initiation of V(D)J recombination. PMID:18775324

  7. l-Histidyl-glycyl-glycyl-l-histidine. Amino-acid structuring of the bleomycin-type pentadentate metal-binding environment capable of efficient double-strand cleavage of plasmid DNA.

    PubMed

    Ida, Satomi; Iwamaru, Kana; Fujita, Mikako; Okamoto, Yoshinari; Kudo, Yuri; Kurosaki, Hiromasa; Otsuka, Masami

    2015-10-01

    A tetrapeptide, l-histidyl-glycyl-glycyl-l-histidine (HGGH), was synthesized and the pUC19 plasmid DNA cleaving activity by copper(II) complex of HGGH (Cu(II)-HGGH) was investigated. Cu(II)-HGGH showed bleomycin-like DNA cleaving activity and, at 50nM, converted a supercoiled DNA efficiently to a linear DNA in the presence of 500μM H2O2/sodium ascorbate through an oxidative pathway. PMID:26159895

  8. Synthesis, spectroscopic characterisation, thermal analysis, DNA interaction and antibacterial activity of copper(I) complexes with N, N‧- disubstituted thiourea

    NASA Astrophysics Data System (ADS)

    Chetana, P. R.; Srinatha, B. S.; Somashekar, M. N.; Policegoudra, R. S.

    2016-02-01

    copper(I) complexes [Cu(4MTU)2Cl] (2), [Cu(4MTU) (B)Cl] (3), [Cu(6MTU)2Cl] (5) and [Cu(6MTU) (B)Cl] (6) where 4MTU = 1-Benzyl-3-(4-methyl-pyridin-2-yl)-thiourea (1) and 6MTU = 1-Benzyl-3-(6-methyl-pyridin-2-yl)-thiourea (4), B is a N,N-donor heterocyclic base, viz. 1,10-phenanthroline (phen 3, 6), were synthesized, characterized by various physico-chemical and spectroscopic techniques. The elemental analysis suggests that the stoichiometry to be 1:2 (metal:ligand) for 2, 5 1:1:1 (metal:ligand:B) for 3, 6. X-ray powder diffraction illustrates that the complexes have crystalline nature. IR data coupled with electronic spectra and molar conductance values suggest that the complex 2, 5 show the presence of a trigonal planar geometry and the complex 3, 6 show the presence of a tetrahedral geometry about the Cu(I) centre. The binding affinity towards calf thymus (CT) DNA was determined using UV-Vis, fluorescence spectroscopic titrations and viscosity studies. These studies showed that the tested phen complexes 3, 6 bind moderately (in the order of 105 M-1) to CT DNA. The complex 2, 5 does not show any apparent binding to the DNA and hence poor cleavage efficiency. Complex 3, 6 shows efficient oxidative cleavage of plasmid DNA in the presence of H2O2 involving hydroxyl radical species as evidenced from the control data showing inhibition of DNA cleavage in the presence of DMSO and KI. The in vitro antibacterial assay indicates that these complexes are good antimicrobial agents against various pathogens. Anti-bacterial activity is higher when thiourea coordinates to metal ion than the thiourea alone.

  9. Regulation of Mutagenic DNA Polymerase V Activation in Space and Time

    PubMed Central

    Robinson, Andrew; McDonald, John P.; Caldas, Victor E. A.; Patel, Meghna; Wood, Elizabeth A.; Punter, Christiaan M.; Ghodke, Harshad; Cox, Michael M.; Woodgate, Roger; Goodman, Myron F.; van Oijen, Antoine M.

    2015-01-01

    Spatial regulation is often encountered as a component of multi-tiered regulatory systems in eukaryotes, where processes are readily segregated by organelle boundaries. Well-characterized examples of spatial regulation are less common in bacteria. Low-fidelity DNA polymerase V (UmuD′2C) is produced in Escherichia coli as part of the bacterial SOS response to DNA damage. Due to the mutagenic potential of this enzyme, pol V activity is controlled by means of an elaborate regulatory system at transcriptional and posttranslational levels. Using single-molecule fluorescence microscopy to visualize UmuC inside living cells in space and time, we now show that pol V is also subject to a novel form of spatial regulation. After an initial delay (~ 45 min) post UV irradiation, UmuC is synthesized, but is not immediately activated. Instead, it is sequestered at the inner cell membrane. The release of UmuC into the cytosol requires the RecA* nucleoprotein filament-mediated cleavage of UmuD→UmuD′. Classic SOS damage response mutants either block [umuD(K97A)] or constitutively stimulate [recA(E38K)] UmuC release from the membrane. Foci of mutagenically active pol V Mut (UmuD′2C-RecA-ATP) formed in the cytosol after UV irradiation do not co-localize with pol III replisomes, suggesting a capacity to promote translesion DNA synthesis at lesions skipped over by DNA polymerase III. In effect, at least three molecular mechanisms limit the amount of time that pol V has to access DNA: (1) transcriptional and posttranslational regulation that initially keep the intracellular levels of pol V to a minimum; (2) spatial regulation via transient sequestration of UmuC at the membrane, which further delays pol V activation; and (3) the hydrolytic activity of a recently discovered pol V Mut ATPase function that limits active polymerase time on the chromosomal template. PMID:26317348

  10. Regulation of Mutagenic DNA Polymerase V Activation in Space and Time.

    PubMed

    Robinson, Andrew; McDonald, John P; Caldas, Victor E A; Patel, Meghna; Wood, Elizabeth A; Punter, Christiaan M; Ghodke, Harshad; Cox, Michael M; Woodgate, Roger; Goodman, Myron F; van Oijen, Antoine M

    2015-08-01

    Spatial regulation is often encountered as a component of multi-tiered regulatory systems in eukaryotes, where processes are readily segregated by organelle boundaries. Well-characterized examples of spatial regulation are less common in bacteria. Low-fidelity DNA polymerase V (UmuD'2C) is produced in Escherichia coli as part of the bacterial SOS response to DNA damage. Due to the mutagenic potential of this enzyme, pol V activity is controlled by means of an elaborate regulatory system at transcriptional and posttranslational levels. Using single-molecule fluorescence microscopy to visualize UmuC inside living cells in space and time, we now show that pol V is also subject to a novel form of spatial regulation. After an initial delay (~ 45 min) post UV irradiation, UmuC is synthesized, but is not immediately activated. Instead, it is sequestered at the inner cell membrane. The release of UmuC into the cytosol requires the RecA* nucleoprotein filament-mediated cleavage of UmuD→UmuD'. Classic SOS damage response mutants either block [umuD(K97A)] or constitutively stimulate [recA(E38K)] UmuC release from the membrane. Foci of mutagenically active pol V Mut (UmuD'2C-RecA-ATP) formed in the cytosol after UV irradiation do not co-localize with pol III replisomes, suggesting a capacity to promote translesion DNA synthesis at lesions skipped over by DNA polymerase III. In effect, at least three molecular mechanisms limit the amount of time that pol V has to access DNA: (1) transcriptional and posttranslational regulation that initially keep the intracellular levels of pol V to a minimum; (2) spatial regulation via transient sequestration of UmuC at the membrane, which further delays pol V activation; and (3) the hydrolytic activity of a recently discovered pol V Mut ATPase function that limits active polymerase time on the chromosomal template. PMID:26317348

  11. Label-Free and Sensitive Fluorescent Detection of Sequence-Specific Single-Strand DNA Based on S1 Nuclease Cleavage Effects

    PubMed Central

    Guan, Zheng; Liu, Jinchuan; Bai, Wenhui; Lv, Zhenzhen; Jiang, Xiaoling; Yang, Shuming; Chen, Ailiang; Lv, Guiyuan

    2014-01-01

    The ability to detect sequence-specific single-strand DNA (ssDNA) in complex, contaminant-ridden samples, using a fluorescent method directly without a DNA extraction and PCR step could simplify the detection of pathogens in the field and in the clinic. Here, we have demonstrated a simple label-free sensing strategy to detect ssDNA by employing its complementary ssDNA, S1 nuclease and nucleic acid fluorescent dyes. Upon clearing away redundant complementary ssDNA and possibly mismatched double strand DNA by using S1 nuclease, the fluorescent signal-to-noise ratio could be increased dramatically. It enabled the method to be adaptable to three different types of DNA fluorescent dyes and the ability to detect target ssDNA in complex, multicomponent samples, like tissue homogenate. The method can distinguish a two-base mismatch from avian influenza A (H1N1) virus. Also, it can detect the appearance of 50 pM target ssDNA in 0.5 µg·mL−1 Lambda DNA, and 50 nM target ssDNA in 5 µg·mL−1 Lambda DNA or in tissue homogenate. It is facile and cost-effective, and could be easily extended to detect other ssDNA with many common nucleic acid fluorescent dyes. PMID:25285445

  12. Label-free and sensitive fluorescent detection of sequence-specific single-strand DNA based on S1 nuclease cleavage effects.

    PubMed

    Guan, Zheng; Liu, Jinchuan; Bai, Wenhui; Lv, Zhenzhen; Jiang, Xiaoling; Yang, Shuming; Chen, Ailiang; Lv, Guiyuan

    2014-01-01

    The ability to detect sequence-specific single-strand DNA (ssDNA) in complex, contaminant-ridden samples, using a fluorescent method directly without a DNA extraction and PCR step could simplify the detection of pathogens in the field and in the clinic. Here, we have demonstrated a simple label-free sensing strategy to detect ssDNA by employing its complementary ssDNA, S1 nuclease and nucleic acid fluorescent dyes. Upon clearing away redundant complementary ssDNA and possibly mismatched double strand DNA by using S1 nuclease, the fluorescent signal-to-noise ratio could be increased dramatically. It enabled the method to be adaptable to three different types of DNA fluorescent dyes and the ability to detect target ssDNA in complex, multicomponent samples, like tissue homogenate. The method can distinguish a two-base mismatch from avian influenza A (H1N1) virus. Also, it can detect the appearance of 50 pM target ssDNA in 0.5 µg · mL(-1) Lambda DNA, and 50 nM target ssDNA in 5 µg · mL(-1) Lambda DNA or in tissue homogenate. It is facile and cost-effective, and could be easily extended to detect other ssDNA with many common nucleic acid fluorescent dyes. PMID:25285445

  13. Opposing effects of pericentrin and microcephalin on the pericentriolar material regulate CHK1 activation in the DNA damage response.

    PubMed

    Antonczak, A K; Mullee, L I; Wang, Y; Comartin, D; Inoue, T; Pelletier, L; Morrison, C G

    2016-04-14

    Genotoxic stresses lead to centrosome amplification, a frequently-observed feature in cancer that may contribute to genome instability and to tumour cell invasion. Here we have explored how the centrosome controls DNA damage responses. For most of the cell cycle, centrosomes consist of two centrioles embedded in the proteinaceous pericentriolar material (PCM). Recent data indicate that the PCM is not an amorphous assembly of proteins, but actually a highly organised scaffold around the centrioles. The large coiled-coil protein, pericentrin, participates in PCM assembly and has been implicated in the control of DNA damage responses (DDRs) through its interactions with checkpoint kinase 1 (CHK1) and microcephalin (MCPH1). CHK1 is required for DNA damage-induced centrosome amplification, whereas MCPH1 deficiency greatly increases the amplification seen after DNA damage. We found that the PCM showed a marked expansion in volume and a noticeable change in higher-order organisation after ionising radiation treatment. PCM expansion was dependent on CHK1 kinase activity and was potentiated by MCPH1 deficiency. Furthermore, pericentrin deficiency or mutation of a separase cleavage site blocked DNA damage-induced PCM expansion. The extent of nuclear CHK1 activation after DNA damage reflected the level of PCM expansion, with a reduction in pericentrin-deficient or separase cleavage site mutant-expressing cells, and an increase in MCPH1-deficient cells that was suppressed by the loss of pericentrin. Deletion of the nuclear export signal of CHK1 led to its hyperphosphorylation after irradiation and reduced centrosome amplification. Deletion of the nuclear localisation signal led to low CHK1 activation and low centrosome amplification. From these data, we propose a feedback loop from the PCM to the nuclear DDR in which CHK1 regulates pericentrin-dependent PCM expansion to control its own activation. PMID:26165835

  14. Purification of total DNA extracted from activated sludge.

    PubMed

    Shan, Guobin; Jin, Wenbiao; Lam, Edward K H; Xing, Xinhui

    2008-01-01

    Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis treatments (lysozyme, sodium dodecyl sulfate (SDS), sonication, mechanical mill and thermal shock) on yield and purity of the total DNA extracted from activated sludge were investigated. It was found that SDS and mechanical mill were the most effective ways for cell lysis, and both gave the highest DNA yields, while by SDS and thermal shock, the purest DNA extract could be obtained. The combination of SDS with other lysis treatment, such as sonication and thermal shock, could apparently increase the DNA yields but also result in severe shearing. For the purification of the crude DNA extract, polyvinyl polypyrrolidone was used for the removal of humic contaminants. Cetyltrimethyl ammonium bromide, potassium acetate and phenol/chloroform were used to remove proteins and polysaccharides from crude DNA. Crude DNA was further purified by isopropanol precipitation. Thus, a suitable protocol was proposed for DNA extraction, yielding about 49.9 mg (total DNA)/g volatile suspended solids, and the DNA extracts were successfully used in PCR amplifications for 16S rDNA and 16S rDNA V3 region. The PCR products of 16S rDNA V3 region allowed the DGGE analysis (denatured gradient gel electrophoresis) to be possible. PMID:18572527

  15. Integrity and Biological Activity of DNA after UV Exposure

    NASA Astrophysics Data System (ADS)

    Lyon, Delina Y.; Monier, Jean-Michel; Dupraz, Sébastien; Freissinet, Caroline; Simonet, Pascal; Vogel, Timothy M.

    2010-04-01

    The field of astrobiology lacks a universal marker with which to indicate the presence of life. This study supports the proposal to use nucleic acids, specifically DNA, as a signature of life (biosignature). In addition to its specificity to living organisms, DNA is a functional molecule that can confer new activities and characteristics to other organisms, following the molecular biology dogma, that is, DNA is transcribed to RNA, which is translated into proteins. Previous criticisms of the use of DNA as a biosignature have asserted that DNA molecules would be destroyed by UV radiation in space. To address this concern, DNA in plasmid form was deposited onto different surfaces and exposed to UVC radiation. The surviving DNA was quantified via the quantitative polymerase chain reaction (qPCR). Results demonstrate increased survivability of DNA attached to surfaces versus non-adsorbed DNA. The DNA was also tested for biological activity via transformation into the bacterium Acinetobacter sp. and assaying for antibiotic resistance conferred by genes encoded by the plasmid. The success of these methods to detect DNA and its gene products after UV exposure (254 nm, 3.5 J/m2s) not only supports the use of the DNA molecule as a biosignature on mineral surfaces but also demonstrates that the DNA retained biological activity.

  16. Integrity and biological activity of DNA after UV exposure.

    PubMed

    Lyon, Delina Y; Monier, Jean-Michel; Dupraz, Sébastien; Freissinet, Caroline; Simonet, Pascal; Vogel, Timothy M

    2010-04-01

    The field of astrobiology lacks a universal marker with which to indicate the presence of life. This study supports the proposal to use nucleic acids, specifically DNA, as a signature of life (biosignature). In addition to its specificity to living organisms, DNA is a functional molecule that can confer new activities and characteristics to other organisms, following the molecular biology dogma, that is, DNA is transcribed to RNA, which is translated into proteins. Previous criticisms of the use of DNA as a biosignature have asserted that DNA molecules would be destroyed by UV radiation in space. To address this concern, DNA in plasmid form was deposited onto different surfaces and exposed to UVC radiation. The surviving DNA was quantified via the quantitative polymerase chain reaction (qPCR). Results demonstrate increased survivability of DNA attached to surfaces versus non-adsorbed DNA. The DNA was also tested for biological activity via transformation into the bacterium Acinetobacter sp. and assaying for antibiotic resistance conferred by genes encoded by the plasmid. The success of these methods to detect DNA and its gene products after UV exposure (254 nm, 3.5 J/m(2)s) not only supports the use of the DNA molecule as a biosignature on mineral surfaces but also demonstrates that the DNA retained biological activity. PMID:20446869

  17. Escherichia coli DnaE Polymerase Couples Pyrophosphatase Activity to DNA Replication

    PubMed Central

    Lapenta, Fabio; Montón Silva, Alejandro; Brandimarti, Renato; Lanzi, Massimiliano; Gratani, Fabio Lino; Vellosillo Gonzalez, Perceval; Perticarari, Sofia; Hochkoeppler, Alejandro

    2016-01-01

    DNA Polymerases generate pyrophosphate every time they catalyze a step of DNA elongation. This elongation reaction is generally believed as thermodynamically favoured by the hydrolysis of pyrophosphate, catalyzed by inorganic pyrophosphatases. However, the specific action of inorganic pyrophosphatases coupled to DNA replication in vivo was never demonstrated. Here we show that the Polymerase-Histidinol-Phosphatase (PHP) domain of Escherichia coli DNA Polymerase III α subunit features pyrophosphatase activity. We also show that this activity is inhibited by fluoride, as commonly observed for inorganic pyrophosphatases, and we identified 3 amino acids of the PHP active site. Remarkably, E. coli cells expressing variants of these catalytic residues of α subunit feature aberrant phenotypes, poor viability, and are subject to high mutation frequencies. Our findings indicate that DNA Polymerases can couple DNA elongation and pyrophosphate hydrolysis, providing a mechanism for the control of DNA extension rate, and suggest a promising target for novel antibiotics. PMID:27050298

  18. Engineered DNA ligases with improved activities in vitro.

    PubMed

    Wilson, Robert H; Morton, Susan K; Deiderick, Heather; Gerth, Monica L; Paul, Hayden A; Gerber, Ilana; Patel, Ankita; Ellington, Andrew D; Hunicke-Smith, Scott P; Patrick, Wayne M

    2013-07-01

    The DNA ligase from bacteriophage T4 is one of the most widely used enzymes in molecular biology. It has evolved to seal single-stranded nicks in double-stranded DNA, but not to join double-stranded fragments with cohesive or blunt ends. Its poor activity in vitro, particularly with blunt-ended substrates, can lead to failed or sub-optimal experimental outcomes. We have fused T4 DNA ligase to seven different DNA-binding proteins, including eukaryotic transcription factors, bacterial DNA repair proteins and archaeal DNA-binding domains. Representatives from each of these classes improved the activity of T4 DNA ligase, by up to 7-fold, in agarose gel-based screens for cohesive- and blunt-ended fragment joining. Overall, the most active variants were p50-ligase (i.e. NF-κB p50 fused to T4 DNA ligase) and ligase-cTF (T4 DNA ligase fused to an artificial, chimeric transcription factor). Ligase-cTF out-performed T4 DNA ligase by ∼160% in blunt end 'vector + insert' cloning assays, and p50-ligase showed an improvement of a similar magnitude when it was used to construct a library for Illumina sequencing. The activity of the Escherichia coli DNA ligase was also enhanced by fusion to p50. Together, these results suggest that our protein design strategy is a generalizable one for engineering improved DNA ligases. PMID:23754529

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

    PubMed Central

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

    2012-01-01

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

  20. Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE

    PubMed Central

    Sen, Doyel; Patel, Gayatri; Patel, Smita S.

    2016-01-01

    A crucial component of the human mitochondrial DNA replisome is the ring-shaped helicase TWINKLE—a phage T7-gene 4-like protein expressed in the nucleus and localized in the human mitochondria. Our previous studies showed that despite being a helicase, TWINKLE has unique DNA annealing activity. At the time, the implications of DNA annealing by TWINKLE were unclear. Herein, we report that TWINKLE uses DNA annealing function to actively catalyze strand-exchange reaction between the unwinding substrate and a homologous single-stranded DNA. Using various biochemical experiments, we demonstrate that the mechanism of strand-exchange involves active coupling of unwinding and annealing reactions by the TWINKLE. Unlike strand-annealing, the strand-exchange reaction requires nucleotide hydrolysis and greatly stimulated by short region of homology between the recombining DNA strands that promote joint molecule formation to initiate strand-exchange. Furthermore, we show that TWINKLE catalyzes branch migration by resolving homologous four-way junction DNA. These four DNA modifying activities of TWINKLE: strand-separation, strand-annealing, strand-exchange and branch migration suggest a dual role of TWINKLE in mitochondrial DNA maintenance. In addition to playing a major role in fork progression during leading strand DNA synthesis, we propose that TWINKLE is involved in recombinational repair of the human mitochondrial DNA. PMID:26887820

  1. Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE.

    PubMed

    Sen, Doyel; Patel, Gayatri; Patel, Smita S

    2016-05-19

    A crucial component of the human mitochondrial DNA replisome is the ring-shaped helicase TWINKLE-a phage T7-gene 4-like protein expressed in the nucleus and localized in the human mitochondria. Our previous studies showed that despite being a helicase, TWINKLE has unique DNA annealing activity. At the time, the implications of DNA annealing by TWINKLE were unclear. Herein, we report that TWINKLE uses DNA annealing function to actively catalyze strand-exchange reaction between the unwinding substrate and a homologous single-stranded DNA. Using various biochemical experiments, we demonstrate that the mechanism of strand-exchange involves active coupling of unwinding and annealing reactions by the TWINKLE. Unlike strand-annealing, the strand-exchange reaction requires nucleotide hydrolysis and greatly stimulated by short region of homology between the recombining DNA strands that promote joint molecule formation to initiate strand-exchange. Furthermore, we show that TWINKLE catalyzes branch migration by resolving homologous four-way junction DNA. These four DNA modifying activities of TWINKLE: strand-separation, strand-annealing, strand-exchange and branch migration suggest a dual role of TWINKLE in mitochondrial DNA maintenance. In addition to playing a major role in fork progression during leading strand DNA synthesis, we propose that TWINKLE is involved in recombinational repair of the human mitochondrial DNA. PMID:26887820

  2. Mapping Homing Endonuclease Cleavage Sites Using In Vitro Generated Protein

    PubMed Central

    Belfort, Marlene

    2015-01-01

    Mapping the precise position of endonucleolytic cleavage sites is a fundamental experimental technique used to describe the function of a homing endonuclease. However, these proteins are often recalcitrant to cloning and over-expression in biological systems because of toxicity induced by spurious DNA cleavage events. In this chapter we outline the steps to successfully express a homing endonuclease in vitro and use this product in nucleotide-resolution cleavage assays. PMID:24510259

  3. Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity.

    PubMed

    Akopian, Tatos; Kandror, Olga; Tsu, Christopher; Lai, Jack H; Wu, Wengen; Liu, Yuxin; Zhao, Peng; Park, Annie; Wolf, Lisa; Dick, Lawrence R; Rubin, Eric J; Bachovchin, William; Goldberg, Alfred L

    2015-04-24

    The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X3X2X1-aminomethylcoumarin) and by determining specificity constants (kcat/Km), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X1 position, basic residues or Trp in the X2 position, and Pro ≫ Ala > Trp in the X3 position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents. PMID:25759383

  4. Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity*

    PubMed Central

    Akopian, Tatos; Kandror, Olga; Tsu, Christopher; Lai, Jack H.; Wu, Wengen; Liu, Yuxin; Zhao, Peng; Park, Annie; Wolf, Lisa; Dick, Lawrence R.; Rubin, Eric J.; Bachovchin, William; Goldberg, Alfred L.

    2015-01-01

    The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X3X2X1-aminomethylcoumarin) and by determining specificity constants (kcat/Km), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X1 position, basic residues or Trp in the X2 position, and Pro ≫ Ala > Trp in the X3 position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents. PMID:25759383

  5. Campylobacter jejuni outer membrane vesicle-associated proteolytic activity promotes bacterial invasion by mediating cleavage of intestinal epithelial cell E-cadherin and occludin.

    PubMed

    Elmi, Abdi; Nasher, Fauzy; Jagatia, Heena; Gundogdu, Ozan; Bajaj-Elliott, Mona; Wren, Brendan; Dorrell, Nick

    2016-04-01

    Outer membrane vesicles (OMVs) play an important role in the pathogenicity of Gram-negative bacteria. Campylobacter jejuni produces OMVs that trigger IL-8, IL-6, hBD-3 and TNF-α responses from T84 intestinal epithelial cells and are cytotoxic to Caco-2 IECs and Galleria mellonella larvae. Proteomic analysis of 11168H OMVs identified the presence of three proteases, HtrA, Cj0511 and Cj1365c. In this study, 11168H OMVs were shown to possess proteolytic activity that was reduced by pretreatment with specific serine protease inhibitors. OMVs isolated from 11168H htrA, Cj0511 or Cj1365c mutants possess significantly reduced proteolytic activity. 11168H OMVs are able to cleave both E-cadherin and occludin, but this cleavage is reduced with OMVs pretreated with serine protease inhibitors and also with OMVs isolated from htrA or Cj1365c mutants. Co-incubation of T84 monolayers with 11168H OMVs results in a visible reduction in both E-cadherin and occludin. The addition of 11168H OMVs to the co-culture of live 11168H bacteria with T84 cells results in enhanced levels of bacterial adhesion and invasion in a time-dependent and dose-dependent manner. Further investigation of the cleavage of host cell structural proteins by C. jejuni OMVs should enhance our understanding of the interactions of this important pathogen with intestinal epithelial cells. PMID:26451973

  6. Analysis of the complement sensitivity of oral treponemes and the potential influence of FH binding, FH cleavage and dentilisin activity on the pathogenesis of periodontal disease

    PubMed Central

    Miller, Daniel P.; McDowell, John V.; Bell, Jessica K.; Goetting-Minesky, Melissa P.; Fenno, J. Christopher; Marconi, Richard T.

    2014-01-01

    SUMMARY Treponema denticola, a periopathogen, evades complement-mediated killing by binding the negative complement regulatory protein factor H (FH) to its surface via the FhbB protein. Paradoxically, bound FH is cleaved by T. denticola’s dentilisin protease, a process hypothesized to trigger localized dysregulation of complement activation in periodontal pockets. The ability of other oral treponemes to evade complement-mediated killing and bind and cleave FH has not been assessed. In this report, we demonstrate that representative isolates of T. socranskii, T. medium, T. pectinovorum and T. maltophilum are also serum resistant while T. vincentii and T. amylovorum are serum sensitive. While T. denticola’s ability to evade complement-mediated killing is strictly dependent on FH binding, other serum resistant treponemal species lack FhbB and do not bind FH indicating an FH-independent mechanism of complement evasion. To assess the influence of FhbB sequence variation on FH binding and cleavage by T. denticola, fhbB sequences were determined for 30 isolates. Three distinct phyletic types were identified. While all T. denticola strains bound FH and were serum resistant, differences in binding kinetics, dentilisin activity, and FH cleavage ability were observed. Based on these analyses, we hypothesize that the composition of the T. denticola population is a determining factor that influences the progression and severity of periodontal disease. PMID:24815960

  7. cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

    PubMed Central

    Hartl, M; Willnow, T; Fanning, E

    1990-01-01

    Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA. Images PMID:2159549

  8. Effect of. gamma. -irradiated DNA on the activity of DNA polymerase. [/sup 60/Co

    SciTech Connect

    Leadon, S.A.; Ward, J.F.

    1981-06-01

    A cell-free assay was developed to measure the effect of ..gamma..-irradiated DNA template on the ability of DNA polymerase to copy unirradiated template. Doses as low as 1 krad were able to decrease (approx. 15%) the activity of both bacterial and mammalian DNA polymerases in the assay. The percentage of polymerase activity decreased as the dose received by the template increased. The reduction in DNA polymerase activity was shown to be due to an inhibition of the enzyme by the irradiated DNA. Irradiated poly(dA-dT) was more effective in reducing polymerase activity than calf thymus DNA. Thus the polymerase-inhibition site(s) appears to be associated with base damage, specifically adenine or thymine. Using a free-radical scavenger, OH radicals were found to be involved in producing the damage sites. The interaction between irradiated DNA and DNA polymerase was found to be specific for the enzyme and not for other proteins present in the assay. The inhibition of DNA polymerase occurred prior to or during the initiation of DNA synthesis rather than after initiation of synthesis, i.e., during elongation.

  9. Recovery of the poisoned topoisomerase II for DNA religation: coordinated motion of the cleavage core revealed with the microsecond atomistic simulation

    PubMed Central

    Huang, Nan-Lan; Lin, Jung-Hsin

    2015-01-01

    Type II topoisomerases resolve topological problems of DNA double helices by passing one duplex through the reversible double-stranded break they generated on another duplex. Despite the wealth of information in the cleaving operation, molecular understanding of the enzymatic DNA ligation remains elusive. Topoisomerase poisons are widely used in anti-cancer and anti-bacterial therapy and have been employed to entrap the intermediates of topoisomerase IIβ with religatable DNA substrate. We removed drug molecules from the structure and conducted molecular dynamics simulations to investigate the enzyme-mediated DNA religation. The drug-unbound intermediate displayed transitions toward the resealing-compliant configuration: closing distance between the cleaved DNA termini, B-to-A transformation of the double helix, and restoration of the metal-binding motif. By mapping the contact configurations and the correlated motions between enzyme and DNA, we identified the indispensable role of the linker preceding winged helix domain (WHD) in coordinating the movements of TOPRIM, the nucleotide-binding motifs, and the bound DNA substrate during gate closure. We observed a nearly vectorial transition in the recovery of the enzyme and identified the previously uncharacterized roles of Asn508 and Arg677 in DNA rejoining. Our findings delineate the dynamic mechanism of the DNA religation conducted by type II topoisomerases. PMID:26150421

  10. Anticancer, antioxidant activities, and DNA affinity of novel monocationic bithiophenes and analogues

    PubMed Central

    Ismail, Mohamed A; Arafa, Reem K; Youssef, Magdy M; El-Sayed, Wael M

    2014-01-01

    A series of 15 monocationic bithiophenes and isosteres were prepared and subjected to in vitro antiproliferative screening using the full National Cancer Institute (NCI)-60 cell line panel, representing nine types of cancer. Among the nine types of cancer involved in a five-dose screen, non-small cell lung and breast cancer cell lines were the most responsive to the antiproliferative effect of the tested compounds, especially cell lines A549/ATCC, NCI-H322M, and NCI-H460, whereas compounds 1a, 1c, 1d, and 7 exhibited potent activity, with GI50 values (drug concentration that causes 50% inhibition of cell growth) from less than 10 nM to 102 nM. In addition, compounds 1c and 1d gave GI50 values of 73 nM and 79 nM, respectively, against the MDA-MB-468 breast cancer cell line. Structure–activity relationship findings indicated that the mononitriles were far less active than their corresponding monoamidines and, within the amidines series, the bioisosteric replacement of a thiophene ring by a furan led to a reduction in antiproliferative activity. Also, molecular manipulations, involving substitution on the phenyl ring, or its replacement by a pyridyl, or alteration of the position of the amidine group, led to significant alteration in antiproliferative activity. On the other hand, DNA studies demonstrated that these monoamidine bichalcophenes have promising ability to cleave the genomic DNA. These monoamidines show a wide range of DNA affinities, as judged from their DNA cleavage effect, which are remarkably sensitive to all kinds of structural modifications. Finally, the novel bichalcophenes were tested for their antioxidant property by the ABTS (2,2′-azino- bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt) assay, as well as lipid and nitric oxide scavenging techniques, and were found to exhibit good-to-potent antioxidant abilities. PMID:25302019

  11. 2-Alkynyl-N-propargyl pyridinium salts: pyridinium-based heterocyclic skipped aza-enediynes that cleave DNA by deoxyribosyl hydrogen-atom abstraction and guanine oxidation.

    PubMed

    Tuesuwan, Bodin; Kerwin, Sean M

    2006-06-13

    Diradical-generating cyclizations such as the enediyne Bergman cyclization and the enyne allene Myers-Saito cyclization have been exploited by nature in the mechanism of DNA cleavage by a series of potent antitumor antibiotics. Alternative diradical-generating cyclizations have been proposed in the design of selective antitumor agents; however, little information is available concerning the utility of these alternative cyclizations in radical-based DNA cleavage chemistry. One such alternative diradical-generating cyclization, the aza-Myers-Saito cyclization of aza-enyne allenes that are derived from base-promoted isomerization of skipped aza-enediynes, has been recently reported. Here, we report the synthesis and DNA cleavage chemistry of a series of pyridinium skipped aza-enediynes (2-alkynyl-N-propargyl pyridinium salts). Efficient DNA cleavage requires the presence of the skipped aza-enediyne functionality, and optimal DNA cleavage occurs at basic pH. Within this series of compounds, the analogue bearing a p-methoxyphenyl group on the pyridinium 2-alkyne substituents was found to be the most effective DNA cleavage agent, displaying significant supercoiled DNA-nicking activity at concentrations as low as 1 microM. Detailed studies of this analogue show that DNA cleavage occurs through 4'-hydrogen-atom abstraction from the DNA backbone and oxidation of guanine bases. This is the first report of enediyne-like radical-based DNA cleavage by an agent designed to undergo an alternative diradical-generating cyclization. PMID:16752915

  12. Double-stranded DNA-templated cleavage of oligonucleotides containing a P3′→N5′ linkage triggered by triplex formation: the effects of chemical modifications and remarkable enhancement in reactivity

    PubMed Central

    Ito, Kosuke Ramon; Kodama, Tetsuya; Tomizu, Masaharu; Negoro, Yoshinori; Orita, Ayako; Osaki, Tomohisa; Hosoki, Noritsugu; Tanaka, Takaya; Imanishi, Takeshi; Obika, Satoshi

    2010-01-01

    We recently reported double-stranded DNA-templated cleavage of oligonucleotides as a sequence-specific DNA-detecting method. In this method, triplex-forming oligonucleotides (TFOs) modified with 5′-amino-2′,4′-BNA were used as a DNA-detecting probe. This modification introduced a P3′→N5′ linkage (P–N linkage) in the backbone of the TFO, which was quickly cleaved under acidic conditions when it formed a triplex. The prompt fission of the P–N linkage was assumed to be driven by a conformational strain placed on the linkage upon triplex formation. Therefore, chemical modifications around the P–N linkage should change the reactivity by altering the microenvironment. We synthesized 5′-aminomethyl type nucleic acids, and incorporated them into TFOs instead of 5′-amino-2′,4′-BNA to investigate the effect of 5′-elongation. In addition, 2′,4′-BNA/LNA or 2′,5′-linked DNA were introduced at the 3′- and/or 5′-neighboring residues of 5′-amino-2′,4′-BNA to reveal neighboring residual effects. We evaluated the triplex stability and reaction properties of these TFOs, and found out that chemical modifications around the P–N linkage greatly affected their reaction properties. Notably, 2′,5′-linked DNA at the 3′ position flanking 5′-amino-2′,4′-BNA brought significantly higher reactivity, and we succeeded in indicating that a TFO with this modification is promising as a DNA analysis tool. PMID:20615902

  13. Peptidyl anthraquinones as potential antineoplastic drugs: synthesis, DNA binding, redox cycling, and biological activity.

    PubMed

    Gatto, B; Zagotto, G; Sissi, C; Cera, C; Uriarte, E; Palù, G; Capranico, G; Palumbo, M

    1996-08-01

    A series of new compounds containing a 9,10-anthracenedione moiety and one or two peptide chains at position 1 and/or 4 have been synthesized. The amino acid residues introduced are glycine (Gly), lysine (Lys), and tryptophan (Trp), the latter two in both the L- and D-configurations. The peptidyl anthraquinones maintain the ability of intercalating efficiently into DNA, even though the orientation within the base-pair pocket may change somewhat with reference to the parent drugs mitoxantrone (MX) and ametantrone (AM). The interaction constants of the mono-, di-, and triglycyl derivatives are well comparable to those found for AM but 5-10 times lower than the value reported for MX. On the other hand, the glycyl-lysyl compounds bind DNA to the same extent as (L-isomer) or even better than (D-isomer) MX. As for the parent drugs without peptidyl chains, the new compounds prefer alternating CG binding sites, although to different extents. The bis-Gly-Lys derivatives are the least sensitive to base composition, which may be due to extensive aspecific charged interactions with the polynucleotide backbone. As far as redox properties are concerned, all peptidyl anthraquinones show a reduction potential very close to that of AM and 60-80 mV less negative than that of MX; hence, they can produce free-radical-damaging species to an extent similar to the parent drugs. The biological activity has been tested in human tumor and murine leukemia cell lines. Most of the test anthraquinones exhibit cytotoxic properties close to those of AM and considerably lower than those of MX. Stimulation of topoisomerase-mediated DNA cleavage is moderately present in representatives of the glycylanthraquinone family, whereas inhibition of the background cleavage occurs when Lys is present in the peptide chain. For most of the test anthraquinones, the toxicity data are in line with the DNA affinity scale and the topoisomerase II stimulation activity. However, in the lysyl derivatives, for which

  14. miR-34 activity is modulated through 5'-end phosphorylation in response to DNA damage.

    PubMed

    Salzman, David W; Nakamura, Kotoka; Nallur, Sunitha; Dookwah, Michelle T; Metheetrairut, Chanatip; Slack, Frank J; Weidhaas, Joanne B

    2016-01-01

    MicroRNA (miRNA) expression is tightly regulated by several mechanisms, including transcription and cleavage of the miRNA precursor RNAs, to generate a mature miRNA, which is thought to be directly correlated with activity. MiR-34 is a tumour-suppressor miRNA important in cell survival, that is transcriptionally upregulated by p53 in response to DNA damage. Here, we show for the first time that there is a pool of mature miR-34 in cells that lacks a 5'-phosphate and is inactive. Following exposure to a DNA-damaging stimulus, the inactive pool of miR-34 is rapidly activated through 5'-end phosphorylation in an ATM- and Clp1-dependent manner, enabling loading into Ago2. Importantly, this mechanism of miR-34 activation occurs faster than, and independently of, de novo p53-mediated transcription and processing. Our study reveals a novel mechanism of rapid miRNA activation in response to environmental stimuli occurring at the mature miRNA level. PMID:26996824

  15. Enzymatic Activities and DNA Substrate Specificity of Mycobacterium tuberculosis DNA Helicase XPB

    PubMed Central

    Balasingham, Seetha V.; Zegeye, Ephrem Debebe; Homberset, Håvard; Rossi, Marie L.; Laerdahl, Jon K.; Bohr, Vilhelm A.; Tønjum, Tone

    2012-01-01

    XPB, also known as ERCC3 and RAD25, is a 3′→5′ DNA repair helicase belonging to the superfamily 2 of helicases. XPB is an essential core subunit of the eukaryotic basal transcription factor complex TFIIH. It has two well-established functions: in the context of damaged DNA, XPB facilitates nucleotide excision repair by unwinding double stranded DNA (dsDNA) surrounding a DNA lesion; while in the context of actively transcribing genes, XPB facilitates initiation of RNA polymerase II transcription at gene promoters. Human and other eukaryotic XPB homologs are relatively well characterized compared to conserved homologs found in mycobacteria and archaea. However, more insight into the function of bacterial helicases is central to understanding the mechanism of DNA metabolism and pathogenesis in general. Here, we characterized Mycobacterium tuberculosis XPB (Mtb XPB), a 3′→5′ DNA helicase with DNA-dependent ATPase activity. Mtb XPB efficiently catalyzed DNA unwinding in the presence of significant excess of enzyme. The unwinding activity was fueled by ATP or dATP in the presence of Mg2+/Mn2+. Consistent with the 3′→5′ polarity of this bacterial XPB helicase, the enzyme required a DNA substrate with a 3′ overhang of 15 nucleotides or more. Although Mtb XPB efficiently unwound DNA model substrates with a 3′ DNA tail, it was not active on substrates containing a 3′ RNA tail. We also found that Mtb XPB efficiently catalyzed ATP-independent annealing of complementary DNA strands. These observations significantly enhance our understanding of the biological roles of Mtb XPB. PMID:22615856

  16. High-Throughput Genotyping of Green Algal Mutants Reveals Random Distribution of Mutagenic Insertion Sites and Endonucleolytic Cleavage of Transforming DNA[W][OPEN

    PubMed Central

    Zhang, Ru; Patena, Weronika; Armbruster, Ute; Gang, Spencer S.; Blum, Sean R.; Jonikas, Martin C.

    2014-01-01

    A high-throughput genetic screening platform in a single-celled photosynthetic eukaryote would be a transformative addition to the plant biology toolbox. Here, we present ChlaMmeSeq (Chlamydomonas MmeI-based insertion site Sequencing), a tool for simultaneous mapping of tens of thousands of mutagenic insertion sites in the eukaryotic unicellular green alga Chlamydomonas reinhardtii. We first validated ChlaMmeSeq by in-depth characterization of individual insertion sites. We then applied ChlaMmeSeq to a mutant pool and mapped 11,478 insertions, covering 39% of annotated protein coding genes. We observe that insertions are distributed in a manner largely indistinguishable from random, indicating that mutants in nearly all genes can be obtained efficiently. The data reveal that sequence-specific endonucleolytic activities cleave the transforming DNA and allow us to propose a simple model to explain the origin of the poorly understood exogenous sequences that sometimes surround insertion sites. ChlaMmeSeq is quantitatively reproducible, enabling its use for pooled enrichment screens and for the generation of indexed mutant libraries. Additionally, ChlaMmeSeq allows genotyping of hits from Chlamydomonas screens on an unprecedented scale, opening the door to comprehensive identification of genes with roles in photosynthesis, algal lipid metabolism, the algal carbon-concentrating mechanism, phototaxis, the biogenesis and function of cilia, and other processes for which C. reinhardtii is a leading model system. PMID:24706510

  17. Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system

    PubMed Central

    Liu, Xiaoxi; Homma, Ayaka; Sayadi, Jamasb; Yang, Shu; Ohashi, Jun; Takumi, Toru

    2016-01-01

    The CRISPR-Cas9 system has recently emerged as a versatile tool for biological and medical research. In this system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. In addition to this targeting function, the sgRNA has also been shown to play a role in activating the endonuclease activity of Cas9. This dual function of the sgRNA likely underlies observations that different sgRNAs have varying on-target activities. Currently, our understanding of the relationship between sequence features of sgRNAs and their on-target cleavage efficiencies remains limited, largely due to difficulties in assessing the cleavage capacity of a large number of sgRNAs. In this study, we evaluated the cleavage activities of 218 sgRNAs using in vitro Surveyor assays. We found that nucleotides at both PAM-distal and PAM-proximal regions of the sgRNA are significantly correlated with on-target efficiency. Furthermore, we also demonstrated that the genomic context of the targeted DNA, the GC percentage, and the secondary structure of sgRNA are critical factors contributing to cleavage efficiency. In summary, our study reveals important parameters for the design of sgRNAs with high on-target efficiencies, especially in the context of high throughput applications. PMID:26813419

  18. Cytotoxic, antibacterial, DNA interaction and superoxide dismutase like activities of sparfloxacin drug based copper(II) complexes with nitrogen donor ligands.

    PubMed

    Patel, Mohan N; Joshi, Hardik N; Patel, Chintan R

    2013-03-01

    The novel neutral mononuclear copper(II) complexes with fluoroquinolone antibacterial drug, sparfloxacin and nitrogen donor heterocyclic ligand have been synthesized and characterized. An antimicrobial efficiency of the complexes has been tested against five different microorganisms and showed diverse biological activity. The interaction of complex with Herring sperm (HS) DNA was investigated using viscosity titration and absorption titration techniques. The results indicate that the complexes bind to DNA by intercalative mode and have rather high DNA-binding constants. DNA cleavage study showed better cleaving ability of the complexes compare to metal salt and standard drug. All the complexes showed good cytotoxic activity with LC(50) values ranging from 4.89 to 11.94 μg mL(-1). Complexes also exhibit SOD-like activity with their IC(50) values ranging from 0.717 to 1.848 μM. PMID:23266675

  19. Cytotoxic, antibacterial, DNA interaction and superoxide dismutase like activities of sparfloxacin drug based copper(II) complexes with nitrogen donor ligands

    NASA Astrophysics Data System (ADS)

    Patel, Mohan N.; Joshi, Hardik N.; Patel, Chintan R.

    2013-03-01

    The novel neutral mononuclear copper(II) complexes with fluoroquinolone antibacterial drug, sparfloxacin and nitrogen donor heterocyclic ligand have been synthesized and characterized. An antimicrobial efficiency of the complexes has been tested against five different microorganisms and showed diverse biological activity. The interaction of complex with Herring sperm (HS) DNA was investigated using viscosity titration and absorption titration techniques. The results indicate that the complexes bind to DNA by intercalative mode and have rather high DNA-binding constants. DNA cleavage study showed better cleaving ability of the complexes compare to metal salt and standard drug. All the complexes showed good cytotoxic activity with LC50 values ranging from 4.89 to 11.94 μg mL-1. Complexes also exhibit SOD-like activity with their IC50 values ranging from 0.717 to 1.848 μM.

  20. EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    PubMed Central

    Serapio-Palacios, Antonio

    2016-01-01

    ABSTRACT Enteropathogenic Escherichia coli (EPEC) has the ability to antagonize host apoptosis during infection through promotion and inhibition of effectors injected by the type III secretion system (T3SS), but the total number of these effectors and the overall functional relationships between these effectors during infection are poorly understood. EspC produced by EPEC cleaves fodrin, paxillin, and focal adhesion kinase (FAK), which are also cleaved by caspases and calpains during apoptosis. Here we show the role of EspC in cell death induced by EPEC. EspC is involved in EPEC-mediated cell death and induces both apoptosis and necrosis in epithelial cells. EspC induces apoptosis through the mitochondrial apoptotic pathway by provoking (i) a decrease in the expression levels of antiapoptotic protein Bcl-2, (ii) translocation of the proapoptotic protein Bax from cytosol to mitochondria, (iii) cytochrome c release from mitochondria to the cytoplasm, (iv) loss of mitochondrial membrane potential, (v) caspase-9 activation, (vi) cleavage of procaspase-3 and (vii) an increase in caspase-3 activity, (viii) PARP proteolysis, and (ix) nuclear fragmentation and an increase in the sub-G1 population. Interestingly, EspC-induced apoptosis was triggered through a dual mechanism involving both independent and dependent functions of its EspC serine protease motif, the direct cleavage of procaspase-3 being dependent on this motif. This is the first report showing a shortcut for induction of apoptosis by the catalytic activity of an EPEC protein. Furthermore, this atypical intrinsic apoptosis appeared to induce necrosis through the activation of calpain and through the increase of intracellular calcium induced by EspC. Our data indicate that EspC plays a relevant role in cell death induced by EPEC. PMID:27329750

  1. kuzbanian-mediated cleavage of Drosophila Notch

    PubMed Central

    Lieber, Toby; Kidd, Simon; Young, Michael W.

    2002-01-01

    Loss of Kuzbanian, a member of the ADAM family of metalloproteases, produces neurogenic phenotypes in Drosophila. It has been suggested that this results from a requirement for kuzbanian-mediated cleavage of the Notch ligand Delta. Using transgenic Drosophila expressing transmembrane Notch proteins, we show that kuzbanian, independent of any role in Delta processing, is required for the cleavage of Notch. We show that Kuzbanian can physically associate with Notch and that removal of kuzbanian activity by RNA-mediated interference in Drosophila tissue culture cells eliminates processing of ligand-independent transmembrane Notch molecules. Our data suggest that in Drosophila, kuzbanian can mediate S2 cleavage of Notch. PMID:11799064

  2. Synthesis, characterization, antibacterial activity, SOD mimic and interaction with DNA of drug based copper(II) complexes

    NASA Astrophysics Data System (ADS)

    Patel, Mohan N.; Dosi, Promise A.; Bhatt, Bhupesh S.; Thakkar, Vasudev R.

    2011-02-01

    Novel metal complexes of the second-generation quinolone antibacterial agent enrofloxacin with copper(II) and neutral bidentate ligands have been prepared and characterized with elemental analysis reflectance, IR and mass spectroscopy. Complexes have been screened for their in-vitro antibacterial activity against two Gram (+ve)Staphylococcus aureus, Bacillus subtilis, and three Gram (-ve)Serratia marcescens, Escherichia coli and Pseudomonas aeruginosa organisms using the double dilution technique. The binding of this complex with CT-DNA has been investigated by absorption titration, salt effect and viscosity measurements. Binding constant is ranging from 1.3 × 10 4-3.7 × 10 4. The cleavage ability of complexes has been assessed by gel electrophoresis using pUC19 DNA. The catalytic activity of the copper(II) complexes towards the superoxide anion (O 2rad -) dismutation was assayed by their ability to inhibit the reduction of nitroblue tetrazolium (NBT).

  3. Synthesis, characterization, antibacterial activity, SOD mimic and interaction with DNA of drug based copper(II) complexes.

    PubMed

    Patel, Mohan N; Dosi, Promise A; Bhatt, Bhupesh S; Thakkar, Vasudev R

    2011-02-01

    Novel metal complexes of the second-generation quinolone antibacterial agent enrofloxacin with copper(II) and neutral bidentate ligands have been prepared and characterized with elemental analysis reflectance, IR and mass spectroscopy. Complexes have been screened for their in-vitro antibacterial activity against two Gram(+ve) Staphylococcus aureus, Bacillus subtilis, and three Gram((-ve)) Serratia marcescens, Escherichia coli and Pseudomonas aeruginosa organisms using the double dilution technique. The binding of this complex with CT-DNA has been investigated by absorption titration, salt effect and viscosity measurements. Binding constant is ranging from 1.3×10(4)-3.7×10(4). The cleavage ability of complexes has been assessed by gel electrophoresis using pUC19 DNA. The catalytic activity of the copper(II) complexes towards the superoxide anion (O2.-) dismutation was assayed by their ability to inhibit the reduction of nitroblue tetrazolium (NBT). PMID:21212015

  4. Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity.

    PubMed Central

    Skerra, A

    1992-01-01

    Two thermostable DNA polymerases with proofreading activity--Vent DNA polymerase and Pfu DNA polymerase--have attracted recent attention, mainly because of their enhanced fidelities during amplification of DNA sequences by the polymerase chain reaction. A severe disadvantage for their practical application, however, results from the observation that due to their 3' to 5' exonuclease activities these enzymes degrade the oligodeoxynucleotides serving as primers for the DNA synthesis. It is demonstrated that this exonucleolytic attack on the primer molecules can be efficiently prevented by the introduction of single phosphorothioate bonds at their 3' termini. This strategy, which can be easily accomplished using routine DNA synthesis methodology, may open the way to a widespread use of these novel enzymes in the polymerase chain reaction. Images PMID:1641322

  5. An arcane role of DNA in transcription activation.

    PubMed Central

    Ryu, S; Garges, S; Adhya, S

    1994-01-01

    The mechanism by which the cAMP receptor protein (CRP) activates transcription has been investigated using the lac promoter of Escherichia coli. For transcription activation, an interaction between DNA-bound CRP and RNA polymerase is not sufficient. CRP must bind to a site in the same DNA and close to the promoter. CRP action requires an intact spacer DNA to provide a rigid support in building a CRP-RNA polymerase protein bridge or to allow a conformational change in the DNA to be transmitted to the lac promoter using the protein bridge as a structural support. Images PMID:7811325

  6. A DNA-recombinogenic activity in human cells.

    PubMed Central

    Kenne, K; Ljungquist, S

    1984-01-01

    A DNA recombining protein has been partly purified from cell lines derived from patients suffering from the hereditary disease, Bloom's syndrome. The protein induces the formation of displacement loops in phi X174 RFI DNA molecules after the addition of single-stranded DNA fragments. A filter binding method and electron microscopy were used to determine the reaction. The recombinogenic protein is dependent on divalent cations and ATP for activity. Images PMID:6232501

  7. Leukocyte Protease Binding to Nucleic Acids Promotes Nuclear Localization and Cleavage of Nucleic Acid Binding Proteins

    PubMed Central

    Thomas, Marshall P.; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-01-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. Here we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein (RBP) targets, while adding RNA to recombinant RBP substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Pre-incubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G (CATG). During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps (NETs), which bind NE and CATG. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and NETs in a DNA-dependent manner. Thus, high affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation. PMID:24771851

  8. Reversible Condensation of DNA using a Redox-Active Surfactant

    PubMed Central

    Hays, Melissa E.; Jewell, Christopher M.; Lynn, David M.; Abbott, Nicholas L.

    2008-01-01

    We report characterization of aqueous solutions of dilute Lambda phage DNA containing the redox-active surfactant (11-ferrocenylundecyl)trimethylammonium bromide (FTMA) as a function of the oxidation state of the FTMA. FTMA undergoes a reversible one-electron oxidation from a reduced state that forms micelles in aqueous solution to an oxidized state (containing the ferrocenium cation) that does not selfassociate in solution. This investigation sought to test the hypothesis that FTMA can be used to achieve reversible control over the conformation of DNA-surfactant complexes in solution. Whereas DNA adopts extended coil conformations in aqueous solutions, our measurements revealed that addition of reduced FTMA (2–5μM) to aqueous solutions of DNA (5 μM in nucleotide units) resulted in coexistence of extended coils and compact globules in solution. At higher concentrations of reduced FTMA (up to 30μM), the DNA was present as compact globules only. In contrast, oxidized FTMA had no measurable effect on the conformation of DNA, allowing DNA to maintain an extended coil state up to a concentration of 75μM oxidized FTMA. We further demonstrate that it is possible to chemically or electrochemically transform the oxidation state of FTMA in preformed complexes of FTMA and DNA, thus achieving in situ control over the conformations of the DNA in solution. These results provide guidance for the design of surfactant systems that permit active control of DNA-surfactant interactions. PMID:17428073

  9. Hepatitis B virus: DNA polymerase activity of deletion mutants.

    PubMed

    Kim, Y; Hong, Y B; Jung, G

    1999-02-01

    The hepadnavirus P gene product is a multifunctional protein with priming, DNA- and RNA-dependent DNA polymerase, and RNase H activities. Nested N- or C-terminal deletion mutations and deletions of domain(s) in human HBV polymerase have been made. Wild-type and deletion forms of MBP-fused HBV polymerase were expressed in E. coli, purified by amylose column chromatography, and the DNA-dependent DNA polymerase activities of the purified proteins were compared. Deletion of the terminal protein or spacer regions reduced enzyme activity to 70%, respectively. However, deletion of the RNase H domain affected polymerase activity more than that of the terminal protein or spacer region. The polymerase domain alone or the N-terminal deletion of the polymerase domain still exhibited enzymatic activity. In this report, it is demonstrated that the minimal domain for the polymerizing activity of the HBV polymerase is smaller than the polymerase domain. PMID:10205676

  10. Sphingosine, a modulator of human translesion DNA polymerase activity.

    PubMed

    Kamath-Loeb, Ashwini S; Balakrishna, Sharath; Whittington, Dale; Shen, Jiang-Cheng; Emond, Mary J; Okabe, Takayoshi; Masutani, Chikahide; Hanaoka, Fumio; Nishimura, Susumu; Loeb, Lawrence A

    2014-08-01

    Translesion (TLS) DNA polymerases are specialized, error-prone enzymes that synthesize DNA across bulky, replication-stalling DNA adducts. In so doing, they facilitate the progression of DNA synthesis and promote cell proliferation. To potentiate the effect of cancer chemotherapeutic regimens, we sought to identify inhibitors of TLS DNA polymerases. We screened five libraries of ∼ 3000 small molecules, including one comprising ∼ 600 nucleoside analogs, for their effect on primer extension activity of DNA polymerase η (Pol η). We serendipitously identified sphingosine, a lipid-signaling molecule that robustly stimulates the activity of Pol η by ∼ 100-fold at low micromolar concentrations but inhibits it at higher concentrations. This effect is specific to the Y-family DNA polymerases, Pols η, κ, and ι. The addition of a single phosphate group on sphingosine completely abrogates this effect. Likewise, the inclusion of other sphingolipids, including ceramide and sphingomyelin to extension reactions does not elicit this response. Sphingosine increases the rate of correct and incorrect nucleotide incorporation while having no effect on polymerase processivity. Endogenous Pol η activity is modulated similarly as the recombinant enzyme. Importantly, sphingosine-treated cells exhibit increased lesion bypass activity, and sphingosine tethered to membrane lipids mimics the effects of free sphingosine. Our studies have uncovered sphingosine as a modulator of TLS DNA polymerase activity; this property of sphingosine may be associated with its known role as a signaling molecule in regulating cell proliferation in response to cellular stress. PMID:24928506

  11. Enhanced DPPH radical scavenging activity and DNA protection effect of litchi pericarp extract by Aspergillus awamori bioconversion

    PubMed Central

    2012-01-01

    Background Litchi (Litchi chinensis Sonn.) pericarp is a major byproduct which contains a significant amount of polyphenol. This study was designed to biotransformation litchi pericarp extract (LPE) by Aspergillus awamori to produce more bioactive compounds with stronger antioxidant activities. Results The study exhibited that the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities significantly (p < 0.05) increased from 15.53% to 18.23% in the water-extracted fraction and from 25.41% to 36.82% in the ethyl acetate-extracted fraction. Application of DNA cleavage assay further demonstrated the enhanced protection effect of the fermented phenolics on DNA damage. It is also noted that the water-extracted fraction of the fermented LPE possessed a much stronger capacity than the ethyl acetate-extracted fraction to prevent from damage of supercoiled DNA. Interestingly, it was found that some new compounds such as catechin and quercetin appeared after of A. awamori fermentation of LPE, which could account for the enhanced antioxidant activity. Conclusion The DPPH radical scavenging activity and DNA protection effect of LPE were increased by Aspergillus awamori bioconversion while some compounds responsible for the enhanced antioxidant activity were identified. This study provided an effective way of utilizing fruit pericarp as a readily accessible source of the natural antioxidants in food industry and, thus, extended the application area such as fruit by-products. PMID:23016522

  12. Metabolic activation of carcinogenic ethylbenzene leads to oxidative DNA damage.

    PubMed

    Midorikawa, Kaoru; Uchida, Takafumi; Okamoto, Yoshinori; Toda, Chitose; Sakai, Yoshie; Ueda, Koji; Hiraku, Yusuke; Murata, Mariko; Kawanishi, Shosuke; Kojima, Nakao

    2004-12-01

    Ethylbenzene is carcinogenic to rats and mice, while it has no mutagenic activity. We have investigated whether ethylbenzene undergoes metabolic activation, leading to DNA damage. Ethylbenzene was metabolized to 1-phenylethanol, acetophenone, 2-ethylphenol and 4-ethylphenol by rat liver microsomes. Furthermore, 2-ethylphenol and 4-ethylphenol were metabolically transformed to ring-dihydroxylated metabolites such as ethylhydroquinone and 4-ethylcatechol, respectively. Experiment with 32P-labeled DNA fragment revealed that both ethylhydroquinone and 4-ethylcatechol caused DNA damage in the presence of Cu(II). These dihydroxylated compounds also induced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in calf thymus DNA in the presence of Cu(II). Catalase, methional and Cu(I)-specific chelator, bathocuproine, significantly (P<0.05) inhibited oxidative DNA damage, whereas free hydroxyl radical scavenger and superoxide dismutase did not. These results suggest that Cu(I) and H2O2 produced via oxidation of ethylhydroquinone and 4-ethylcatechol are involved in oxidative DNA damage. Addition of an endogenous reductant NADH dramatically enhanced 4-ethylcatechol-induced oxidative DNA damage, whereas ethylhydroquinone-induced DNA damage was slightly enhanced. Enhancing effect of NADH on oxidative DNA damage by 4-ethylcatechol may be explained by assuming that reactive species are generated from the redox cycle. In conclusion, these active dihydroxylated metabolites would be involved in the mechanism of carcinogenesis by ethylbenzene. PMID:15560893

  13. Mutation, DNA strand cleavage and nitric oxide formation caused by N-nitrosoproline with sunlight: a possible mechanism of UVA carcinogenicity.

    PubMed

    Arimoto-Kobayashi, Sakae; Ando, Yoshiko; Horai, Yumi; Okamoto, Keinosuke; Hayatsu, Hikoya; Lowe, Jillian E; Green, Michael H L

    2002-09-01

    N-Nitrosoproline (NPRO) is endogenously formed from proline and nitrite. NPRO has been reported to be nonmutagenic and noncarcinogenic. In this study, we have detected the direct mutagenicity of NPRO plus natural sunlight towards Salmonella typhimurium. Furthermore, formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a mutagenic lesion, was observed in calf thymus DNA treated with NPRO plus simulated sunlight. The treatment with NPRO and sunlight induced single strand breaks in the superhelical replicative form of phage M13mp2 DNA. Single-strand DNA breaks also occurred in the human fibroblast cells on treatment with NPRO plus UVA, as detected by the comet assay. An analysis using scavengers suggested that both reactive oxygen species and NO radical mediate the strand breaks. The formation of nitric oxide was observed in NPRO solution irradiated with UVA. We analyzed the photodynamic spectrum of mutation induction and DNA breakage using monochromatic radiation at a series of wavelengths between 300 and 400 nm. Both mutation frequencies and DNA breakage were highest at the absorption maximum of NPRO, 340 nm. The co-mutagenic and co-toxic actions of NPRO and sunlight merit attention as possible mechanisms increasing the carcinogenic risk from UVA irradiation. PMID:12189198

  14. DNA-gold nanoparticles network based electrochemical biosensors for DNA MTase activity.

    PubMed

    Hong, Lu; Wan, Jing; Zhang, Xiaojun; Wang, Guangfeng

    2016-05-15

    In this work, a highly sensitive electrochemical DNA methyltransferase (MTase) activity assay was fabricated with DNA-gold nanoparticles (Au NPs) network as signal amplification unit and an easy assembly method by the linkage of benzenedithiol bridge. By two complementary AuNPs modified single-stranded DNA, DNA-gold nanoparticles network was self-assembled. With the linkage of benzenedithiol bridge, the DNA network structure was immobilized on the surface of gold electrode through the covalent Au-S bond. In the presence of Dam MTase, the special sites of DNA-AuNPs network were methylated and could not be digested by restriction endonuclease Mbo I. Thus the loaded electrochemical indicator Methylene blue (MB) was MB molecules still remained on the DNA-Au NPs network. The electrochemical response depended on the methylated degree, which could be used to detect MTase activity. By the differential pulse voltammetry (DPV), it was demonstrated that a linear relationship between the DPV response and logarithm of Dam concentration ranged from 0.075 to 30U/mL, achieving a low detection limit of 0.02U/mL. The use of benzenedithiol avoided the direct incubation of the solid electrode with the capture DNA probe under complex and harsh conditions. Therefore the immobilization of DNA-AuNPs network was easy to be carried out, which is favorable for the specially high stability and reproducibility of the electrochemical biosensor. PMID:26992515

  15. Inhibition of DNA damage repair by artificial activation of PARP with siDNA.

    PubMed

    Croset, Amelie; Cordelières, Fabrice P; Berthault, Nathalie; Buhler, Cyril; Sun, Jian-Sheng; Quanz, Maria; Dutreix, Marie

    2013-08-01

    One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations. PMID:23761435

  16. Synthesis, characterization and crystal structure of cobalt(III) complexes containing 2-acetylpyridine thiosemicarbazones: DNA/protein interaction, radical scavenging and cytotoxic activities.

    PubMed

    Manikandan, Rajendran; Viswanathamurthi, Periasamy; Velmurugan, Krishnaswamy; Nandhakumar, Raju; Hashimoto, Takeshi; Endo, Akira

    2014-01-01

    The synthesis, structure and biological studies of cobalt(III) complexes supported by NNS-tridentate ligands are reported. Reactions of 2-acetylpyridine N-substituted thiosemicarbazone (HL(1-3)) with [CoCl2(PPh3)2] resulted [Co(L(1-3))2]Cl (1-3) which were characterized by elemental analysis and various spectral studies. The molecular structure of the complex 1 has been determined by single crystal X-ray diffraction studies. In vitro DNA binding studies of complexes 1-3 carried out by fluorescence studies and the results revealed the binding of complexes to DNA via intercalation. The binding constant (Kb) values of complexes 1-3 from fluorescence experiments showed that the complex 3 has greater binding propensity for DNA. The DNA cleavage activity of the complexes 1 and 3 were ascertained by gel electrophoresis assay which revealed that the complexes are good DNA cleavage agents. Further, the interactions of the complexes with bovine serum albumin (BSA) were also investigated using fluorescence spectroscopic method, which showed that the complexes 1-3 could bind strongly with BSA. The antioxidant property of the complexes was evaluated to test their free-radical scavenging ability. Furthermore, in vitro cytotoxicity of the complexes against MCF-7 and A431 cell lines was assayed which showed higher activity and efficiently vanished the cancer cells even at low concentrations. PMID:24342132

  17. General Base Catalysis for Cleavage by the Active-Site Cytosine of the Hepatitis Delta Virus Ribozyme: QM/MM Calculations Establish Chemical Feasibility

    PubMed Central

    Banáš, Pavel; Rulíšek, Lubomír; Hánošová, Veronika; Svozil, Daniel; Walter, Nils G.

    2008-01-01

    The hepatitis delta virus (HDV) ribozyme is an RNA motif embedded in human pathogenic HDV RNA. Previous experimental studies have established that the active-site nucleotide C75 is essential for self-cleavage of the ribozyme, although its exact catalytic role in the process remains debated. Structural data from X-ray crystallography generally indicate that C75 acts as the general base that initiates catalysis by deprotonating the 2′-OH nucleophile at the cleavage site, while a hydrated magnesium ion likely protonates the 5′-oxygen leaving group. In contrast, some mechanistic studies support the role of C75 acting as general acid and thus being protonated before the reaction. We report combined quantum chemical/molecular mechanical calculations for the C75 general base pathway, utilizing the available structural data for the wild type HDV genomic ribozyme as a starting point. Several starting configurations differing in magnesium ion placement were considered and both one-dimensional and two-dimensional potential energy surface scans were used to explore plausible reaction paths. Our calculations show that C75 is readily capable of acting as the general base, in concert with the hydrated magnesium ion as the general acid. We identify a most likely position for the magnesium ion, which also suggests it acts as a Lewis acid. The calculated energy barrier of the proposed mechanism, ~20 kcal/mol, would lower the reaction barrier by ~15 kcal/mol compared to the uncatalyzed reaction and is in good agreement with experimental data. PMID:18686993

  18. Positioning Effects of KillerRed inside of Cells correlate with DNA Strand Breaks after Activation with Visible Light

    PubMed Central

    Waldeck, Waldemar; Mueller, Gabriele; Wiessler, Manfred; Tóth, Katalin; Braun, Klaus

    2011-01-01

    Fluorescent proteins (FPs) are established tools for new applications, not-restricted to the cell biological research. They could also be ideal in surgery enhancing the precision to differentiate between the target tissue and the surrounding healthy tissue. FPs like the KillerRed (KRED), used here, can be activated by excitation with visible day-light for emitting active electrons which produce reactive oxygen species (ROS) resulting in photokilling processes. It is a given that the extent of the KRED's cell toxicity depends on its subcellular localization. Evidences are documented that the nuclear lamina as well as especially the chromatin are critical targets for KRED-mediated ROS-based DNA damaging. Here we investigated the damaging effects of the KRED protein fused to the nuclear lamina and to the histone H2A DNA-binding protein. We detected a frequency of DNA strand breaks, dependent first on the illumination time, and second on the spatial distance between the localization at the chromatin and the site of ROS production. As a consequence we could identify defined DNA bands with 200, 400 and (600) bps as most prominent degradation products, presumably representing an internucleosomal DNA cleavage induced by KRED. These findings are not restricted to the detection of programmed cell death processes in the therapeutic field like PDT, but they can also contribute to a better understanding of the structure-function relations in the epigenomic world. PMID:21278894

  19. DNA interaction, SOD, peroxidase and nuclease activity studies of iron complex having ligand with carboxamido nitrogen donors

    NASA Astrophysics Data System (ADS)

    Ghosh, Kaushik; Tyagi, Nidhi; Kumar, Hemant; Rathi, Sweety

    2015-07-01

    Complex (Et3HN)[FeIII(bpb)Cl2], 1 {where H2bpb: N,N‧-(1,2-phenylene)bis(pyridine-2-carboxamide)} was synthesized and characterized by reported procedure (Yang et al., 1991). Complex 1 was found to be effective in superoxide scavenging activity and an IC50 value of 4.1 μM was obtained in xanthine-xanthine oxidase nitro blue tetrazolium assay. Peroxidase-like activity of this complex was determined by the oxidation of 2,2‧-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). DNA interaction studies of complex 1 showed binding of DNA through external or groove binding. Complex 1 exhibited chemical nuclease activity in the presence of hydrogen peroxide and cleaved supercoiled pBR322 DNA to its linear and nicked circular form at physiological pH. Mechanistic studies indicated possible role of hydroxyl radical (radOH) species in DNA cleavage activity via hydroperoxo intermediate: [FeIIIsbnd OOH-]2+ → [FeIVdbnd O]2+ + radOH.

  20. Inactivation efficiencies of radical reactions with biologically active DNA

    NASA Astrophysics Data System (ADS)

    Lafleur, M. V. M.; Retèl, J.; Loman, H.

    Dilute aqueous solutions of biologically active θX174 DNA may serve as a simplified model system of the cell. Damage to the DNA after irradiation with γ-rays, may be ascribed to reactions with .OH, .H and e -aq or secondary radicals, arising from reactions of water radicals with added scavengers. Conversion of primary (water) radicals into secondary (scavenger) radicals leads to a considerable protection of the DNA, which, however, would have been larger if these secondary radicals did not contribute to DNA inactivation. The inactivation yield due to isopropanol or formate (secondary) radicals depends on dose rate as well as DNA concentration. Furthermore the inactivation efficiencies of the reactions of both the primary and the secondary radicals with single-stranded DNA could be established.

  1. Requirement for End-Joining and Checkpoint Functions, but Not RAD52-Mediated Recombination, after EcoRI Endonuclease Cleavage of Saccharomyces cerevisiae DNA

    PubMed Central

    Lewis, L. Kevin; Kirchner, Jakob M.; Resnick, Michael A.

    1998-01-01

    RAD52 and RAD9 are required for the repair of double-strand breaks (DSBs) induced by physical and chemical DNA-damaging agents in Saccharomyces cerevisiae. Analysis of EcoRI endonuclease expression in vivo revealed that, in contrast to DSBs containing damaged or modified termini, chromosomal DSBs retaining complementary ends could be repaired in rad52 mutants and in G1-phase Rad+ cells. Continuous EcoRI-induced scission of chromosomal DNA blocked the growth of rad52 mutants, with most cells arrested in G2 phase. Surprisingly, rad52 mutants were not more sensitive to EcoRI-induced cell killing than wild-type strains. In contrast, endonuclease expression was lethal in cells deficient in Ku-mediated end joining. Checkpoint-defective rad9 mutants did not arrest cell cycling and lost viability rapidly when EcoRI was expressed. Synthesis of the endonuclease produced extensive breakage of nuclear DNA and stimulated interchromosomal recombination. These results and those of additional experiments indicate that cohesive ended DSBs in chromosomal DNA can be accurately repaired by RAD52-mediated recombination and by recombination-independent complementary end joining in yeast cells. PMID:9528760

  2. C1q-TNF-related protein-9, a novel cardioprotetcive cardiokine, requires proteolytic cleavage to generate a biologically active globular domain isoform.

    PubMed

    Yuan, Yuexing; Lau, Wayne Bond; Su, Hui; Sun, Yang; Yi, Wei; Du, Yunhui; Christopher, Theodore; Lopez, Bernard; Wang, Yajing; Ma, Xin-Liang

    2015-05-15

    Prevalence and severity of postmyocardial infarction heart failure continually escalate in type 2 diabetes via incompletely understood mechanisms. The discovery of the cardiac secretomes, collectively known as "cardiokines", has significantly enhanced appreciation of the local microenvironment's influence on disease development. Recent studies demonstrated that C1q-TNF-related protein-9 (CTRP9), a newly discovered adiponectin (APN) paralog, is highly expressed in the heart. However, its relationship with APN (concerning diabetic cardiovascular injury in particular) remains unknown. Plasma CTRP9 levels are elevated in APN knockout and reduced in diabetic mice. In contrast to APN, which circulates as full-length multimers, CTRP9 circulates in the plasma primarily in the globular domain isoform (gCTRP9). Recombinant full-length CTRP9 (fCTRP9) was cleaved when incubated with cardiac tissue extracts, generating gCTRP9, a process inhibited by protease inhibitor cocktail. gCTRP9 rapidly activates cardiac survival kinases, including AMPK, Akt, and endothelial NOS. However, fCTRP9-mediated kinase activation is much less potent and significantly delayed. Kinase activation by fCTRP9, but not gCTRP9, is inhibited by protease inhibitor cocktail. These results demonstrate for the first time that the novel cardiokine CTRP9 undergoes proteolytic cleavage to generate gCTRP9, the dominant circulatory and actively cardioprotective isoform. Enhancing cardiac CTRP9 production and/or its proteolytic posttranslational modification are of therapeutic potential, attenuating diabetic cardiac injury. PMID:25783894

  3. Activation and Regulation of DNA-Driven Immune Responses

    PubMed Central

    2015-01-01

    SUMMARY The innate immune system provides early defense against infections and also plays a key role in monitoring alterations of homeostasis in the body. DNA is highly immunostimulatory, and recent advances in this field have led to the identification of the innate immune sensors responsible for the recognition of DNA as well as the downstream pathways that are activated. Moreover, information on how cells regulate DNA-driven immune responses to avoid excessive inflammation is now emerging. Finally, several reports have demonstrated how defects in DNA sensing, signaling, and regulation are associated with susceptibility to infections or inflammatory diseases in humans and model organisms. In this review, the current literature on DNA-stimulated innate immune activation is discussed, and important new questions facing this field are proposed. PMID:25926682

  4. Cytotoxic activity and DNA-binding properties of isoeuxanthone derivatives.

    PubMed

    Wang, Hui Fang; Yan, Hong; Gao, Xianghua; Niu, Baolong; Guo, Ruijie; Wei, Liqiao; Xu, Bingshe; Tang, Ning

    2014-01-01

    In this study, the interactions of different groups substituted isoeuxanthone derivatives with calf thymus DNA (ct DNA) were investigated by spectrophotometric methods and viscosity measurements. Results indicated that the xanthone derivatives could intercalate into the DNA base pairs by the plane of xanthone ring and the various substituents may influence the binding affinity with DNA according to the calculated quenching constant values. Furthermore, two tumor cell lines including the human cervical cancer cell line (HeLa) and human hepatocellular liver carcinoma cell line (HepG2) were used to evaluate the cytotoxic activities of xanthone derivatives by acid phosphatase assay. Analyses showed that the oxiranylmethoxy substituted xanthone exhibited more effective cytotoxic activity against the cancer cells than the other substituted xanthones. The effects on the inhibition of tumor cells in vitro agreed with the studies of DNA-binding. PMID:24583780

  5. Centralspindlin in Rappaport's cleavage signaling.

    PubMed

    Mishima, Masanori

    2016-05-01

    Cleavage furrow in animal cell cytokinesis is formed by cortical constriction driven by contraction of an actomyosin network activated by Rho GTPase. Although the role of the mitotic apparatus in furrow induction has been well established, there remain discussions about the detailed molecular mechanisms of the cleavage signaling. While experiments in large echinoderm embryos highlighted the role of astral microtubules, data in smaller cells indicate the role of central spindle. Centralspindlin is a constitutive heterotetramer of MKLP1 kinesin and the non-motor CYK4 subunit and plays crucial roles in formation of the central spindle and recruitment of the downstream cytokinesis factors including ECT2, the major activator of Rho during cytokinesis, to the site of division. Recent reports have revealed a role of this centralspindlin-ECT2 pathway in furrow induction both by the central spindle and by the astral microtubules. Here, a unified view of the stimulation of cortical contractility by this pathway is discussed. Cytokinesis, the division of the whole cytoplasm, is an essential process for cell proliferation and embryonic development. In animal cells, cytokinesis is executed using a contractile network of actin filaments driven by a myosin-II motor that constricts the cell cortex (cleavage furrow ingression) into a narrow channel between the two daughter cells, which is resolved by scission (abscission) [1-3]. The anaphase-specific organization of the mitotic apparatus (MA, spindle with chromosomes plus asters) positions the cleavage furrow and plays a major role in spatial coupling between mitosis and cytokinesis [4-6]. The nucleus and chromosomes are dispensable for furrow specification [7-10], although they contribute to persistent furrowing and robust completion in some cell types [11,12]. Likewise, centrosomes are not essential for cytokinesis, but they contribute to the general fidelity of cell division [10,13-15]. Here, classical models of cleavage furrow

  6. DNA hybridization activity of single-stranded DNA-conjugated gold nanoparticles used as probes for DNA detection

    NASA Astrophysics Data System (ADS)

    Kira, Atsushi; Matsuo, Kosuke; Nakajima, Shin-ichiro

    2016-02-01

    Colloidal nanoparticles (NPs) have potential applications in bio-sensing technologies as labels or signal enhancers. In order to meet demands for a development of biomolecular assays by a quantitative understanding of single-molecule, it is necessary to regulate accuracy of the NPs probes modified with biomolecules to optimize the characteristics of NPs. However, to our knowledge, there is little information about the structural effect of conjugated biomolecules to the NPs. In this study, we investigated the contribution of a density of single-stranded DNA (ssDNA) conjugating gold NP to hybridization activity. Hybridization activity decreased in accordance with increases in the density of attached ssDNAs, likely due to electrostatic repulsion generated by negatively charged phosphate groups in the ssDNA backbone. These results highlight the importance of controlling the density of ssDNAs attached to the surface of NPs used as DNA detection probes.

  7. Rapid restriction enzyme free detection of DNA methyltransferase activity based on DNA-templated silver nanoclusters.

    PubMed

    Kermani, Hanie Ahmadzade; Hosseini, Morteza; Dadmehr, Mehdi; Ganjali, Mohammad Reza

    2016-06-01

    DNA methylation has significant roles in gene regulation. DNA methyltransferase (MTase) enzyme characterizes DNA methylation and also induces an aberrant methylation pattern that is related to many diseases, especially cancers. Thus, it is required to develop a method to detect the DNA MTase activity. In this study, we developed a new sensitive and reliable method for methyltransferase activity assay by employing DNA-templated silver nanoclusters (DNA/Ag NCs) without using restriction enzymes. The Ag NCs have been utilized for the determination of M.SssI MTase activity and its inhibition. We designed an oligonucleotide probe which contained an inserted six-cytosine loop as Ag NCs formation template. The changes in fluorescence intensity were monitored to quantify the M.SssI activity. The fluorescence spectra showed a linear decrease in the range of 0.4 to 20 U/ml with a detection limit of 0.1 U/ml, which was significant compared with previous reports. The proposed method was applied successfully for demonstrating the Gentamicin effect as MTase inhibitor. The proposed method showed convenient reproducibility and sensitivity indicating its potential for the determination of methyltransferase activity. PMID:27052776

  8. Screening the DNA interaction ability and antimicrobial activity of a few novel bioactive complexes tethering N-((2-aminophenyl)(phenyl)methylene)-4-nitroaniline.

    PubMed

    Muniyandi, Vellaichamy; Raman, Natarajan

    2016-11-01

    Few novel transition metal complexes having N-((2-aminophenyl)(phenyl)methylene)-4-nitroaniline were synthesized and characterized by elemental analyses, IR, (1)H and (13)C NMR, electronic, EPR and mass spectra, conductivity and magnetic susceptibility measurements. All the metal complexes adopted square planar geometrical arrangements. The DNA-binding properties of the metal(II) complexes have been investigated by electronic absorption, fluorescence, CD spectra, cyclic voltammetry, differential pulse voltammogram and viscosity measurements. The results obtained indicate that these complexes bind to DNA via an intercalation binding mode. DNA cleavage activities indicate that the metal complexes exhibit greater activity than the ligand. The antimicrobial screening reveals that all the metal complexes exhibit better activity than the free ligand. PMID:27524020

  9. Physical map of polyoma viral DNA fragments produced by cleavage with a restriction enzyme from Haemophilus aegyptius, endonuclease R-HaeIII.

    PubMed Central

    Summers, J

    1975-01-01

    Digestion of polyoma viral DNA with a restriction enzyme from Haemophilus aegyptius generates at least 22 unique fragments. The fragments have been characterized with respect to size and physical order on the polyoma genome, and the 5' to 3' orientation of the (+) and (-) strands has been determined. A method for specific radiolabeling of adjacent fragments was employed to establish the fragment order. This technique may be useful for ordering the fragments produced by digestion of complex DNAs. Images PMID:163927

  10. The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C-H Bond Cleavage and Oxygen Atom Transfer Reactions.

    PubMed

    Hartmann, Tobias; Schrapers, Peer; Utesch, Tillmann; Nimtz, Manfred; Rippers, Yvonne; Dau, Holger; Mroginski, Maria Andrea; Haumann, Michael; Leimkühler, Silke

    2016-04-26

    Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal-containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pKa of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase. PMID:27054466

  11. Cleavage of interleukin 1 beta (IL-1 beta) precursor to produce active IL-1 beta by a conserved extracellular cysteine protease from Streptococcus pyogenes.

    PubMed Central

    Kapur, V; Majesky, M W; Li, L L; Black, R A; Musser, J M

    1993-01-01

    Streptococcal pyrogenic exotoxin B (SPE B), a conserved extracellular cysteine protease expressed by the human pathogenic bacterium Streptococcus pyogenes, was purified and shown to cleave inactive human interleukin 1 beta precursor (pIL-1 beta) to produce biologically active IL-1 beta. SPE B cleaves pIL-1 beta one residue amino-terminal to the site where a recently characterized endogenous human cysteine protease acts. IL-1 beta resulting from cleavage of pIL-1 beta by SPE B induced nitric oxide synthase activity in vascular smooth muscle cells and killed of the human melanoma A375 line. Two additional naturally occurring SPE B variants cleaved pIL-1 beta in a similar fashion. By demonstrating that SPE B catalyzes the formation of biologically active IL-1 beta from inactive pIL-1 beta, our data add a further dimension to an emerging theme in microbial pathogenesis that bacterial and viral virulence factors act directly on host cytokine pathways. The data also contribute to an enlarging literature demonstrating that microbial extracellular cysteine proteases are important in host-parasite interactions. Images Fig. 1 Fig. 2 Fig. 4 PMID:7689226

  12. Metal ion cooperativity in ribozyme cleavage of RNA

    PubMed Central

    Brännvall, Mathias; Kirsebom, Leif A.

    2001-01-01

    Combinations of chemical and genetic approaches were used to study the function of divalent metal ions in cleavage of RNA by the ribozyme RNase P RNA. We show that different divalent metal ions have differential effects on cleavage site recognition and rescue of cleavage activity by mixing divalent metal ions that do not promote cleavage by themselves. We conclude that efficient and correct cleavage is the result of cooperativity between divalent metal ions bound at different sites in the RNase P RNA-substrate complex. Complementation of a mutant RNase P RNA phenotype as a result of divalent metal ion replacement is demonstrated also. This finding together with other data indicate that one of the metal ions involved in this cooperativity is positioned near the cleavage site. The possibility that the Mg2+/Ca2+ ratio might regulate the activity of biocatalysts that depend on RNA for activity is discussed. PMID:11606743

  13. Fibrinogen cleavage by the Streptococcus pyogenes extracellular cysteine protease and generation of antibodies that inhibit enzyme proteolytic activity.

    PubMed

    Matsuka, Y V; Pillai, S; Gubba, S; Musser, J M; Olmsted, S B

    1999-09-01

    The extracellular cysteine protease from Streptococcus pyogenes is a virulence factor that plays a significant role in host-pathogen interaction. Streptococcal protease is expressed as an inactive 40-kDa precursor that is autocatalytically converted into a 28-kDa mature (active) enzyme. Replacement of the single cysteine residue involved in formation of the enzyme active site with serine (C192S mutation) abolished detectable proteolytic activity and eliminated autocatalytic processing of zymogen to the mature form. In the present study, we investigated activity of the wild-type (wt) streptococcal protease toward human fibrinogen and bovine casein. The former is involved in blood coagulation, wound healing, and other aspects of hemostasis. Treatment with streptococcal protease resulted in degradation of the COOH-terminal region of fibrinogen alpha chain, indicating that fibrinogen may serve as an important substrate for this enzyme during the course of human infection. Polyclonal antibodies generated against recombinant 40- and 28-kDa (r40- and r28-kDa) forms of the C192S streptococcal protease mutant exhibited high enzyme-linked immunosorbent assay titers but demonstrated different inhibition activities toward proteolytic action of the wt enzyme. Activity of the wt protease was readily inhibited when the reaction was carried out in the presence of antibodies generated against r28-kDa C192S mutant. Antibodies produced against r40-kDa C192S mutant had no significant effect on proteolysis. These data suggest that the presence of the NH(2)-terminal prosegment prevents generation of functionally active antibodies and indicate that inhibition activity of antibodies most likely depends on their ability to bind the active-site region epitope(s) of the protein. PMID:10456870

  14. Fibrinogen Cleavage by the Streptococcus pyogenes Extracellular Cysteine Protease and Generation of Antibodies That Inhibit Enzyme Proteolytic Activity

    PubMed Central

    Matsuka, Yury V.; Pillai, Subramonia; Gubba, Siddeswar; Musser, James M.; Olmsted, Stephen B.

    1999-01-01

    The extracellular cysteine protease from Streptococcus pyogenes is a virulence factor that plays a significant role in host-pathogen interaction. Streptococcal protease is expressed as an inactive 40-kDa precursor that is autocatalytically converted into a 28-kDa mature (active) enzyme. Replacement of the single cysteine residue involved in formation of the enzyme active site with serine (C192S mutation) abolished detectable proteolytic activity and eliminated autocatalytic processing of zymogen to the mature form. In the present study, we investigated activity of the wild-type (wt) streptococcal protease toward human fibrinogen and bovine casein. The former is involved in blood coagulation, wound healing, and other aspects of hemostasis. Treatment with streptococcal protease resulted in degradation of the COOH-terminal region of fibrinogen α chain, indicating that fibrinogen may serve as an important substrate for this enzyme during the course of human infection. Polyclonal antibodies generated against recombinant 40- and 28-kDa (r40- and r28-kDa) forms of the C192S streptococcal protease mutant exhibited high enzyme-linked immunosorbent assay titers but demonstrated different inhibition activities toward proteolytic action of the wt enzyme. Activity of the wt protease was readily inhibited when the reaction was carried out in the presence of antibodies generated against r28-kDa C192S mutant. Antibodies produced against r40-kDa C192S mutant had no significant effect on proteolysis. These data suggest that the presence of the NH2-terminal prosegment prevents generation of functionally active antibodies and indicate that inhibition activity of antibodies most likely depends on their ability to bind the active-site region epitope(s) of the protein. PMID:10456870

  15. Identification of the catalytic and DNA-binding region of the human immunodeficiency virus type I integrase protein.

    PubMed

    Vink, C; Oude Groeneger, A M; Plasterk, R H

    1993-03-25

    The integrase (IN) protein of the human immunodeficiency virus (HIV) is required for specific cleavage of the viral DNA termini, and subsequent integration of the viral DNA into target DNA. To identify the various domains of the IN protein we generated a series of IN deletion mutants as fusions to maltose-binding protein (MBP). The deletion mutants were tested for their ability to bind DNA, to mediate site-specific cleavage of the viral DNA ends, and to carry out integration and disintegration reactions. We found that the DNA-binding region resides between amino acids 200 and 270 of the 288-residues HIV-1 IN protein. The catalytic domain of the protein was mapped between amino acids 50 and 194. For the specific activities of IN, cleavage of the viral DNA and integration, both the DNA-binding domain and the conserved amino-terminal region of IN are required. These regions are dispensable however, for disintegration activity. PMID:8464733

  16. The loss of c-Jun N-terminal protein kinase activity prevents the amyloidogenic cleavage of amyloid precursor protein and the formation of amyloid plaques in vivo.

    PubMed

    Mazzitelli, Sonia; Xu, Ping; Ferrer, Isidre; Davis, Roger J; Tournier, Cathy

    2011-11-23

    Phosphorylation plays a central role in the dynamic regulation of the processing of the amyloid precursor protein (APP) and the production of amyloid-β (Aβ), one of the clinically most important factors that determine the onset of Alzheimer's disease (AD). This has led to the hypothesis that aberrant Aβ production associated with AD results from regulatory defects in signal transduction. However, conflicting findings have raised a debate over the identity of the signaling pathway that controls APP metabolism. Here, we demonstrate that activation of the c-Jun N-terminal protein kinase (JNK) is essential for mediating the apoptotic response of neurons to Aβ. Furthermore, we discovered that the functional loss of JNK signaling in neurons significantly decreased the number of amyloid plaques present in the brain of mice carrying familial AD-linked mutant genes. This correlated with a reduction in Aβ production. Biochemical analyses indicate that the phosphorylation of APP at threonine 668 by JNK is required for γ-mediated cleavage of the C-terminal fragment of APP produced by β-secretase. Overall, this study provides genetic evidence that JNK signaling is required for the formation of amyloid plaques in vivo. Therefore, inhibition of increased JNK activity associated with aging or with a pathological condition constitutes a potential strategy for the treatment of AD. PMID:22114267

  17. A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide

    PubMed Central

    Sun, J; Fass, D N; Viss, M A; Hummel, A M; Tang, H; Homburger, H A; Specks, U

    1998-01-01

    ANCA directed against PR3 are highly specific for Wegener's granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3-ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N-terminal activation dipeptide of PR3 is required for the binding of PR3-ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N-terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3-S176A and δ-rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, δ-rPR3-S176A does not. Culture supernatants of rPR3-S176A and δ-rPR3-S176A expressing 293 cells were used as sources of target antigen for PR3-ANCA testing by capture ELISA. Forty unselected consecutive PR3-ANCA+ sera were tested. With δ-rPR3-S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3-S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the N-terminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3-ANCA. However, a substantial proportion of PR3-ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N-terminal processing. In 15% of sera this PR3-ANCA subset occurred exclusively. PR3-ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non-haematopoietic mammalian cells without regulated secretory pathway can be used for their expression. PMID:9822293

  18. Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification.

    PubMed

    Cheng, Rui; Tao, Mangjuan; Shi, Zhilu; Zhang, Xiafei; Jin, Yan; Li, Baoxin

    2015-11-15

    Several fluorescence signal amplification strategies have been developed for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity, but they need fluorescence dye labeled DNA probe. We have addressed the limitation and report here a label-free strategy for sensitive detection of PNK activity by coupling DNA strand displacement reaction with enzymatic-aided amplification. A hairpin oligonucleotide (hpDNA) with blunt ends was used as the substrate for T4 PNK phosphorylation. In the presence of T4 PNK, the stem of hpDNA was phosphorylated and further degraded by lambda exonuclease (λ exo) from 5' to 3' direction to release a single-stranded DNA as a trigger of DNA strand displacement reaction (SDR). The trigger DNA can continuously displace DNA P2 from P1/P2 hybrid with the help of specific cleavage of nicking endonuclease (Nt.BbvCI). Then, DNA P2 can form G-quadruplex in the presence of potassium ions and quadruplex-selective fluorphore, N-methyl mesoporphyrin IX (NMM), resulting in a significant increase in fluorescence intensity of NMM. Thus, the accumulative release of DNA P2 led to fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10(-4) U/mL, which is superior or comparative with established approaches. By ingeniously utilizing T4 PNK-triggered DNA SDR, T4 PNK activity can be specifically and facilely studied in homogeneous solution containing complex matrix without any external fluorescence labeling. Moreover, the influence of different inhibitors on the T4 PNK activity revealed that it also can be explored to screen T4 PNK inhibitors. Therefore, this label-free amplification strategy presents a facile and cost-effective approach for nucleic acid phosphorylation related research. PMID:26057733

  19. The Zymogen-Enteropeptidase System: A Practical Approach to Study the Regulation of Enzyme Activity by Proteolytic Cleavage

    ERIC Educational Resources Information Center

    Pizauro, Joao M., Jr.; Ferro, Jesus A.; de Lima, Andrea C. F.; Routman, Karina S.; Portella, Maria Celia

    2004-01-01

    The present research describes an efficient procedure to obtain high levels of trypsinogen and chymotrypsinogen by using a simple, rapid, and easily reproducible method. The extraction process and the time-course of activation of zymogens can be carried out in a single laboratory period, without sophisticated equipment. The main objective was to…

  20. Removal of cell surface heparan sulfate increases TACE activity and cleavage of ErbB4 receptor

    PubMed Central

    Määttä, Jorma A; Olli, Kaisa; Henttinen, Tiina; Tuittila, Minna T; Elenius, Klaus; Salmivirta, Markku

    2009-01-01

    Background Nuclear localization of proteolytically formed intracellular fragment of ErbB4 receptor tyrosine kinase has been shown to promote cell survival, and nuclear localization of ErbB4 receptor has been described in human breast cancer. Tumor necrosis factor alpha converting enzyme (TACE) initiates the proteolytic cascade leading to ErbB4 intracellular domain formation. Interactions between matrix metalloproteases and heparan sulfate have been described, but the effect of cell surface heparan sulfate on TACE activity has not been previously described. Results As indicated by immunodetection of increased ErbB4 intracellular domain formation and direct enzyme activity analysis, TACE activity was substantially amplified by enzymatic removal of cell surface heparan sulfate but not chondroitin sulfate. Conclusion In this communication, we suggest a novel role for cell surface heparan sulfate. Removal of cell surface heparan sulfate led to increased formation of ErbB4 intracellular domain. As ErbB4 intracellular domain has previously been shown to promote cell survival this finding may indicate a novel mechanism how HS degradation active in tumor tissue may favor cell survival. PMID:19171023

  1. Drug targeting of HIV-1 RNA.DNA hybrid structures: thermodynamics of recognition and impact on reverse transcriptase-mediated ribonuclease H activity and viral replication.

    PubMed

    Li, Tsai-Kun; Barbieri, Christopher M; Lin, Hsin-Chin; Rabson, Arnold B; Yang, Gengcheng; Fan, Yupeng; Gaffney, Barbara L; Jones, Roger A; Pilch, Daniel S

    2004-08-01

    RNA degradation via the ribonuclease H (RNase H) activity of human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) is a critical component of the reverse transcription process. In this connection, mutations of RT that inactivate RNase H activity result in noninfectious virus particles. Thus, interfering with the RNase H activity of RT represents a potential vehicle for the inhibition of HIV-1 replication. Here, we demonstrate an approach for inhibiting the RNase H activity of HIV-1 RT by targeting its RNA.DNA hybrid substrates. Specifically, we show that the binding of the 4,5-disubstituted 2-deoxystreptamine aminoglycosides, neomycin, paromomycin, and ribostamycin, to two different chimeric RNA-DNA duplexes, which mimic two distinct intermediates in the reverse transcription process, inhibits specific RT-mediated RNase H cleavage, with this inhibition being competitive in nature. UV melting and isothermal titration calorimetry studies reveal a correlation between the relative binding affinities of the three drugs for each of the chimeric RNA-DNA host duplexes and the relative extents to which the drugs inhibit RT-mediated RNase H cleavage of the duplexes. Significantly, this correlation also extends to the relative efficacies with which the drugs inhibit HIV-1 replication. In the aggregate, our results highlight a potential strategy for AIDS chemotherapy that should not be compromised by the unusual genetic diversity of HIV-1. PMID:15274628

  2. Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota

    PubMed Central

    Makarova, Alena V.; Grabow, Corinn; Gening, Leonid V.; Tarantul, Vyacheslav Z.; Tahirov, Tahir H.; Bessho, Tadayoshi; Pavlov, Youri I.

    2011-01-01

    Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts. PMID:21304950

  3. Cleavage of Chordin by Xolloid Metalloprotease Suggests a Role for Proteolytic Processing in the Regulation of Spemann Organizer Activity

    PubMed Central

    Piccolo, Stefano; Agius, Eric; Lu, Bin; Goodman, Shelley; Dale, Leslie

    2011-01-01

    Summary The Xolloid secreted metalloprotease, a tolloid-related protein, was found to cleave Chordin and Chordin/BMP-4 complexes at two specific sites in biochemical experiments. Xolloid mRNA blocks secondary axes caused by chordin, but not by noggin, follistatin, or dominant-negative BMP receptor, mRNA injection. Xolloid-treated Chordin protein was unable to antagonize BMP activity. Furthermore, Xolloid digestion released biologically active BMPs from Chordin/BMP inactive complexes. Injection of dominant-negative Xolloid mRNA indicated that the in vivo function of Xolloid is to limit the extent of Spemann’s organizer field. We propose that Xolloid regulates organizer function by a novel proteolytic mechanism involving a double inhibition pathway required to pattern the dorsoventral axis: XOLL⊣CHD⊣BMPs→BMPR PMID:9363949

  4. Activation of the DNA Damage Response by RNA Viruses.

    PubMed

    Ryan, Ellis L; Hollingworth, Robert; Grand, Roger J

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  5. Using DNA devices to track anticancer drug activity.

    PubMed

    Kahanda, Dimithree; Chakrabarti, Gaurab; Mcwilliams, Marc A; Boothman, David A; Slinker, Jason D

    2016-06-15

    It is beneficial to develop systems that reproduce complex reactions of biological systems while maintaining control over specific factors involved in such processes. We demonstrated a DNA device for following the repair of DNA damage produced by a redox-cycling anticancer drug, beta-lapachone (β-lap). These chips supported ß-lap-induced biological redox cycle and tracked subsequent DNA damage repair activity with redox-modified DNA monolayers on gold. We observed drug-specific changes in square wave voltammetry from these chips at therapeutic ß-lap concentrations of high statistical significance over drug-free control. We also demonstrated a high correlation of this change with the specific ß-lap-induced redox cycle using rational controls. The concentration dependence of ß-lap revealed significant signal changes at levels of high clinical significance as well as sensitivity to sub-lethal levels of ß-lap. Catalase, an enzyme decomposing peroxide, was found to suppress DNA damage at a NQO1/catalase ratio found in healthy cells, but was clearly overcome at a higher NQO1/catalase ratio consistent with cancer cells. We found that it was necessary to reproduce key features of the cellular environment to observe this activity. Thus, this chip-based platform enabled tracking of ß-lap-induced DNA damage repair when biological criteria were met, providing a unique synthetic platform for uncovering activity normally confined to inside cells. PMID:26901461

  6. Activation of the DNA Damage Response by RNA Viruses

    PubMed Central

    Ryan, Ellis L.; Hollingworth, Robert; Grand, Roger J.

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  7. Human Immunodeficiency Virus Integration Protein Expressed in Escherichia Coli Possesses Selective DNA Cleaving Activity

    NASA Astrophysics Data System (ADS)

    Sherman, Paula A.; Fyfe, James A.

    1990-07-01

    The human immunodeficiency virus (HIV) integration protein, a potential target for selective antiviral therapy, was expressed in Escherichia coli. The purified protein, free of detectable contaminating endonucleases, selectively cleaved double-stranded DNA oligonucleotides that mimic the U3 and the U5 termini of linear HIV DNA. Two nucleotides were removed from the 3' ends of both the U5 plus strand and the U3 minus strand; in both cases, cleavage was adjacent to a conserved CA dinucleotide. The reaction was metal-ion dependent, with a preference for Mn2+ over Mg2+. Reaction selectivity was further demonstrated by the lack of cleavage of an HIV U5 substrate on the complementary (minus) strand, an analogous substrate that mimics the U3 terminus of an avian retrovirus, and an HIV U5 substrate in which the conserved CA dinucleotide was replaced with a TA dinucleotide. Such an integration protein-mediated cleavage reaction is expected to occur as part of the integration event in the retroviral life cycle, in which a double-stranded DNA copy of the viral RNA genome is inserted into the host cell DNA.

  8. The γ-secretase cleavage product of polycystin-1 regulates TCF and CHOP-mediated transcriptional activation through a p300-dependent mechanism.

    PubMed

    Merrick, David; Chapin, Hannah; Baggs, Julie E; Yu, Zhiheng; Somlo, Stefan; Sun, Zhaoxia; Hogenesch, John B; Caplan, Michael J

    2012-01-17

    Mutations in Pkd1, encoding polycystin-1 (PC1), cause autosomal-dominant polycystic kidney disease (ADPKD). We show that the carboxy-terminal tail (CTT) of PC1 is released by γ-secretase-mediated cleavage and regulates the Wnt and CHOP pathways by binding the transcription factors TCF and CHOP, disrupting their interaction with the common transcriptional coactivator p300. Loss of PC1 causes increased proliferation and apoptosis, while reintroducing PC1-CTT into cultured Pkd1 null cells reestablishes normal growth rate, suppresses apoptosis, and prevents cyst formation. Inhibition of γ-secretase activity impairs the ability of PC1 to suppress growth and apoptosis and leads to cyst formation in cultured renal epithelial cells. Expression of the PC1-CTT is sufficient to rescue the dorsal body curvature phenotype in zebrafish embryos resulting from either γ-secretase inhibition or suppression of Pkd1 expression. Thus, γ-secretase-dependent release of the PC1-CTT creates a protein fragment whose expression is sufficient to suppress ADPKD-related phenotypes in vitro and in vivo. PMID:22178500

  9. A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System.

    PubMed

    Pompey, Justine M; Foda, Bardees; Singh, Upinder

    2015-01-01

    Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway. PMID:26230096

  10. A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System

    PubMed Central

    Singh, Upinder

    2015-01-01

    Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway. PMID:26230096

  11. Requirement for the Kinase Activity of Human DNA-Dependent Protein Kinase Catalytic Subunit in DNA Strand Break Rejoining

    PubMed Central

    Kurimasa, Akihiro; Kumano, Satoshi; Boubnov, Nikolai V.; Story, Michael D.; Tung, Chang-Shung; Peterson, Scott R.; Chen, David J.

    1999-01-01

    The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an enormous, 470-kDa protein serine/threonine kinase that has homology with members of the phosphatidylinositol (PI) 3-kinase superfamily. This protein contributes to the repair of DNA double-strand breaks (DSBs) by assembling broken ends of DNA molecules in combination with the DNA-binding factors Ku70 and Ku80. It may also serve as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks. This study attempts to better define the role of protein kinase activity in the repair of DNA DSBs. We constructed a contiguous 14-kb human DNA-PKcs cDNA and demonstrated that it can complement the DNA DSB repair defects of two mutant cell lines known to be deficient in DNA-PKcs (M059J and V3). We then created deletion and site-directed mutations within the conserved PI 3-kinase domain of the DNA-PKcs gene to test the importance of protein kinase activity for DSB rejoining. These DNA-PKcs mutant constructs are able to express the protein but fail to complement the DNA DSB or V(D)J recombination defects of DNA-PKcs mutant cells. These results indicate that the protein kinase activity of DNA-PKcs is essential for the rejoining of DNA DSBs in mammalian cells. We have also determined a model structure for the DNA-PKcs kinase domain based on comparisons to the crystallographic structure of a cyclic AMP-dependent protein kinase. This structure gives some insight into which amino acid residues are crucial for the kinase activity in DNA-PKcs. PMID:10207111

  12. Complex Negative Regulation of TLR9 by Multiple Proteolytic Cleavage Events.

    PubMed

    Sinha, Siddhartha S; Cameron, Jody; Brooks, James C; Leifer, Cynthia A

    2016-08-15

    TLR9 is an innate immune receptor important for recognizing DNA of host and foreign origin. A mechanism proposed to prevent excessive response to host DNA is the requirement for proteolytic cleavage of TLR9 in endosomes to generate a mature form of the receptor (TLR9(471-1032)). We previously described another cleavage event in the juxtamembrane region of the ectodomain that generated a dominant-negative form of TLR9. Thus, there are at least two independent cleavage events that regulate TLR9. In this study, we investigated whether an N-terminal fragment of TLR9 could be responsible for regulation of the mature or negative-regulatory form. We show that TLR9(471-1032), corresponding to the proteolytically cleaved form, does not function on its own. Furthermore, activity is not rescued by coexpression of the N-terminal fragment (TLR9(1-440)), inclusion of the hinge region (TLR9(441-1032)), or overexpression of UNC93B1, the last of which is critical for trafficking and cleavage of TLR9. TLR9(1-440) coimmunoprecipitates with full-length TLR9 and TLR9(471-1032) but does not rescue the native glycosylation pattern; thus, inappropriate trafficking likely explains why TLR9(471-1032) is nonfunctional. Lastly, we show that TLR9(471-1032) is also a dominant-negative regulator of TLR9 signaling. Together, these data provide a new perspective on the complexity of TLR9 regulation by proteolytic cleavage and offer potential ways to inhibit activity through this receptor, which may dampen autoimmune inflammation. PMID:27421483

  13. Plasma-activated air mediates plasmid DNA delivery in vivo.

    PubMed

    Edelblute, Chelsea M; Heller, Loree C; Malik, Muhammad A; Bulysheva, Anna; Heller, Richard

    2016-01-01

    Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs