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Sample records for dna cleavage activities

  1. Quercetin-Iron Complex: Synthesis, Characterization, Antioxidant, DNA Binding, DNA Cleavage, and Antibacterial Activity Studies.

    PubMed

    Raza, Aun; Xu, Xiuquan; Xia, Li; Xia, Changkun; Tang, Jian; Ouyang, Zhen

    2016-11-01

    Quercetin-iron (II) complex was synthesized and characterized by elemental analysis, ultraviolet-visible spectrophotometry, fourier transform infrared spectroscopy, mass spectrometry, proton nuclear magnetic resonance spectroscopy, thermogravimetry and differential scanning calorimetry, scanning electron micrography and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal:ligand) of the complex. Antioxidant study of the quercetin and its metal complex against 2, 2-di-phenyl-1-picryl hydrazyl radical showed that the complex has much more radical scavenging activity than free quercetin. The interaction of quercetin-iron (II) complex with DNA was determined using ultraviolet visible spectra, fluorescence spectra and agarose gel electrophoresis. The results showed that quercetin-iron (II) complex can intercalate moderately with DNA, quench a strong intercalator ethidium bromide and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form to nicked circular form and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was an oxidative cleavage pathway. These results revealed the potential nuclease activity of complex to cleave DNA. In addition, antibacterial activity of complex on E.coli and S. aureus was also investigated. The results showed that complex has higher antibacterial activity than ligand.

  2. Rutin-Nickel Complex: Synthesis, Characterization, Antioxidant, DNA Binding, and DNA Cleavage Activities.

    PubMed

    Raza, Aun; Bano, Shumaila; Xu, Xiuquan; Zhang, Rong Xian; Khalid, Haider; Iqbal, Furqan Muhammad; Xia, Changkun; Tang, Jian; Ouyang, Zhen

    2016-12-17

    The rutin-nickel (II) complex (RN) was synthesized and characterized by elemental analysis, UV-visible spectroscopy, IR, mass spectrometry, (1)H NMR, TG-DSC, SEM, and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal/ligand) of the complex. An antioxidant study of rutin and its metal complex against DPPH radical showed that the complex has more radical scavenging activity than free rutin. The interaction of complex RN with DNA was determined using fluorescence spectra and agarose gel electrophoresis. The results showed that RN can intercalate moderately with DNA, quench a strong intercalator ethidium bromide (EB), and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form (SC) to nicked circular form (NC), and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was a hydrolytic cleavage pathway. These results revealed the potential nuclease activity of the complex to cleave DNA.

  3. 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

  4. Indirect DNA Sequence Recognition and Its Impact on Nuclease Cleavage Activity.

    PubMed

    Lambert, Abigail R; Hallinan, Jazmine P; Shen, Betty W; Chik, Jennifer K; Bolduc, Jill M; Kulshina, Nadia; Robins, Lori I; Kaiser, Brett K; Jarjour, Jordan; Havens, Kyle; Scharenberg, Andrew M; Stoddard, Barry L

    2016-06-07

    LAGLIDADG meganucleases are DNA cleaving enzymes used for genome engineering. While their cleavage specificity can be altered using several protein engineering and selection strategies, their overall targetability is limited by highly specific indirect recognition of the central four base pairs within their recognition sites. In order to examine the physical basis of indirect sequence recognition and to expand the number of such nucleases available for genome engineering, we have determined the target sites, DNA-bound structures, and central four cleavage fidelities of nine related enzymes. Subsequent crystallographic analyses of a meganuclease bound to two noncleavable target sites, each containing a single inactivating base pair substitution at its center, indicates that a localized slip of the mutated base pair causes a small change in the DNA backbone conformation that results in a loss of metal occupancy at one binding site, eliminating cleavage activity.

  5. Design, synthesis, cytotoxicities and DNA cleavage activities of dibenzoxepine and isoquinoline derivatives starting from dehydroabietylamine.

    PubMed

    Liu, Chao-Xiang; Lin, Zhong-Xiang; Zhou, Ai-Min

    2016-12-01

    A series of novel hexahydrodibenzoxepine and quinazoline derivatives were designed and synthesized starting from dehydroabietylamine. The cytotoxicities of the compounds against L02 and HepG2 cell lines were investigated. Meanwhile, the plasmid DNA (Escherichia coli) cleavage of several heterocyclic derivatives was studied. These compounds exhibit remarkable activities on plasmid DNA pBR322. Our study provides useful information for developing new and more potent antitumor agents.

  6. DNA cleavage activity of V IV O(acac)2 and derivatives.

    PubMed

    Butenko, Nataliya; Tomaz, Ana Isabel; Nouri, Ofelia; Escribano, Esther; Moreno, Virtudes; Gama, Sofia; Ribeiro, Vera; Telo, João Paulo; Pesssoa, João Costa; Cavaco, Isabel

    2009-04-01

    The DNA cleavage activity of several beta-diketonate vanadyl complexes is examined. Vanadyl acetylacetonate, V(IV)O(acac)(2), 1, shows a remarkable activity in degrading plasmid DNA in the absence of any activating agents, air and photoirradiation. The cleaving activity of several related complexes V(IV)O(hd)(2) (2, Hhd=3,5-heptanedione), V(IV)O(acac-NH(2))(2) (3, Hacac-NH(2)=acetoacetamide) and V(IV)O(acac-NMe(2))(2) (4, Hacac-NMe(2)=N,N-dimethylacetoacetamide) is also evaluated. It is shown that 2 exhibits an activity similar to 1, while 3 and 4 are much less efficient cleaving agents. The different activity of the complexes is related to their stability towards hydrolysis in aqueous solution, which follows the order 1 approximately 2>3 approximately 4. The nature of the pH buffer was also found to be determinant in the nuclease activity of 1 and 2. In a phosphate buffered medium DNA cleavage by these agents is much more efficient than in tris, hepes, mes or mops buffers. The reaction seems to take place through a mixed mechanism, involving the formation of reactive oxygen species (ROS), namely OH radicals, and possibly also direct cleavage at phosphodiester linkages induced by the vanadium complexes.

  7. The syntheses, characterization, antimicrobial, DNA cleavage and cytotoxic activities of novel terephthalato complexes

    NASA Astrophysics Data System (ADS)

    Yıldız, Özge; Çolak, Alper Tolga; Yılmaz, Murat; İça, Tuba; Oztopcu-Vatan, Pinar; Topaloǧlu, Emel; Çolak, Ferdaǧ

    2017-01-01

    [Cu(tp)(dmpd)2] (1), [Cu(tp)(pen)2] (2), [Cu(tp)(dmen)2] (3), [Cu(tp)(deen)2]·4H2O (4), [Cu(tp)(mpen)2]·2H2O (5) and [Cu(tp)(amp)2]·3H2O (6) (H2tp = Benzene-1,4-dicarboxylic acid or terephthalic acid, dmpd = 2,2-dimethyl-1,3-propanediamine, pen = 1,3-propanediamine, dmen = N,N-dimethylethylenediamine, deen = N,N-diethylethylenediamine, mpen = N-methyl-1,3-propanediamine and amp = 2-aminomethylpyridine) were synthesised and characterised by elemental analysis, spectroscopic measurements (UV-vis. and FT-IR spectra) and thermal analysis technique. These complexes have been screened for antimicrobial activities and DNA cleavage. Antimicrobial activity of compounds 1-6 were evaluated by the agar diffusion method. The DNA cleavage activities of the complexes were evaluated by agarose gel electrophoresis. In addition, cytotoxic activities of all complexes were performed prostate carcinoma cells LNCaP and DU145 by MTT assay for 24 and 48 h. Especially after 24 h treatment, 6 complex increased the apoptotic and necrotic cell death in both cell lines in a concentration dependent manner. Particularly, 6 complex good show antimicrobial, nuclease and cytotoxic activity.

  8. Synthesis, characterization, DNA binding, cleavage activity and cytotoxicity of copper(II) complexes.

    PubMed

    Li, Mei-Jin; Lan, Tao-Yu; Cao, Xiu-Hui; Yang, Huang-Hao; Shi, Yupeng; Yi, Changqing; Chen, Guo-Nan

    2014-02-21

    Three new mononuclear copper(II) complexes, [Cu(L2)](2+) (1), [Cu(acac)(L)](+) (2), and [Cu(acac-Cl)(L)](+) (3) (L = 2-(4-pyridine)oxazo[4,5-f]1,10-phenanthroline (4-PDOP); acac = acetylacetone; acac-Cl = 3-chloroacetylacetone), have been synthesized and characterized by elemental analysis, high resolution mass spectrometry (Q-TOF), and IR spectroscopy. Two of the complexes were structurally characterized by single-crystal X-ray diffraction techniques. Their interactions with DNA were studied by UV-vis absorption and emission spectra, viscosity, thermal melting, DNA unwinding assay and CD spectroscopy. The nucleolytic cleavage activity of the compounds was carried out on double stranded pBR322 circular plasmid DNA by using a gel electrophoresis experiment in the presence and absence of an oxidant (H2O2). Active oxygen intermediates such as hydroxyl radicals and hydrogen peroxide generated in the presence of L and complexes 1-3 may act as active species for the DNA scission. The cytotoxicity of the complexes against HepG2 cancer cells was also studied.

  9. Homodinuclear lanthanide complexes of phenylthiopropionic acid: synthesis, characterization, cytotoxicity, DNA cleavage, and antimicrobial activity.

    PubMed

    Shiju, C; Arish, D; Kumaresan, S

    2013-03-15

    Lanthanide complexes of La(III), Pr(III), Nd(III), Sm(III), and Ho(III) with phenylthiopropionic acid were synthesized and characterized by elemental analysis, mass, IR, electronic spectra, molar conductance, TGA, and powder XRD. The results show that the lanthanide complexes are homodinuclear in nature. The two lanthanide ions are bridged by eight oxygen atoms from four carboxylate groups. Thermal decomposition profiles are consistent with the proposed formulations. Powder XRD studies show that all the complexes are amorphous in nature. Antimicrobial studies indicate that these complexes exhibit more activity than the ligand itself. The DNA cleavage activity of the ligand and its complexes were assayed on Escherichia coli DNA using gel electrophoresis in the presence of H(2)O(2). The result shows that the Pr(III) and Nd(III) complexes have completely cleaved the DNA. The anticancer activities of the complexes have also been studied towards human cervical cancer cell line (HeLa) and colon cancer cells (HCT116) and it was found that the La(III) and Nd(III) complexes are more active than the corresponding Pr(III), Sm(III), Ho(III) complexes, and the free ligand on both the cancer cells.

  10. 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

    2016-06-04

    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.

  11. Synthesis, DNA cleavage and antimicrobial activity of 4-thiazolidinones-benzothiazole conjugates.

    PubMed

    Singh, Meenakshi; Gangwar, Mayank; Nath, Gopal; Singh, Sushil K

    2014-11-01

    Antimicrobial screening of several novel 4-thiazolidinones with benzothiazole moiety has been performed. These compounds were evaluated for antimicrobial activity against a panel of bacterial and fungal strains. The strains were treated with these benzothiazole derivatives at varying concentrations, and MIC's were calculated. Structures of these compounds have been determined by spectroscopic studies viz., FT-IR, 1H NMR, 13C NMR and elemental analysis. Significant antimicrobial activity was observed for some members of the series, and compounds viz. 3-(4-(benzo[d]thiazol-2-yl) phenyl-2-(4-methoxyphenyl)thiazolidin-4-one and 3-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-hydroxy phenyl)thiazolidin-4-one were found to be the most active against E.coli and C. albicans with MIC values in the range of 15.6-125 microg/ml. Preliminary study of the structure-activity relationship revealed that electron donating groups associated with thiazolidine bearing benzothiazole rings had a great effect on the antimicrobial activity of these compounds and contributes positively for the action. DNA cleavage experiments gave valuable hints with supporting evidence for describing the mechanism of action and hence showed a good correlation between their calculated MIC's and its lethality.

  12. Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system

    PubMed Central

    Elmore, Joshua R.; Sheppard, Nolan F.; Ramia, Nancy; Deighan, Trace; Li, Hong; Terns, Rebecca M.; Terns, Michael P.

    2016-01-01

    CRISPR–Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (CRISPR-associated Rossman fold [CARF] superfamily) protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2 (Cas10 superfamily) protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a rPAM. PMID:26848045

  13. Synthesis and DNA cleavage activities of mononuclear macrocyclic polyamine zinc(II), copper(II), cobalt(II) complexes which linked with uracil.

    PubMed

    Wang, Xiao-Yan; Zhang, Ji; Li, Kun; Jiang, Ning; Chen, Shan-Yong; Lin, Hong-Hui; Huang, Yu; Ma, Li-Jian; Yu, Xiao-Qi

    2006-10-01

    Mononuclear macrocyclic polyamine zinc(II), copper(II), cobalt(II) complexes, which could attach to peptide nucleic acid (PNA), were synthesized as DNA cleavage agents. The structures of these new mononuclear complexes were identified by MS and (1)H NMR spectroscopy. The catalytic activities on DNA cleavage of these mononuclear complexes with different central metals were subsequently studied, which showed that copper complex was better catalyst in the DNA cleavage process than zinc and cobalt complexes. The effects of reaction time, concentration of complexes were also investigated. The results indicated that the copper(II) complexes could catalyze the cleavage of supercoiled DNA (pUC 19 plasmid DNA) (Form I) under physiological conditions to produce selectively nicked DNA (Form II, no Form III produced) with high yields. The mechanism of the cleavage process was also studied.

  14. Altered DNA-cleavage activity of topoisomerase II from WEHI-3B leukemia cells with specific resistance to ciprofloxacin.

    PubMed

    Pessina, A; Raimondi, A; Croera, C; Acchini, M; Mineo, E; Foti, P; Neri, M G

    2001-06-01

    In order to investigate the mechanisms of drug resistance arising in tumor cells, we investigated the capacity of fluoroquinolones to inhibit the in vitro growth of WEHI-3B monomyelocytic leukemia cells and then we established a variant of this line (currently maintained in the absence of drug). The line, named WEHI-3B/CPX, expresses a specific resistance to ciprofloxacin (CPX; resistance index=17.3+/-2.2), and does not show cross-resistance with other fluoroquinolones, camptothecin and topoisomerase II inhibitors such as doxorubicin, etoposide and teniposide. Although a little decrease in intracellular accumulation of CPX is observed in WEHI-3B/CPX cells, these cells do not express MDR or LRP markers, and the resistance is not circumvented by verapamil. Purified nuclear extracts from WEHI-3B and WEHI-3B/CPX cells were tested for topoisomerase I catalytic activity and checking in vitro topoisomerase I sensitivity to CPX and camptothecin inhibition, but no difference was observed. As the treatment with CPX showed that the resistant cell line suffers a significantly lower number of breaks in the DNA molecule we also addressed our investigations to the topoisomerase II-dependent DNA cleavage that, in the resistant clone, was found dramatically less susceptible to be enhanced by CPX both in pre-strand and post-strand DNA passage conditions. WEHI-3B/CPX cells do not express any character of multidrug resistance and represent a rare case of specific drug resistance to CPX. The specific resistance to CPX observed in these cells is related to a functional decrease of topoisomerase II cleavage activity. It could be consequent to a decreased binding affinity of CPX for the topoisomerase II--DNA complex or to a decreased affinity or specificity of topoisomerase II for its DNA cleavage sites.

  15. Metal Ion Interactions in the DNA Cleavage/Ligation Active Site of Human Topoisomerase IIα†

    PubMed Central

    Deweese, Joseph E.; Guengerich, F. Peter; Burgin, Alex B.; Osheroff, Neil

    2009-01-01

    Human topoisomerase IIα utilizes a two-metal-ion mechanism for DNA cleavage. One of the metal ions (M12+) is believed to make a critical interaction with the 3′-bridging atom of the scissile phosphate, while the other (M22+) is believed to interact with a non-bridging oxygen of the scissile phosphate. Based on structural and mutagenesis studies of prokaryotic nucleic acid enzymes, it has been proposed that the active site divalent metal ions interact with type II topoisomerases through a series of conserved acidic amino acid residues. The homologous residues in human topoisomerase IIα are E461, D541, D543, and D545. To address the validity of these assignments and to delineate interactions between individual amino acids and M12+ and M22+, we individually mutated each of these acidic amino acid residues in topoisomerase IIα to either cysteine or alanine. Mutant enzymes displayed a marked loss of catalytic and DNA cleavage activity as well as a reduced affinity for divalent metal ions. Additional experiments determined the ability of wild-type and mutant topoisomerase IIα enzymes to cleave an oligonucleotide substrate that contained a sulfur atom in place of the 3′-bridging oxygen of the scissile phosphate in the presence of Mg2+, Mn2+, or Ca2+. Based on the results of these studies, we conclude that the four acidic amino acid residues interact with metal ions in the DNA cleavage/ligation active site of topoisomerase IIα. Furthermore, we propose that M12+ interacts with E461, D543, and D545 and M22+ interacts with E461 and D541. PMID:19697956

  16. Poly(ADP-ribose)polymerase 1 stimulates the AP-site cleavage activity of tyrosyl-DNA phosphodiesterase 1.

    PubMed

    Lebedeva, Natalia A; Anarbaev, Rashid O; Sukhanova, Maria; Vasil'eva, Inna A; Rechkunova, Nadejda I; Lavrik, Olga I

    2015-06-15

    The influence of poly(ADP-ribose)polymerase 1 (PARP1) on the apurinic/apyrimidinic (AP)-site cleavage activity of tyrosyl-DNA phosphodiesterase 1 (TDP1) and interaction of PARP1 and TDP1 were studied. The efficiency of single or clustered AP-site hydrolysis catalysed by TDP1 was estimated. It was shown that the efficiency of AP-site cleavage increases in the presence of an additional AP-site in the opposite DNA strand depending on its position. PARP1 stimulates TDP1; the stimulation effect was abolished in the presence of NAD(+). The interaction of these two proteins was characterized quantitatively by measuring the dissociation constant for the TDP1-PARP1 complex using fluorescently-labelled proteins. The distance between the N-termini of the proteins within the complex was estimated using FRET. The data obtained suggest that PARP1 and TDP1 bind in an antiparallel orientation; the N-terminus of the former protein interacts with the C-terminal domain of the latter. The functional significance of PARP1 and TDP1 interaction in the process of DNA repair was demonstrated for the first time.

  17. 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

  18. Novel metal-based pharmacologically dynamic agents of transition metal(II) complexes: Designing, synthesis, structural elucidation, DNA binding and photo-induced DNA cleavage activity

    NASA Astrophysics Data System (ADS)

    Raman, N.; Jeyamurugan, R.; Sakthivel, A.; Mitu, L.

    2010-01-01

    Novel Schiff base Cu(II), Ni(II), Co(II) and Zn(II) complexes have been designed and synthesized using the macrocyclic ligand derived from the condensation of diethylphthalate with Schiff base, obtained from benzene-1,2-diamine and 3-benzylidene-pentane-2,4-dione. The ligand and its complexes have been characterized by analytical and spectral techniques. DNA binding properties of these complexes have been investigated by UV-vis, viscosity measurements, cyclic voltammetric and differential pulse voltammogram studies. The intrinsic binding constants for Co(II), Ni(II), Cu(II) and Zn(II) complexes are 1.6 × 10 6, 1.8 × 10 6, 2.0 × 10 6 and 1.5 × 10 6 M -1 respectively which are obtained from electronic absorption experiment. Control DNA cleavage experiments using pUC19 supercoiled (SC) DNA and minor groove binder (distamycin) suggest the major groove binding tendency for the synthesized complexes. In the presence of a reducing agent like 3-mercaptopropionic acid (MPA), the synthesized complexes show chemical nuclease activity under dark reaction condition. The complexes also show efficient photo-induced DNA cleavage activity on irradiation with a monochromatic UV light of 360 nm in the presence of inhibitors. Control experiments show inhibition of cleavage in the presence of singlet oxygen quencher like sodium azide and enhancement of cleavage in D 2O, suggesting the formation of singlet oxygen as a reactive species in a type-II process.

  19. Enhancing cell nucleus accumulation and DNA cleavage activity of anti-cancer drug via graphene quantum dots.

    PubMed

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

    2013-10-04

    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.

  20. Photoactivated DNA cleavage and anticancer activity of pyrenyl-terpyridine lanthanide complexes.

    PubMed

    Hussain, Akhtar; Gadadhar, Sudarshan; Goswami, Tridib K; Karande, Anjali A; Chakravarty, Akhil R

    2012-04-01

    Lanthanide(III) complexes [Ln(R-tpy)(acac)(NO(3))(2)] (Ln = La(III) in 1, 2; Gd(III) in 4, 5) and [Ln(py-tpy)(sacac)(NO(3))(2)] (Ln = La(III), 3; Gd(III), 6), where R-tpy is 4'-phenyl-2,2':6',2″-terpyridine (ph-tpy in 1, 4), 4'-(1-pyrenyl)-2,2':6',2″-terpyridine (py-tpy in 2, 3, 5 and 6), acac is acetylacetonate and sacac is 4-hydroxy-6-{4-[(β-d-glucopyranoside)oxy]phenyl}hex-3,5-dien-2-onate, were prepared to study their DNA photocleavage activity and photocytotoxicity. Complexes [La(ph-tpy)(acac)(EtOH)(NO(3))(2)] (1a) and [Gd(ph-tpy)(acac)(NO(3))(2)] (4) were characterized by X-ray crystallography. The 1:1 electrolytic complexes bind to calf thymus DNA. The py-tpy complexes cleave pUC19 DNA and exhibit remarkable photocytotoxicity in HeLa cells in UV-A light of 365 nm with apoptotic cell death (IC(50): ∼40 nM in light, >200 μM in dark). Confocal microscopy using HeLa cells reveal primarily cytosolic localization of the complexes.

  1. 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.

  2. 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

  3. Fluoroquinolones stimulate the DNA cleavage activity of topoisomerase IV by promoting the binding of Mg2+ to the second metal binding site

    PubMed Central

    Oppegard, Lisa M.; Schwanz, Heidi A.; Towle, Tyrell R.; Kerns, Robert J.; Hiasa, Hiroshi

    2016-01-01

    Background Fluoroquinolones target bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Fluoroquinolones trap a topoisomerase-DNA covalent complex as a topoisomerase-fluoroquinolone-DNA ternary complex and ternary complex formation is critical for their cytotoxicity. A divalent metal ion is required for type IIA topoisomerase-catalyzed strand breakage and religation reactions. Recent studies have suggested that type IIA topoisomerases use two metal ions, one structural and one catalytic, to carry out the strand breakage reaction. Methods We conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg2+-, Mn2+-, or Ca2+-supported DNA cleavage activity of Esherichia coli Topo IV. Results In the absence of any drug, 20–30 mM Mg2+ was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1 mM of either Mn2+ or Ca2+ was sufficient to support the maximum levels of the DNA cleavage activity of Topo IV. Fluoroquinolones promoted the Topo IV-catalyzed strand breakage reaction at low Mg2+ concentrations where Topo IV alone could not efficiently cleave DNA. Conclusions and General Significance At low Mg2+ concentrations, fluoroquinolones may stimulate the Topo IV-catalyzed strand breakage reaction by promoting Mg2+ binding to metal binding site B through the structural distortion in DNA. As Mg2+ concentration increases, fluoroquinolones may inhibit the religation reaction by either stabilizing Mg2+ at site B or inhibition the binding of Mg2+ to site A. This study provides a molecular basis of how fluoroquinolones stimulate the Topo IV-catalyzed strand breakage reaction by modulating Mg2+ binding. PMID:26723176

  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.

  5. Synthesis of new heterometallic macromolecules: Their DNA binding, cleavage activity and in vitro model electrochemotherapy study

    NASA Astrophysics Data System (ADS)

    Tabassum, Sartaj; Bhat, Irshad-ul-Haq; Arjmand, Farukh

    2009-12-01

    The homodinuclear C 16H 30N 8O 5Sn 2Cl 4 ( 1), heterotetranuclear C 16H 38N 8O 9Sn 2Cu 2Cl 8 ( 2) and C 16H 38N 8O 9Sn 2Mn 2Cl 8 ( 3) macrocyclic complexes were synthesized and characterized by elemental analysis, spectroscopic techniques and molar conductance measurements. The interaction studies of 1-3 with calf thymus DNA (CT-DNA) were carried out by UV-vis titration, fluorescence, cyclic voltammetry and viscosity measurements. These results were further authenticated by carrying out interaction studies of 1-3 with plasmid pBR322 DNA employing gel electrophoresis. To overcome the dose resistance, auto toxicity of the drugs, a model study based on electrochemotherapy (ECT) was carried out and the results were compared in the presence and in the absence of the applied electrical potential.

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

    PubMed

    Meluzzi, Dario; Arya, Gaurav

    2016-01-08

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

  7. Intramolecular nucleophilic activation promoting efficient hydrolytic cleavage of DNA by (aqua)bis(dipyridoquinoxaline)copper(II) complex.

    PubMed

    Dhar, Shanta; Reddy, Pattubala A N; Chakravarty, Akhil R

    2004-03-07

    The axial aqua bound copper(II) complex [Cu(dpq)2(H2O)](ClO4)2, having a planar NN-donor heterocyclic base dipyridoquinoxaline (dpq) as the DNA minor groove binder, shows efficient hydrolytic cleavage of supercoiled DNA in the dark and in the absence of any external reagents, as evidenced from T4 ligase experiments, with a rate of 5.58 +/- 0.4 h(-1) and a rate enhancement of 1.55 x 10(8).

  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. Hydrolytic cleavage of DNA by quercetin manganese(II) complexes.

    PubMed

    Jun, Tan; Bochu, Wang; Liancai, Zhu

    2007-04-01

    Quercetin manganese(II) complexes were investigated focusing on its DNA hydrolytic activity. The complexes successfully promote the cleavage of plasmid DNA, producing single and double DNA strand breaks. The amount of conversion of supercoiled form (SC) of plasmid DNA to the nicked circular form (NC) depends on the concentration of the complex as well as the duration of incubation of the complexes with DNA. The maximum rate of conversion of the supercoiled form to the nicked circular form at pH 7.2 in the presence of 100 microM of the complexes is found to be 1.32 x 10(-4) s(-1). The hydrolytic cleavage of DNA by the complexes was supported by the evidence from free radical quenching, thiobarbituric acid-reactive substances (TBARS) assay and T4 ligase ligation.

  10. 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.

  11. Evaluation of DNA cleavage, antimicrobial and anti-tubercular activities of potentially active transition metal complexes derived from 2,6-di(benzofuran-2-carbohydrazono)-4-methylphenol

    NASA Astrophysics Data System (ADS)

    Kokare, Dhoolesh Gangaram; Kamat, Vinayak; Naik, Krishna; Nevrekar, Anupama; Kotian, Avinash; Revankar, Vidyanand K.

    2017-01-01

    A 2,6-diformyl-4-methyl phenol based multidentate novel symmetric ligand and it is late first-row transition metal complexes have been prepared. The ligand and metal complexes were characterized by different spectroscopic techniques. The ligand shows a symmetric polydentate coordination mode through the phenoxide bimetallic bridge, two azomethine nitrogen atoms and two carbonyl oxygen atoms. All the complexes appear to be binuclear with octahedral geometry and nonelectrolytic nature. Complexes have shown significant growth inhibitory activity against tested bacterial and fungal strains as compared to that of ligand. The cobalt complex exhibited better antifungal potency than the standard used. Copper complex exhibits good antifungal activity whereas cobalt and zinc complexes are found to be good antibacterial agents. Ligand and complexes have shown excellent anti-tubercular activity and Calf Thymus-DNA cleavage property.

  12. 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.

  13. Hydrolytic cleavage of DNA by quercetin zinc(II) complex.

    PubMed

    Jun, Tan; Bochu, Wang; Liancai, Zhu

    2007-03-01

    Quercetin zinc(II) complex was investigated focusing on its hydrolytic activity toward DNA. The complex successfully promotes the cleavage of plasmid DNA, producing single and double DNA strand breaks. The amount of conversion of supercoiled form (SC) of plasmid to the nicked circular form (NC) depends on the concentration of the complex as well as the duration of incubation of the complex with DNA. The rate of conversion of SC to NC is 1.68x10(-4) s(-1) at pH 7.2 in the presence of 100 microM of the complex. The hydrolytic cleavage of DNA by the complex is supported by the evidence from free radical quenching, thiobarbituric acid-reactive substances (TBARS) assay, and T4 ligase ligation.

  14. PEG-mediated one-pot multicomponent reactions for the efficient synthesis of functionalized dihydropyridines and their functional group dependent DNA cleavage activity.

    PubMed

    Pal, Suman; Singh, Vandana; Das, Prolay; Choudhury, Lokman H

    2013-06-01

    Polyethylene glycol (PEG) has been found to be an inexpensive, non-toxic and useful medium for the one pot synthesis of highly functionalized dihydropyridines using multicomponent reactions (MCRs) at room temperature under catalyst free conditions. The notable features of this protocol are: mild reaction condition, applicability to wide range of substrates, reusability of the PEG and good yields. The interaction of the synthesized compounds with pUC19 plasmid DNA was also analyzed. Some of the synthesized compounds showed interesting functional group dependent nuclease activity for plasmid DNA cleavage under physiological conditions.

  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. "Self-activating" chemical nuclease: ferrocenyl cyclen Cu(II) complexes act as efficient DNA cleavage reagents in the absence of reductant.

    PubMed

    Li, Kun; Zhou, Li-Hong; Zhang, Ji; Chen, Shan-Yong; Zhang, Zhong-Wei; Zhang, Jing-Jing; Lin, Hong-Hui; Yu, Xiao-Qi

    2009-04-01

    The interactions of cyclen Cu(II) complexes functionalized by ferrocenyl group with plasmid DNA indicated that these complexes have high cleavage efficiency via an oxidative mechanism in the absence of any reductant or oxidant.

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

    PubMed

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

    2017-01-12

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

  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.

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

    PubMed

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

    2014-03-25

    The mononuclear copper(II) complexes (1&2) of ligands L(1) [N,N'-bis(2-hydroxy-5-methylbenzyl)-1,4-bis(3-iminopropyl)piperazine] or L(2) [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 L(1) and L(2) 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.

  2. Two novel ternary dicopper(II) μ-guanazole complexes with aromatic amines strongly activated by quantum dots for DNA cleavage.

    PubMed

    Hernández-Gil, Javier; Ferrer, Sacramento; Castiñeiras, Alfonso; Liu-González, Malva; Lloret, Francesc; Ribes, Angela; Coga, Lucija; Bernecker, Anja; Mareque-Rivas, Juan C

    2014-01-06

    Two novel (μ-guanazole)-bridged binuclear copper(II) complexes with 1,10-phenanthroline (phen) or 2,2'-bipyridine (bipy), [Cu2(μ-N2,N4-Hdatrz)(phen)2(H2O)(NO3)4] (1) and [Cu2(μ-N1,N2-datrz)2(μ-OH2)(bipy)2](ClO4)2 (2) (Hdatrz = 3,5-diamino-1,2,4-triazole = guanazole), have been prepared and characterized by X-ray diffraction, spectroscopy, and susceptibility measurements. Compounds 1 and 2 differ in the aromatic amine, which acts as a coligand, and in the Cu···Cu'-bridging system. Compound 1, which contains two mono-bridged copper ions, represents the first example of a discrete Cu-(NCN-trz)-Cu' complex. Compound 2, with two triply bridged copper ions, is one of the few compounds featuring a Cu-[(NN-trz)2 + (O-aquo)]-Cu' unit. Both compounds display antiferromagnetic coupling but of different magnitude: J (μ2,4-triazole) = -52 cm(-1) for 1 and J (μ1,2-triazolate) = -115 cm(-1) for 2. The DNA binding and cleavage properties of the two compounds have been investigated. Fluorescence, viscosimetry, and thermal denaturation studies reveal that both complexes have high affinity for DNA (1 > 2) and that only 1 acts as an intercalator. In the presence of a reducing agent like 3-mercaptopropionic acid, 1 produces significant oxidative DNA cleavage, whereas 2 is inactive. However, in the presence of very small quantities of micelles filled with core-shell CdSe-ZnS quantum dots (15 nM), 1 and 2 are considerably more active and become highly efficient nucleases as a result of the different possible mechanisms for promoting cooperative catalysis (metal-metal, metal-hydrogen bonding, metal-intercalation, and metal-nanoparticle). Electrophoresis DNA-cleavage inhibition experiments, X-ray photoelectron spectroscopy studies, and fluorescence ethidium bromide displacement assays reveal that in these novel nucleases the QDs act as redox-active protein-like nanoparticle structures that bind to the DNA and deliver electrons to the copper(II) centers for the generation of Cu

  3. DNA cleavage activity of Fe(II)N4Py under photo irradiation in the presence of 1,8-naphthalimide and 9-aminoacridine: unexpected effects of reactive oxygen species scavengers.

    PubMed

    Li, Qian; Browne, Wesley R; Roelfes, Gerard

    2011-09-05

    The DNA cleavage activity of the iron(II) complex of the ligand N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine (N4Py) was investigated in the presence of the chromophores 1,8-naphthalimide (NI) and 9-aminoacridine (AA) under photo irradiation at 355 and 400.8 nm and compared to the activity of the complex without the chromophores. Whereas in most cases no synergistic effect of the added chromophores on DNA cleavage efficiency was observed, it was found that for Fe(II)N4Py, in combination with NI under irradiation at 355 nm, the DNA cleavage activity was increased. Surprisingly, it was found that the addition of reactive oxygen species (ROS) scavengers gave rise to significantly increased DNA cleavage efficiency, which is a highly counterintuitive observation since ROS are needed to achieve DNA cleavage. A hypothesis is put forward to explain, at least partly, these results. It is proposed that the addition of scavengers inhibits quenching of (3)NI*, thus making photo-induced electron transfer between (3)NI* and Fe(III)N4Py more efficient. This results in reduction of Fe(III)N4Py to Fe(II)N4Py, which can then react with ROS giving rise to DNA cleavage. Hence the role of the scavengers is to maintain a close to optimal concentration of ROS. The present study serves as an illustration of the care that needs to be exercised in interpreting the results of experiments using standard ROS scavengers, since especially in complex systems such as presented here they can give rise to unexpected phenomena. In the presence of 1,8-naphthalimide or 9-aminoacridine, ROS scavengers can increase the DNA cleavage efficiency of Fe(II)N4Py complex under photo irradiation.

  4. New modulated design and synthesis of chiral CuII/SnIV bimetallic potential anticancer drug entity: In vitro DNA binding and pBR322 DNA cleavage activity

    NASA Astrophysics Data System (ADS)

    Tabassum, Sartaj; Sharma, Girish Chandra; Arjmand, Farukh

    2012-05-01

    A new chiral ligand scaffold L derived from (R)-2-amino-2-phenyl ethanol and diethyl oxalate was isolated and thoroughly characterized by various spectroscopic methods. The ligand L was allowed to react with CuCl2·2H2O and NiCl2·6H2O to achieve monometallic complexes 1 and 2, respectively. Subsequently modulation of 1 and 2 was carried out in the presence of SnCl4·5H2O to obtain heterobimetallic potential drug candidates 3 and 4 possessing (CuII/SnIV and NiII/SnIV) metallic cores, respectively and characterized by elemental analysis and spectroscopic data including 1H, 13C and 119Sn NMR in case of 3 and 4. In vitro DNA binding studies revealed that complex 3 avidly binds to DNA as quantified by Kb and Ksv values. Complex 3 exhibits a remarkable DNA cleavage activity (concentration dependent) with pBR322 DNA and the cleavage activity of 3 was significantly enhanced in the presence of activators and follows the order H2O2 > Asc > MPA > GSH. Complex 3 cleave pBR322 DNA via hydrolytic pathway and accessible to major groove of DNA.

  5. Quinoxaline based bio-active mixed ligand transition metal complexes: Synthesis, characterization, electrochemical, antimicrobial, DNA binding, cleavage, antioxidant and molecular docking studies.

    PubMed

    Dhanaraj, C Justin; Johnson, Jijo

    2015-10-01

    Co(II), Ni(II), Cu(II) and Zn(II) mixed ligand complexes have been synthesized from N(2), N(3)-bis(4-nitrophenyl)quinoxaline-2,3-diamine and 1,10-phenanthroline. The compounds were characterized by elemental analyses, molar conductance, magnetic susceptibility, IR, UV-Vis., (1)H NMR, mass and ESR spectra. Octahedral geometry has been assigned for Co(II), Ni(II) and Zn(II) complexes and distorted octahedral geometry for Cu(II) complex. Electrochemical behavior of the synthesized complexes was studied using cyclic voltammetry. Grain size and surface morphologies of the complexes were determined by powder XRD and SEM analyses. The mixed ligand metal complexes were screened for antimicrobial activity against bacterial species Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus; fungal species Aspergillus niger, and Candida albicans by disc diffusion method. The DNA binding and DNA cleavage activities of the compounds were determined using electronic absorption titration and agarose gel electrophoresis respectively. The superoxide radical scavenging and free radical scavenging activities of the Cu(II) complex was also evaluated. Molecular docking studies of the synthesized mixed ligand metal complexes were carried out against B-DNA dodecamer and the protein Plasmodium falciparum dihydrofolate reductase (pf DHFR).

  6. 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.

  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.

  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. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage

    PubMed Central

    Jiang, Fuguo; Taylor, David W.; Chen, Janice S.; Kornfeld, Jack E.; Zhou, Kaihong; Thompson, Aubri J.; Nogales, Eva; Doudna, Jennifer A.

    2016-01-01

    Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)–associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation. PMID:26841432

  10. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage.

    PubMed

    Jiang, Fuguo; Taylor, David W; Chen, Janice S; Kornfeld, Jack E; Zhou, Kaihong; Thompson, Aubri J; Nogales, Eva; Doudna, Jennifer A

    2016-02-19

    Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation.

  11. 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.

  12. DNA sequence and structure requirements for cleavage of V(D)J recombination signal sequences.

    PubMed Central

    Cuomo, C A; Mundy, C L; Oettinger, M A

    1996-01-01

    Purified RAG1 and RAG2 proteins can cleave DNA at V(D)J recombination signals. In dissecting the DNA sequence and structural requirements for cleavage, we find that the heptamer and nonamer motifs of the recombination signal sequence can independently direct both steps of the cleavage reaction. Proper helical spacing between these two elements greatly enhances the efficiency of cleavage, whereas improper spacing can lead to interference between the two elements. The signal sequences are surprisingly tolerant of structural variation and function efficiently when nicks, gaps, and mismatched bases are introduced or even when the signal sequence is completely single stranded. Sequence alterations that facilitate unpairing of the bases at the signal/coding border activate the cleavage reaction, suggesting that DNA distortion is critical for V(D)J recombination. PMID:8816481

  13. Copper(II) Complexes of Phenanthroline and Histidine Containing Ligands: Synthesis, Characterization and Evaluation of their DNA Cleavage and Cytotoxic Activity.

    PubMed

    Leite, Sílvia M G; Lima, Luís M P; Gama, Sofia; Mendes, Filipa; Orio, Maylis; Bento, Isabel; Paulo, António; Delgado, Rita; Iranzo, Olga

    2016-11-21

    Copper(II) complexes have been intensely investigated in a variety of diseases and pathological conditions due to their therapeutic potential. The development of these complexes requires a good knowledge of metal coordination chemistry and ligand design to control species distribution in solution and tailor the copper(II) centers in the right environment for the desired biological activity. Herein we present the synthesis and characterization of two ligands HL1 and H2L2 containing a phenanthroline unit (phen) attached to the amino group of histidine (His). Their copper(II) coordination properties were studied using potentiometry, spectroscopy techniques (UV-vis and EPR), mass spectrometry (ESI-MS) and DFT calculations. The data showed the formation of single copper complexes, [CuL1](+) and [CuL2], with high stability within a large pH range (from 3.0 to 9.0 for [CuL1](+) and from 4.5 to 10.0 for [CuL2]). In both complexes the Cu(2+) ion is bound to the phen unit, the imidazole ring and the deprotonated amide group, and displays a distorted square pyramidal geometry as confirmed by single crystal X-ray crystallography. Interestingly, despite having similar structures, these copper complexes show different redox potentials, DNA cleavage properties and cytotoxic activity against different cancer cell lines (human ovarian (A2780), its cisplatin-resistant variant (A2780cisR) and human breast (MCF7) cancer cell lines). The [CuL2] complex has lower reduction potential (Epc= -0.722 V vs -0.452 V for [CuL1](+)) but higher biological activity. These results highlight the effect of different pendant functional groups (carboxylate vs amide), placed out of the coordination sphere, in the properties of these copper complexes.

  14. Synthesis, Spectral Characterization, SEM, Antimicrobial, Antioxidative Activity Evaluation, DNA Binding and DNA Cleavage Investigation of Transition Metal(II) Complexes Derived from a tetradentate Schiff base bearing thiophene moiety.

    PubMed

    Abdel Aziz, Ayman A; Seda, Sabry H

    2017-03-01

    A novel series of Co(II), Ni(II), Cu(II) and Zn(II) mononuclear complexes have been synthesized involving a potentially tetradentate Schiff base ligand, which was obtained by condensation of 2-aminophenol with 2,5-thiophene-dicarboxaldehyde. The complexes were synthesized via reflux reaction of methanolic solution of the appropriate Schiff base ligand with one equivalent of corresponding metal acetate salt. Based on different techniques including micro analysis, FT-IR, NMR, UV-Vis, ESR, ESI-mass and conductivity measurements, four-coordinated geometry was assigned for all complexes. Spectroscopic data have shown that, the reported Schiff base coordinated to metal ions as a dibasic tetradentate ligand through the phenolic oxygen and the azomethine nitrogen. The antimicrobial activities of the parent ligand and its complexes were investigated by using the agar disk diffusion method. Antioxidation properties of the novel complexes were investigated and it was found that all the complexes have good radical scavenging properties. The binding of complexes to calf thymus DNA (CT-DNA) was investigated by absorption, emission and viscosity measurements. Binding studies have shown that all the complexes interacted with CT-DNA via intercalation mode and the binding affinity varies with relative order as Cu(II) complex > Co(II) complex > Zn(II) complex > Ni(II) complex. Furthermore, DNA cleavage properties of the metal complexes were also investigated. The results suggested the possible utilization of novel complexes for pharmaceutical applications.

  15. The water-soluble Roussin's red ester acting as a potential photochemical NO-delivery agent: photolysis reactions, DNA cleavage and anticancer activity.

    PubMed

    Chang, Han-Hun; Huang, Hung-Jen; Ho, Yun-Lung; Wen, Yu-Der; Huang, Wei-Ning; Chiou, Show-Jen

    2009-08-28

    The water-soluble Roussin's red ester [(NO)(2)Fe(mu-SCH(2)CH(2)P(O)(CH(2)OH)(2))(2)Fe(NO)(2)] (1), a potential photochemical prodrug of an NO precursor, was synthesized from the reaction of HSCH(2)CH(2)P(O)(CH(2)OH)(2) (F) and [Fe(CO)(2)(NO)(2)]. The IR v(NO) stretching frequencies of complex 1 appear at 1759 (s), 1784 (s) and 1816 (w) cm(-1) in buffer (pH = 7.4). NO was released with a stoichiometry ratio Delta[NO]/Delta[1] = 3.6 +/- 0.2 when complex 1 was exposed to UV in deaerated aqueous phosphate buffer solution. Here light acts as an On/Off switch for NO release. Incubation of pBR322 supercoiled DNA with complex 1, followed by irradiation, produced DNA strand breakage. In contrast to the addition of carboxy-PTIO (NO radical scavenger), DNA strand breakage was not inhibited when the scavengers of hydroxyl radical and singlet oxygen were added. Complex 1 irradiated under a N(2) atmosphere exhibited the same cleavage efficiency as complex 1 irradiated under air. The results show that DNA strand cleavage efficiency depends on the concentration of complex 1, the pH value of the buffer, and the duration of the photolysis of complex 1. The conversion rate from supercoiled (SC form) to nicked circular (NC form) of complex 1 was 2.96 x 10(-2) s(-1). The results of a T4 ligase enzymatic assay reveals the nonhydrolytic DNA breakage mechanism. The NO-release ability of complexes 1, 2, and 3 follows the order 1 > 2 > 3. Upon UV-irradiation, complex 1 exhibits cytotoxicity against B16-F10 mouse melanoma cells.

  16. Efficient, Mg(2+)-dependent photochemical DNA cleavage by the antitumor quinobenzoxazine (S)-A-62176.

    PubMed

    Yu, H; Kwok, Y; Hurley, L H; Kerwin, S M

    2000-08-22

    The quinobenzoxazines, a group of structural analogues of the antibacterial fluoroquinolones, are topoisomerase II inhibitors that have demonstrated promising anticancer activity in mice. It has been proposed that the quinobenzoxazines form a 2:2 drug-Mg(2+) self-assembly complex on DNA. The quinobenzoxazine (S)-A-62176 is photochemically unstable and undergoes a DNA-accelerated photochemical reaction to afford a highly fluorescent photoproduct. Here we report that the irradiation of both supercoiled DNA and DNA oligonucleotides in the presence of (S)-A-62176 results in photochemical cleavage of the DNA. The (S)-A-62176-mediated DNA photocleavage reaction requires Mg(2+). Photochemical cleavage of supercoiled DNA by (S)-A-62176 is much more efficient that the DNA photocleavage reactions of the fluoroquinolones norfloxacin, ciprofloxacin, and enoxacin. The photocleavage of supercoiled DNA by (S)-A-62176 is unaffected by the presence of SOD, catalase, or other reactive oxygen scavengers, but is inhibited by deoxygenation. The photochemical cleavage of supercoiled DNA is also inhibited by 1 mM KI. Photochemical cleavage of DNA oligonucleotides by (S)-A-62176 occurs most extensively at DNA sites bound by drug, as determined by DNase I footprinting, and especially at certain G and T residues. The nature of the DNA photoproducts, and inhibition studies, indicate that the photocleavage reaction occurs by a free radical mechanism initiated by abstraction of the 4'- and 1'-hydrogens from the DNA minor groove. These results lend further support for the proposed DNA binding model for the quinobenzoxazine 2:2 drug-Mg(2+) complex and serve to define the position of this complex on the minor groove of DNA.

  17. Relaxase DNA binding and cleavage are two distinguishable steps in conjugative DNA processing that involve different sequence elements of the nic site.

    PubMed

    Lucas, María; González-Pérez, Blanca; Cabezas, Matilde; Moncalian, Gabriel; Rivas, Germán; de la Cruz, Fernando

    2010-03-19

    TrwC, the relaxase of plasmid R388, catalyzes a series of concerted DNA cleavage and strand transfer reactions on a specific site (nic) of its origin of transfer (oriT). nic contains the cleavage site and an adjacent inverted repeat (IR(2)). Mutation analysis in the nic region indicated that recognition of the IR(2) proximal arm and the nucleotides located between IR(2) and the cleavage site were essential for supercoiled DNA processing, as judged either by in vitro nic cleavage or by mobilization of a plasmid containing oriT. Formation of the IR(2) cruciform and recognition of the distal IR(2) arm and loop were not necessary for these reactions to take place. On the other hand, IR(2) was not involved in TrwC single-stranded DNA processing in vitro. For single-stranded DNA nic cleavage, TrwC recognized a sequence embracing six nucleotides upstream of the cleavage site and two nucleotides downstream. This suggests that TrwC DNA binding and cleavage are two distinguishable steps in conjugative DNA processing and that different sequence elements are recognized by TrwC in each step. IR(2)-proximal arm recognition was crucial for the initial supercoiled DNA binding. Subsequent recognition of the adjacent single-stranded DNA binding site was required to position the cleavage site in the active center of the protein so that the nic cleavage reaction could take place.

  18. Conformational control of DNA target cleavage by CRISPR-Cas9.

    PubMed

    Sternberg, Samuel H; LaFrance, Benjamin; Kaplan, Matias; Doudna, Jennifer A

    2015-11-05

    Cas9 is an RNA-guided DNA endonuclease that targets foreign DNA for destruction as part of a bacterial adaptive immune system mediated by clustered regularly interspaced short palindromic repeats (CRISPR). Together with single-guide RNAs, Cas9 also functions as a powerful genome engineering tool in plants and animals, and efforts are underway to increase the efficiency and specificity of DNA targeting for potential therapeutic applications. Studies of off-target effects have shown that DNA binding is far more promiscuous than DNA cleavage, yet the molecular cues that govern strand scission have not been elucidated. Here we show that the conformational state of the HNH nuclease domain directly controls DNA cleavage activity. Using intramolecular Förster resonance energy transfer experiments to detect relative orientations of the Cas9 catalytic domains when associated with on- and off-target DNA, we find that DNA cleavage efficiencies scale with the extent to which the HNH domain samples an activated conformation. We furthermore uncover a surprising mode of allosteric communication that ensures concerted firing of both Cas9 nuclease domains. Our results highlight a proofreading mechanism beyond initial protospacer adjacent motif (PAM) recognition and RNA-DNA base-pairing that serves as a final specificity checkpoint before DNA double-strand break formation.

  19. Conformational control of DNA target cleavage by CRISPR–Cas9

    PubMed Central

    Sternberg, Samuel H.; LaFrance, Benjamin; Kaplan, Matias; Doudna, Jennifer A.

    2015-01-01

    Cas9 is an RNA-guided DNA endonuclease that targets foreign DNA for destruction as part of a bacterial adaptive immune system mediated by CRISPR (clustered regularly interspaced short palindromic repeats)1,2. Together with single-guide RNAs (sgRNA)3, Cas9 also functions as a powerful genome engineering tool in plants and animals4–6, and efforts are underway to increase the efficiency and specificity of DNA targeting for potential therapeutic applications7,8. Studies of off-target effects have shown that DNA binding is far more promiscuous than DNA cleavage9–11, yet the molecular cues that govern strand scission have not been elucidated. Here we show that the conformational state of the HNH nuclease domain directly controls DNA cleavage activity. Using intramolecular Förster resonance energy transfer (FRET) experiments to detect relative orientations of the Cas9 catalytic domains when associated with on- and off-target DNA, we find that DNA cleavage efficiencies scale with the extent to which the HNH domain samples an activated conformation. We furthermore uncover a surprising mode of allosteric communication that ensures concerted firing of both Cas9 nuclease domains. Our results highlight a proofreading mechanism beyond initial PAM recognition12 and RNA–DNA base-pairing3 that serves as a final specificity checkpoint before DNA double-strand break formation. PMID:26524520

  20. Hydrolytic cleavage of DNA-model substrates promoted by polyoxovanadates.

    PubMed

    Steens, Nele; Ramadan, Ahmed M; Absillis, Gregory; Parac-Vogt, Tatjana N

    2010-01-14

    Hydrolysis of 4-nitrophenyl phosphate (NPP) and bis-4-nitrophenyl phosphate (BNPP), two commonly used DNA model substrates, was examined in vanadate solutions by means of (1)H, (31)P and (51)V NMR spectroscopy. The hydrolysis of the phosphoester bond in NPP at 50 degrees C and pH 5.0 proceeds with a rate constant of 1.74 x 10(-5) s(-1). The cleavage of the phosphoester bond in BNPP at 70 degrees C and pH 5.0 proceeds with a rate constant of 3.32 x 10(-6) s(-1), representing an acceleration of four orders of magnitude compared to the uncatalyzed cleavage. Inorganic phosphate and nitrophenol (NP) were the only products of hydrolysis. The NMR spectra did not show evidence of any paramagnetic species, excluding the possibility of V(V) reduction to V(IV), indicating that the cleavage of the phosphoester bond is purely hydrolytic. The pH dependence of k(obs) revealed that the hydrolysis proceeds fastest in solutions of pH 5.5. Comparison of the rate profile with the concentration profile of polyoxovanadates shows a striking overlap of the k(obs) profile with the concentration of decavanadate (V(10)). Kinetic experiments at 37 degrees C using a fixed amount of NPP and increasing amounts of V(10) permitted the calculation of catalytic (k(c) = 5.67 x 10(-6) s(-1)) and formation constants for the NPP-V(10) complex (K(f) = 71.53 M(-1)). Variable temperature (31)P NMR spectra of a reaction mixture revealed broadening and shifting of the (31)P resonance upon addition of increasing amounts of decavanadate and upon increasing temperature, implying the dynamic exchange process between free and bound NPP at higher temperatures. The origin of the hydrolytic activity of V(10) is most likely due its high lability and its dissociation into smaller fragments which may allow the attachment of NPP and BNPP into the polyoxovanadate framework.

  1. A ribozyme with DNA in the hybridising arms displays enhanced cleavage ability.

    PubMed Central

    Hendry, P; McCall, M J; Santiago, F S; Jennings, P A

    1992-01-01

    Hammerhead ribozymes cleave RNA substrates containing the UX sequence, where X = U, C or A, embedded within sequences which are complementary to the hybridising 'arms' of the ribozyme. In this study we have replaced the RNA in the hybridising arms of the ribozyme with DNA, and the resulting ribozyme is many times more active than its precursor. In turnover-kinetics experiments with a 13-mer RNA substrate, the kcat/Km ratios are 10 and 150 microM-1min-1 for the RNA- and DNA-armed ribozymes, respectively. The effect is due mainly to differences in kcat. In independent experiments where the cleavage step is rate-limiting, the DNA-armed ribozyme cleaves the substrate with a rate constant more than 3 times greater than the all-RNA ribozyme. DNA substrates containing a ribocytidine at the cleavage site have been shown to be cleaved less efficiently than their all-RNA analogues; again however, the DNA-armed ribozyme is more effective than the all-RNA ribozyme against such DNA substrates. These results demonstrate that there are no 2'-hydroxyl groups in the arms of the ribozyme that are required for cleavage; and that the structure of the complex formed by the DNA-armed ribozyme with its substrate is more favourable for cleavage than that formed by the all-RNA ribozyme and its substrate. PMID:1280808

  2. Studies on cleavage of DNA by N-phosphoryl branched peptides.

    PubMed

    Feng, Yuping; Cao, Shengli; Xiao, Anshan; Xie, Wenjun; Li, Yanmei; Zhao, Yufen

    2006-06-01

    It was found that Nalpha,Nepsilon-di[N-(O,O-diisopropyl)phosphoryl-L-leucy]-L-lysyl-methyl ester (1) and Nalpha,Nepsilon-di[N-(O,O-diisopropyl)phosphoryl-L-phenylalanyl]-L-lysyl-methyl ester (2) could cleave supercoiled DNA such as PUC19 efficiently in 40 mM Britton-Robinson buffer. The cleavage activities for both were investigated by agarose gel electrophoresis. The T4 ligase experiments implied that the cleavage of DNA occurs via a hydrolytic path. The results showed that the cleavage reaction of DNA is dependent on the value of pH and ionic strength in the solution. DNA cleavage is more efficient by N-phosphoryl branched peptide 2 than by N-phosphoryl branched peptide 1. The experiments also show that hydrolysis of DNA by N-phosphoryl branched peptide 1 was accelerated in the presence of Mg2+ or Zn2+ ions. The interactions of DNA with N-phosphoryl branched peptides were also characterized by melting temperature measurements and circular dichroism (CD) techniques. On the basis of experimental data, the possible mechanism of interactions between DNA with N-phosphoryl branched peptides was discussed.

  3. Binding, Electrochemical Activation and Cleavage of DNA by Cobalt(II)tetrakis-N-Methylpyridyl Porphyrin and its β-Pyrrole Brominated Derivative

    PubMed Central

    Yellappa, Shivaraj; Seetharamappa, Jaldappagari; Rogers, Lisa M.; Chitta, Raghu; Singhal, Ram P.; D’Souza, Francis

    2008-01-01

    The binding of nucleic acids by water soluble cobalt(II) tetrakis-N-methylpyridyl porphyrin, (TMPyP)Co and its highly electron deficient derivative, cobalt(II) tetrakis-N-methyl pyridyl-β-octabromoporphyrin, (Br8TMPyP)Co was investigated by UV-visible absorption, circular dichroism (CD), electrochemical and gel electrophoresis methods. The changes of the absorption spectra during the titration of these complexes with polynucleotides revealed a shift in the absorption maxima and a hypochromicity of the porphyrin Soret bands. The intrinsic binding constants were found to be in the range of 105 – 106 M−1. These values were higher for more electron deficient (Br8TMPyP)Co. Induced CD bands were noticed in the Soret region of the complexes due to the interaction of these complexes with different polynucleotides and an analysis of the CD spectra supported mainly external mode of binding. Electrochemical studies revealed the cleavage of polynucleotide by (TMPyP)Co and (Br8TMPyP)Co in the presence of oxygen preferentially at the A-T base pair region. Gel electrophoresis experiments further supported the cleavage of nucleic acids. The results indicate that the β-pyrrole brominated porphyrin, (Br8TMPyP)Co binds strongly and cleaves nucleic acids efficiently as compared to (TMPyP)Co. This electrolytic procedure offers a unique tool in biotechnology for cleaving double-stranded DNA with specificity at the A-T regions. PMID:17105219

  4. 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.

  5. Guide-independent DNA cleavage by archaeal Argonaute from Methanocaldococcus jannaschii.

    PubMed

    Zander, Adrian; Willkomm, Sarah; Ofer, Sapir; van Wolferen, Marleen; Egert, Luisa; Buchmeier, Sabine; Stöckl, Sarah; Tinnefeld, Philip; Schneider, Sabine; Klingl, Andreas; Albers, Sonja-Verena; Werner, Finn; Grohmann, Dina

    2017-03-20

    Prokaryotic Argonaute proteins acquire guide strands derived from invading or mobile genetic elements, via an unknown pathway, to direct guide-dependent cleavage of foreign DNA. Here, we report that Argonaute from the archaeal organism Methanocaldococcus jannaschii (MjAgo) possesses two modes of action: the canonical guide-dependent endonuclease activity and a non-guided DNA endonuclease activity. The latter allows MjAgo to process long double-stranded DNAs, including circular plasmid DNAs and genomic DNAs. Degradation of substrates in a guide-independent fashion primes MjAgo for subsequent rounds of DNA cleavage. Chromatinized genomic DNA is resistant to MjAgo degradation, and recombinant histones protect DNA from cleavage in vitro. Mutational analysis shows that key residues important for guide-dependent target processing are also involved in guide-independent MjAgo function. This is the first characterization of guide-independent cleavage activity for an Argonaute protein potentially serving as a guide biogenesis pathway in a prokaryotic system.

  6. Disruption of a Topoisomerase-DNA Cleavage Complex by a DNA Helicase

    NASA Astrophysics Data System (ADS)

    Howard, Michael T.; Neece, Sue H.; Matson, Steven W.; Kreuzer, Kenneth N.

    1994-12-01

    The type II DNA topoisomerases are targets for a variety of chemotherapeutic agents, including the antibacterial quinolones and several families of antitumor drugs. These agents stabilize an enzyme-DNA cleavage complex that consists of the topoisomerase covalently linked to the 5' phosphates of a double-stranded DNA break. Although the drug-stabilized cleavage complex is readily reversible, it can result in cell death by a mechanism that remains uncertain. Here we demonstrate that the action of a DNA helicase can convert the cleavage complex into a nonreversible DNA break by displacing DNA strands from the complex. Formation of a nonreversible DNA break, induced by a DNA helicase, could explain the cytotoxicity of these topoisomerase poisons.

  7. 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.

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

    PubMed

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

    2015-04-15

    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 3.2×10(4) M(-1) and 2.9×10(4) M(-1) and acted as proficient photocleavers of SC pUC19 DNA in UV-A light, forming (1)O2 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.

  9. Differential induction of Leishmania donovani bi-subunit topoisomerase I-DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I.

    PubMed

    Das, Benu Brata; Sen, Nilkantha; Roy, Amit; Dasgupta, Somdeb Bose; Ganguly, Agneyo; Mohanta, Bikash Chandra; Dinda, Biswanath; Majumder, Hemanta K

    2006-01-01

    Emergence of the bi-subunit topoisomerase I in the kinetoplastid family (Trypanosoma and Leishmania) has brought a new twist in topoisomerase research related to evolution, functional conservation and preferential sensitivities to the specific inhibitors of type IB topoisomerase family. In the present study, we describe that naturally occurring flavones baicalein, luteolin and quercetin are potent inhibitors of the recombinant Leishmania donovani topoisomerase I. These compounds bind to the free enzyme and also intercalate into the DNA at a very high concentration (300 microM) without binding to the minor grove. Here, we show that inhibition of topoisomerase I by these flavones is due to stabilization of topoisomerase I-DNA cleavage complexes, which subsequently inhibit the religation step. Their ability to stabilize the covalent topoisomerase I-DNA complex in vitro and in living cells is similar to that of the known topoisomerase I inhibitor camptothecin (CPT). However, in contrast to CPT, baicalein and luteolin failed to inhibit the religation step when the drugs were added to pre-formed enzyme substrate binary complex. This differential mechanism to induce the stabilization of cleavable complex with topoisomerase I and DNA by these selected flavones and CPT led us to investigate the effect of baicalein and luteolin on CPT-resistant mutant enzyme LdTOP1Delta39LS lacking 1-39 amino acids of the large subunit [B. B. Das, N. Sen, S. B. Dasgupta, A. Ganguly and H. K. Majumder (2005) J. Biol. Chem. 280, 16335-16344]. Baicalein and luteolin stabilize duplex oligonucleotide cleavage with LdTOP1Delta39LS. This observation was further supported by the stabilization of in vivo cleavable complex by baicalein and luteolin with highly CPT-resistant L.donovani strain. Taken together, our data suggest that the interacting amino acid residues of topoisomerase I may be partially overlapping or different for flavones and CPT. This study illuminates new properties of the flavones

  10. Differential induction of Leishmania donovani bi-subunit topoisomerase I–DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I

    PubMed Central

    Das, Benu Brata; Sen, Nilkantha; Roy, Amit; Dasgupta, Somdeb Bose; Ganguly, Agneyo; Mohanta, Bikash Chandra; Dinda, Biswanath; Majumder, Hemanta K.

    2006-01-01

    Emergence of the bi-subunit topoisomerase I in the kinetoplastid family (Trypanosoma and Leishmania) has brought a new twist in topoisomerase research related to evolution, functional conservation and preferential sensitivities to the specific inhibitors of type IB topoisomerase family. In the present study, we describe that naturally occurring flavones baicalein, luteolin and quercetin are potent inhibitors of the recombinant Leishmania donovani topoisomerase I. These compounds bind to the free enzyme and also intercalate into the DNA at a very high concentration (300 µM) without binding to the minor grove. Here, we show that inhibition of topoisomerase I by these flavones is due to stabilization of topoisomerase I–DNA cleavage complexes, which subsequently inhibit the religation step. Their ability to stabilize the covalent topoisomerase I–DNA complex in vitro and in living cells is similar to that of the known topoisomerase I inhibitor camptothecin (CPT). However, in contrast to CPT, baicalein and luteolin failed to inhibit the religation step when the drugs were added to pre-formed enzyme substrate binary complex. This differential mechanism to induce the stabilization of cleavable complex with topoisomerase I and DNA by these selected flavones and CPT led us to investigate the effect of baicalein and luteolin on CPT-resistant mutant enzyme LdTOP1Δ39LS lacking 1–39 amino acids of the large subunit [B. B. Das, N. Sen, S. B. Dasgupta, A. Ganguly and H. K. Majumder (2005) J. Biol. Chem. 280, 16335–16344]. Baicalein and luteolin stabilize duplex oligonucleotide cleavage with LdTOP1Δ39LS. This observation was further supported by the stabilization of in vivo cleavable complex by baicalein and luteolin with highly CPT-resistant L.donovani strain. Taken together, our data suggest that the interacting amino acid residues of topoisomerase I may be partially overlapping or different for flavones and CPT. This study illuminates new properties of the flavones

  11. Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases.

    PubMed

    Volná, Petra; Jarjour, Jordan; Baxter, Sarah; Roffler, Steve R; Monnat, Raymond J; Stoddard, Barry L; Scharenberg, Andrew M

    2007-01-01

    LAGLIDADG homing endonucleases (LHEs) cleave 18-24 bp DNA sequences and are promising enzymes for applications requiring sequence-specific DNA cleavage amongst genome-sized DNA backgrounds. Here, we report a method for cell surface display of LHEs, which facilitates analysis of their DNA binding and cleavage properties by flow cytometry. Cells expressing surface LHEs can be stained with fluorescently conjugated double-stranded oligonucleotides (dsOligos) containing their respective target sequences. The signal is absolutely sequence specific and undetectable with dsOligos carrying single base-pair substitutions. LHE-dsOligo interactions facilitate rapid enrichment and viable recovery of rare LHE expressing cells by both fluorescence-activated cell sorting (FACS) and magnetic cell sorting (MACS). Additionally, dsOligos conjugated with unique fluorophores at opposite termini can be tethered to the cell surface and used to detect DNA cleavage. Recapitulation of DNA binding and cleavage by surface-displayed LHEs provides a high-throughput approach to library screening that should facilitate rapid identification and analysis of enzymes with novel sequence specificities.

  12. DNA binding, DNA cleavage, and cytotoxicity studies of two new copper (II) complexes.

    PubMed

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

    2011-05-01

    The DNA binding behavior of [Cu(phen)(phen-dione)Cl]Cl (1) and [Cu(bpy)(phen-dione)Cl]Cl (2) was studied with a series of techniques including UV-vis absorption, circular dichroism spectroscopy, and viscometric methods. Cytotoxicity effect and DNA unwinding properties were also investigated. The results indicate that the Cu(II) complexes interact with calf-thymus DNA by both partially intercalative and hydrogen binding. These findings have been further substantiated by the determination of intrinsic binding constants spectrophotometrically, 12.5 × 10(5) and 5 × 10(5) for 1 and 2, respectively. Our findings suggest that the type of ligands and structure of complexes have marked effect on the binding affinity of complexes involving CT-DNA. Circular dichroism results show that complex 1 causes considerable increase in base stacking of DNA, whereas 2 decreases the base stacking, which is related to more extended aromatic area of 1,10-phenanthroline in 1 rather than bipyridine in 2. Slow decrease in DNA viscosity indicates partially intercalative binding in addition to hydrogen binding on the surface of DNA. The second binding mode was also confirmed by additional tests: interaction in denaturation condition and acidic pH. Also, these new complexes induced cleavage in pUC18 plasmid DNA as indicated in gel electrophoresis and showed excellent antitumor activity against K562 (human chronic myeloid leukemia) cells.

  13. Activity dependent CAM cleavage and neurotransmission

    PubMed Central

    Conant, Katherine; Allen, Megan; Lim, Seung T.

    2015-01-01

    Spatially localized proteolysis represents an elegant means by which neuronal activity dependent changes in synaptic structure, and thus experience dependent learning and memory, can be achieved. In vitro and in vivo studies suggest that matrix metalloproteinase and adamalysin activity is concentrated at the cell surface, and emerging evidence suggests that increased peri-synaptic expression, release and/or activation of these proteinases occurs with enhanced excitatory neurotransmission. Synaptically expressed cell adhesion molecules (CAMs) could therefore represent important targets for neuronal activity-dependent proteolysis. Several CAM subtypes are expressed at the synapse, and their cleavage can influence the efficacy of synaptic transmission through a variety of non-mutually exclusive mechanisms. In the following review, we discuss mechanisms that regulate neuronal activity-dependent synaptic CAM shedding, including those that may be calcium dependent. We also highlight CAM targets of activity-dependent proteolysis including neuroligin and intercellular adhesion molecule-5 (ICAM-5). We include discussion focused on potential consequences of synaptic CAM shedding, with an emphasis on interactions between soluble CAM cleavage products and specific pre- and post-synaptic receptors. PMID:26321910

  14. Cleavage at Arg-1689 influences heavy chain cleavages during thrombin-catalyzed activation of factor VIII.

    PubMed

    Newell, Jennifer L; Fay, Philip J

    2009-04-24

    The procofactor, factor VIII, is activated by thrombin or factor Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689. The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at either Arg-1689 or Arg-372 and influences reaction rates at these sites. Because cleavage at Arg-372 appears rate-limiting and dependent upon initial cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences catalysis at this step. Recombinant B-domainless factor VIII mutants, R1689H and R1689Q were prepared and stably expressed to slow and eliminate cleavage, respectively. Specific activity values for the His and Gln mutations were approximately 50 and approximately 10%, respectively, that of wild type. Thrombin activation of the R1689H variant showed an approximately 340-fold reduction in the rate of Arg-1689 cleavage, whereas the R1689Q variant was resistant to thrombin cleavage at this site. Examination of heavy chain cleavages showed approximately 4- and 11-fold reductions in A2 subunit generation and approximately 3- and 7-fold reductions in A1 subunit generation for the R1689H and R1689Q mutants, respectively. These results suggest a linkage between light chain cleavage and cleavages in heavy chain. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed modest rate reductions (<5-fold) in generating A2 and A1 subunits and in cleaving light chain at Arg-1721 from either variant, suggesting little dependence upon prior cleavage at residue 1689 as compared with thrombin. Overall, these results are consistent with a competition between heavy and light chains for thrombin exosite binding and subsequent proteolysis with binding of the former chain preferred.

  15. 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.

  16. Regulation of DNA Replication in Early Embryonic Cleavages

    PubMed Central

    Kermi, Chames; Lo Furno, Elena; Maiorano, Domenico

    2017-01-01

    Early embryonic cleavages are characterized by short and highly synchronous cell cycles made of alternating S- and M-phases with virtually absent gap phases. In this contracted cell cycle, the duration of DNA synthesis can be extraordinarily short. Depending on the organism, the whole genome of an embryo is replicated at a speed that is between 20 to 60 times faster than that of a somatic cell. Because transcription in the early embryo is repressed, DNA synthesis relies on a large stockpile of maternally supplied proteins stored in the egg representing most, if not all, cellular genes. In addition, in early embryonic cell cycles, both replication and DNA damage checkpoints are inefficient. In this article, we will review current knowledge on how DNA synthesis is regulated in early embryos and discuss possible consequences of replicating chromosomes with little or no quality control. PMID:28106858

  17. DNA photoreacts by nucleobase ring cleavage to form labile isocyanates.

    PubMed

    Buschhaus, Laura; Rolf, Josefin; Kleinermanns, Karl

    2013-11-14

    Differential infrared absorption spectroscopy was used to study the formation of isocyanates and further photo-products in the oligonucleotides dG10, dC10 and dT10 and in their mononucleosides by ultraviolet light at 266 nm. We find that α-cleavage takes place in oligonucleotides and mononucleosides both in films and in solution. The very intense and spectrally isolated isocyanate (N=C=O) asymmetric stretch vibration at 2277 cm(-1) is used as a spectroscopic marker for detection of the photo-product. The band disappears upon reaction with small amounts of water vapour as expected for isocyanates. Quantum yields for isocyanate formation by nucleobase ring cleavage in the α-position to the carbonyl group are ∼5 × 10(-5) in the mononucleosides and up to 5 × 10(-4) in the oligonucleotides. In the mixed oligonucleotides dG10/dC10 and dA10/dT10 the quantum yield of α-cleavage drops by a factor of 10 compared to the single oligonucleotides. Implications for DNA repair and photo-induced DNA-protein cross-linking via isocyanate reaction with NH2 groups of amino acids are discussed.

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

    PubMed Central

    Woo, Christina M.; Li, Zhenwu; Herzon, Seth B.

    2016-01-01

    (–)-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

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

    PubMed Central

    Zuo, Zhicheng; Liu, Jin

    2016-01-01

    The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends. PMID:27874072

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

    NASA Astrophysics Data System (ADS)

    Zuo, Zhicheng; Liu, Jin

    2016-11-01

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

  1. DNA Enzyme-Decorated DNA Nanoladders as Enhancer for Peptide Cleavage-Based Electrochemical Biosensor.

    PubMed

    Kou, Bei-Bei; Zhang, Li; Xie, Hua; Wang, Ding; Yuan, Ya-Li; Chai, Ya-Qin; Yuan, Ruo

    2016-09-07

    Herein, we developed a label-free electrochemical biosensor for sensitive detection of matrix metalloproteinase-7 (MMP-7) based on DNA enzyme-decorated DNA nanoladders as enhancer. A peptide and single-stranded DNA S1-modified platinum nanoparticles (P1-PtNPs-S1), which served as recognition nanoprobes, were first immobilized on electrode. When target MMP-7 specifically recognized and cleaved the peptide, the PtNPs-S1 bioconjugates were successfully released from electrode. The remaining S1 on electrode then hybridized with ssDNA1 (I1) and ssDNA2 (I2), which could synchronously trigger two hybridization chain reactions (HCRs), resulting in the in situ formation of DNA nanoladders. The desired DNA nanoladders not only were employed as ideal nanocarriers for enzyme loading, but also maintained its catalytic activity. With the help of hydrogen peroxide (H2O2), manganese porphyrin (MnPP) with peroxidase-like activity accelerated the 4-chloro-1-naphthol (4-CN) oxidation with generation of insoluble precipitation on electrode, causing a very low differential pulse voltammetry (DPV) signal for quantitative determination of MMP-7. Under optimal conditions, the developed biosensor exhibited a wide linear ranging from 0.2 pg/mL to 20 ng/mL, and the detection limit was 0.05 pg/mL. This work successfully realized the combination of DNA signal amplification technique with artificial mimetic enzyme-catalyzed precipitation reaction in peptide cleavage-based protein detection, offering a promising avenue for the detection of other proteases.

  2. Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes.

    PubMed

    Ma, Enbo; Harrington, Lucas B; O'Connell, Mitchell R; Zhou, Kaihong; Doudna, Jennifer A

    2015-11-05

    Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9-guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family.

  3. Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

    PubMed Central

    Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

    2015-01-01

    Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

  4. 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

  5. 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.

  6. Histidine-Based Lipopeptides Enhance Cleavage of Nucleic Acids: Interactions with DNA and Hydrolytic Properties.

    PubMed

    Bélières, M; Déjugnat, C; Chouini-Lalanne, N

    2015-12-16

    Interaction studies and cleavage activity experiments were carried out between plasmid DNA and a series of histidine-based lipopeptides. Specific fluorescent probes (ethidium bromide, Hoechst 33342, and pyrene) were used to monitor intercalation, minor groove binding, and self-assembly of lipopeptides, respectively. Association between DNA and lipopeptides was thus evidenced, highlighting the importance of both histidine and hydrophobic tail in the interaction process. DNA cleavage in the presence of lipopeptides was then detected by gel electrophoresis and quantified, showing the importance of histidine and the involvement of its side-chain imidazole in the hydrolysis mechanism. These systems could then be developed as synthetic nucleases while raising concern of introducing histidine in the design of lipopeptide-based transfection vectors.

  7. Clerocidin interacts with the cleavage complex of Streptococcus pneumoniae topoisomerase IV to induce selective irreversible DNA damage.

    PubMed

    Richter, Sara N; Leo, Elisabetta; Giaretta, Giulia; Gatto, Barbara; Fisher, L Mark; Palumbo, Manlio

    2006-01-01

    Clerocidin (CL), a diterpenoid natural product, alkylates DNA through its epoxide moiety and exhibits both anticancer and antibacterial activities. We have examined CL action in the presence of topoisomerase IV from Streptococcus pneumoniae. CL promoted irreversible enzyme-mediated DNA cleavage leading to single- and double-stranded DNA breaks at specific sites. Reaction required the diterpenoid function: no cleavage was seen using a naphthalene-substituted analogue. Moreover, drug-induced DNA breakage was not observed using a mutant topoisomerase IV (ParC Y118F) unable to form a cleavage complex with DNA. Sequence analysis of 102 single-stranded DNA breaks and 79 double-stranded breaks revealed an overwhelming preference for G at the -1 position, i.e. immediately 5' of the enzyme DNA scission site. This specificity contrasts with that of topoisomerase IV cleavage with antibacterial quinolones. Indeed, CL stimulated DNA breakage by a quinolone-resistant topoisomerase IV (ParC S79F). Overall, the results indicate that topoisomerase IV facilitates selective irreversible CL attack at guanine and that its cleavage complex differs markedly from that of mammalian topoisomerase II which promotes both irreversible and reversible CL attack at guanine and cytosine, respectively. The unique ability to form exclusively irreversible DNA breaks suggests topoisomerase IV may be a key intracellular target of CL in bacteria.

  8. Enantioselective cleavage of supercoiled plasmid DNA catalyzed by chiral macrocyclic lanthanide(III) complexes.

    PubMed

    Krężel, Artur; Lisowski, Jerzy

    2012-02-01

    The enantiomers of the Sm (III), Eu (III) and Yb (III) complexes [LnL(NO(3))(2)](NO(3)) of a chiral hexaazamacrocycle were tested as catalysts for the hydrolytic cleavage of supercoiled plasmid DNA. The catalytic activity was remarkably enantioselective; while the [LnL(SSSS)(NO(3))(2)](NO(3)) enantiomers promoted the cleavage of plasmid pBR322 from the supercoiled form (SC) to the nicked form (NC), the [LnL(RRRR)(NO(3))(2)](NO(3)) enantiomers were inactive. Kinetics of plasmid DNA hydrolysis was also investigated by agarose electrophoresis and it indicated typical single-exponential cleavage reaction. The hydrolytic mechanism of DNA cleavage was confirmed by the successful ligation of hydrolysis product by T4 ligase. The NMR study of the solutions of the complexes in various buffers indicated that the complexes exist as monomeric cationic complexes [LnL(H(2)O)(3)](3+) in slightly acidic solutions and as dimeric cationic complexes [Ln(2)L(2)(μ-OH)(2)(H(2)O)(2)](4+) in slightly basic 8mM solutions, with the latter form being a possible catalyst for hydrolysis of phosphodiester bonds.

  9. Hybrid TiO II nanoparticles: an approach for developing site specific DNA cleavage

    NASA Astrophysics Data System (ADS)

    Liu, J.; Saponjic, Z.; Dimitrijevic, N. M.; Luo, S.; Preuss, D.; Rajh, T.

    2006-02-01

    We have developed hybrid light responsive TiO II nanoparticles electronically linked to PNA oligonucleotides that site specifically bind to double stranded target DNA. This opens a new opportunity for the development of a highly efficient "artificial restriction enzyme" whose activity can be controlled by using light. The work focuses on the use of TiO II nanocomposites as analogs of restriction enzymes with unique specificity that does not exist in current biological approaches. TiO II nanoparticles electronically linked to DNA or PNA adapters have been site-specifically attached along double stranded λ DNA vectors. Illumination of this assembly results in selective oxidation of DNA at the deepest "thermodynamic traps" located closest to the nanoparticle surface, causing DNA cleavage. We investigate the effect of the sequence and length of DNA and PNA adapters on the specificity of DNA cleavage. Related to this issue, the potential use of TiO II/DNA nanocomposites as "rare cutters" that cleave DNA in the places not achieved with existing protein-based enzymes is investigated.

  10. Unusual reactivity in a commercial chromium supplement compared to baseline DNA cleavage with synthetic chromium complexes.

    PubMed

    Chaudhary, Shveta; Pinkston, Joel; Rabile, M Mohamed; Van Horn, J David

    2005-03-01

    Commercially available chromium supplements were tested for their DNA cleavage ability compared with synthetic chromium(III) complexes, including chromium(III) tris-picolinate [Cr(pic)3], basic chromium acetate [Cr3O(OAc)6]+, model complexes, and recently patented Cr-complexes for use in supplements or therapy. Four different supplements (P1-P4) were tested for their DNA cleaving activity in the presence and the absence of H2O2, dithiothreitol (DTT) or ascorbate. One supplement, P1, showed nicking of DNA in the absence of oxidant or reductant at 120 microM metal concentration. Different lot numbers of P1 were also tested for DNA cleavage activity with similar results. Commercial supplements containing Cr(pic)3 nicked DNA at 120 microM metal concentrations in the presence of 5 mM ascorbate or with excess hydrogen peroxide, analogous to reactions with synthetic Cr(pic)3 reported elsewhere. Another chromium (non-Cr(pic)3) supplement, P2, behaves in a comparable manner to simple Cr(III) salts in the DNA nicking assay. Chromium(III) malonate [Cr(mal)2] and chromium(III) acetate [Cr(OAc)] can nick DNA in the presence of ascorbate or hydrogen peroxide, respectively, only at higher metal concentrations. The Cr(III) complexes of histidine, succinate or N-acetyl-L-glutamate do not nick DNA to a significant degree.

  11. The Ciona intestinalis cleavage clock is independent of DNA methylation.

    PubMed

    Suzuki, Miho M; Mori, Tomoko; Satoh, Noriyuki

    2016-10-01

    The initiation of embryonic gene expression in ascidian embryos appears to be tightly regulated by the number of DNA replication cycles. DNA methylation is thought to contribute to the clock mechanism that counts the rounds of DNA replication. We used mass spectrometry and whole genome bisulfite sequencing to characterize DNA methylation changes that occur in early developmental stages of the ascidian, Ciona intestinalis. We found that global DNA methylation in early Ciona development was static, and a base-wise comparison between the genomes of consecutive developmental stages found no DNA demethylation that was related to zygotic gene activation. Additionally, 5hmC was hardly detected by mass spectrometry in the developmental samples, suggesting a lack of demethylation mediated by ten eleven translocation (TET) methylcytosine dioxygenase in C. intestinalis. We conclude that DNA methylation is not involved in regulating DNA replication-dependent transcriptional activation.

  12. Copper(I) and nickel(II) complexes with 1:1 vs. 1:2 coordination of ferrocenyl hydrazone ligands: do the geometry and composition of complexes affect DNA binding/cleavage, protein binding, antioxidant and cytotoxic activities?

    PubMed

    Krishnamoorthy, Paramasivam; Sathyadevi, Palanisamy; Butorac, Rachel R; Cowley, Alan H; Bhuvanesh, Nattamai S P; Dharmaraj, Nallasamy

    2012-04-21

    A new series of geometrically different complexes containing ferrocenyl hydrazone ligands were synthesised by reacting suitable precursor complex [MCl(2)(PPh(3))(2)] with the ligands HL(1) or HL(2) (where M = Cu(II) or Ni(II); HL(1) = [Cp(2)Fe(CH=N-NH-CO-C(6)H(5))] (1) and HL(2) = [Cp(2)Fe(CH=N-NH-CO-C(5)H(4)N)]) (2). The new complexes of the composition [Cu(L(1))(PPh(3))(2)], (3) [Cu(L(2))(PPh(3))(2)] (4), [Ni(L(1))(2)] (5) and [Ni(L(2))(2)] (6) were characterised by various spectral studies. Among them, complexes 3 and 5 characterised by single crystal X-ray diffraction showed a distorted tetrahedral structure for the former with 1:1 metal-ligand stoichiometry, but a distorted square planar geometry with 1:2 metal-ligand stoichiometry in the case of the latter. Systematic biological investigations like DNA binding, DNA cleavage, protein binding, free radical scavenging and cytotoxicity activities were carried out using all the synthesised compounds and the results obtained were explained on the basis of structure-activity relationships. The binding constant (K(b)) values of the synthesised compounds are found to be in the order of magnitude 10(3)-10(5) M(-1) and also they exhibit significant cleavage of supercoiled (SC) pUC19 DNA in the presence of H(2)O(2) as co-oxidant. The conformational changes of bovine serum albumin (BSA) upon binding with the above complexes were also studied. In addition, concentration dependent free radical scavenging potential of all the synthesised compounds (1-6) was also carried out under in vitro conditions. Assays on the cytotoxicity of the above complexes against HeLa and A431 tumor cells and NIH 3T3 normal cells were also carried out.

  13. Synthesis, characterization, and photoactivated DNA cleavage by copper (II)/cobalt (II) mediated macrocyclic complexes.

    PubMed

    Naik, H R Prakash; Naik, H S Bhojya; Aravinda, T; Lamani, D S

    2010-01-01

    We report the synthesis of new photonuclease consisting of two Co(II)/Cu(II) complexes of macrocyclic fused quinoline. Metal complexes are [MLX(2)], type where M = Co(II) (5), Cu(II) (6), and X = Cl, and are well characterized by elemental analysis, Fourier transform infrared spectroscopy, (1)H-NMR and electronic spectra. We have shown that photocleavage of plasmid DNA is markedly enhanced when this ligand is irradiated in the presence of Cu(II), and more so than that of cobalt. The chemistry of ternary and binary Co(II) complexes showing efficient light induced (360 nm) DNA cleavage activity is summarized. The role of the metal in photoinduced DNA cleavage reactions is explored by designing complex molecules having macrocyclic structure. The mechanistic pathways are found to be concentration dependent on Co(II)/Cu(II) complexes and the photoexcitation energy photoredox chemistry. Highly effective DNA cleavage ability of 6 is attributed to the effective cooperation of the metal moiety.

  14. Coupling between ATP Binding and DNA Cleavage by DNA Topoisomerase II

    PubMed Central

    Mueller-Planitz, Felix; Herschlag, Daniel

    2008-01-01

    DNA topoisomerase II is a molecular machine that couples ATP hydrolysis to the transport of one DNA segment through a transient break in another segment. To learn about the energetic connectivity that underlies this coupling, we investigated how the ATPase domains exert control over DNA cleavage. We dissected the DNA cleavage reaction by measuring rate and equilibrium constants for the individual reaction steps utilizing defined DNA duplexes in the presence and absence of the nonhydrolyzable ATP analog 5′-adenylyl-β,γ-imidodiphosphate (AMPPNP). Our results revealed the existence of two enzyme conformations whose relative abundance is sensitive to the presence of nucleotides. The predominant species in the absence of nucleotides binds DNA at a diffusion limited rate but cannot efficiently cleave DNA. In the presence of AMPPNP, most of the enzyme is converted to a state in which DNA binding and release is extremely slow but which allows DNA cleavage. A minimal kinetic and thermodynamic framework is established that accounts for the cooperativity of cleavage of the two DNA strands in the presence and absence of bound AMPPNP and includes conformational steps revealed in the kinetic studies. The model unifies available kinetic, thermodynamic, and structural data to provide a description for the reaction in terms of the order and rate of individual reaction steps and the physical nature of the species on the reaction path. Furthermore, this reaction framework provides a foundation for a future in-depth analysis of energy transduction by topoisomerase II, for guiding and interpreting future structural studies, and for analyzing the mechanism of drugs that convert topoisomerase into a cellular poison. PMID:18403371

  15. Use of Divalent Metal Ions in the DNA Cleavage Reaction of Human Type II Topoisomerases†

    PubMed Central

    Deweese, Joseph E.; Burch, Amber M.; Burgin, Alex B.; Osheroff, Neil

    2009-01-01

    All type II topoisomerases require divalent metal ions in order to cleave and ligate DNA. In order to further elucidate the mechanistic basis for these critical enzyme-mediated events, the role of the metal ion in the DNA cleavage reaction of human topoisomerase IIβ was characterized and compared to that of topoisomerase IIα. The present study utilized divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that substituted a sulfur atom for the 3′-bridging oxygen or the non-bridging oxygens of the scissile phosphate. Based on time courses of DNA cleavage, cation titrations, and metal ion mixing experiments, we propose the following model for the use of divalent metal ions by human type II topoisomerases. First, both enzymes employ a two-metal-ion mechanism to support DNA cleavage. Second, an interaction between one divalent metal ion and the 3′-bridging atom of the scissile phosphate greatly enhances enzyme-mediated DNA cleavage, most likely by stabilizing the leaving 3′-oxygen. Third, there is an important interaction between a divalent second metal ion and a non-bridging atom of the scissile phosphate that stimulates DNA cleavage mediated by topoisomerase IIβ. If this interaction exists in topoisomerase IIα, its effects on DNA cleavage are equivocal. This last aspect of the model highlights a difference in metal ion utilization during DNA cleavage mediated by human topoisomerase IIα and IIβ. PMID:19222228

  16. Enthused research on DNA-binding and DNA-cleavage aptitude of mixed ligand metal complexes

    NASA Astrophysics Data System (ADS)

    Mahalakshmi, Rajkumar; Raman, Natarajan

    2013-08-01

    Five new Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) mixed ligand complexes have been synthesized using a Schiff base precursor (obtained by the condensation of N-(4-aminophenyl)acetamide and 4-chlorobenzaldehyde) as main ligand and 1,10-phenanthroline as co-ligand. They have been characterized by microanalytical data, IR, UV-Vis, magnetic moment values, conductivity and electrochemical measurements. The spectral data reveal that all the complexes exhibit octahedral geometry. The high electrical conductance of the complexes supports their electrolytic nature. The monomeric nature of the complexes has been assessed from their magnetic susceptibility values. These complexes are better antimicrobial active agents than the free ligands. DNA (CT) binding properties of these complexes have been explored by UV-Vis., viscosity measurements, cyclic voltammetry, and differential pulse voltammetry measurements. The oxidative cleavage activity of the complexes has been studied using supercoiled pUC19 DNA by gel electrophoresis. The experimental results show that the complexes are good intercalators.

  17. Enthused research on DNA-binding and DNA-cleavage aptitude of mixed ligand metal complexes.

    PubMed

    Mahalakshmi, Rajkumar; Raman, Natarajan

    2013-08-01

    Five new Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) mixed ligand complexes have been synthesized using a Schiff base precursor (obtained by the condensation of N-(4-aminophenyl)acetamide and 4-chlorobenzaldehyde) as main ligand and 1,10-phenanthroline as co-ligand. They have been characterized by microanalytical data, IR, UV-Vis, magnetic moment values, conductivity and electrochemical measurements. The spectral data reveal that all the complexes exhibit octahedral geometry. The high electrical conductance of the complexes supports their electrolytic nature. The monomeric nature of the complexes has been assessed from their magnetic susceptibility values. These complexes are better antimicrobial active agents than the free ligands. DNA (CT) binding properties of these complexes have been explored by UV-Vis., viscosity measurements, cyclic voltammetry, and differential pulse voltammetry measurements. The oxidative cleavage activity of the complexes has been studied using supercoiled pUC19 DNA by gel electrophoresis. The experimental results show that the complexes are good intercalators.

  18. 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.

  19. DNA microstructural requirements for neocarzinostatin chromophore-induced direct strand cleavage.

    PubMed Central

    Lee, S H; Thivierge, J O; Goldberg, I H

    1989-01-01

    The microstructural requirements for optimal interaction of neocarzinostatin chromophore (NCS-C) with DNA have been investigated using a series of hexadeoxyribonucleotides with modified bases such as O6-methyl G (MeG), I, 5-methyl C (MeC), U, or 5-Bromo U (BrU) at specific sites in its preferred trinucleotide 5'GNaNb3':5'Na,Nb,C3' (Na = A, C, or T). Results show that MeG:C and G:MeC in place of G:C improve direct strand cleavage at the target Nb (Nb = T greater than A much greater than C greater than G), whereas MeC:G and C:MeG in place of Na:Nb, hinder cleavage. The optimal base target at Nb appears to be determined by its ability to form T:A type base pairing instead of C:G type. The observed differences in DNA strand cleavage patterns can be rationalized by induced changes in target site structure and are compatible with a model for NCS-C:DNA interaction in which the naphthoate moiety intercalates between 5'GNa3', and the activated tetrahydro-s-indacene, lying in the minor groove, abstracts a hydrogen atom from C-5' of Nb. PMID:2527356

  20. Exploring the active site of the Streptococcus pneumoniae topoisomerase IV–DNA cleavage complex with novel 7,8-bridged fluoroquinolones

    PubMed Central

    Laponogov, Ivan; Pan, Xiao-Su; Veselkov, Dennis A.; Cirz, Ryan T.; Wagman, Allan; Moser, Heinz E.

    2016-01-01

    As part of a programme of synthesizing and investigating the biological properties of new fluoroquinolone antibacterials and their targeting of topoisomerase IV from Streptococcus pneumoniae, we have solved the X-ray structure of the complexes of two new 7,8-bridged fluoroquinolones (with restricted C7 group rotation favouring tight binding) in complex with the topoisomerase IV from S. pneumoniae and an 18-base-pair DNA binding site—the E-site—found by our DNA mapping studies to bind drug strongly in the presence of topoisomerase IV (Leo et al. 2005 J. Biol. Chem. 280, 14 252–14 263, doi:10.1074/jbc.M500156200). Although the degree of antibiotic resistance towards fluoroquinolones is much lower than that of β-lactams and a range of ribosome-bound antibiotics, there is a pressing need to increase the diversity of members of this successful clinically used class of drugs. The quinolone moiety of the new 7,8-bridged agents ACHN-245 and ACHN-454 binds similarly to that of clinafloxocin, levofloxacin, moxifloxacin and trovofloxacin but the cyclic scaffold offers the possibility of chemical modification to produce interactions with other topoisomerase residues at the active site. PMID:27655731

  1. Exploring the active site of the Streptococcus pneumoniae topoisomerase IV-DNA cleavage complex with novel 7,8-bridged fluoroquinolones.

    PubMed

    Laponogov, Ivan; Pan, Xiao-Su; Veselkov, Dennis A; Cirz, Ryan T; Wagman, Allan; Moser, Heinz E; Fisher, L Mark; Sanderson, Mark R

    2016-09-01

    As part of a programme of synthesizing and investigating the biological properties of new fluoroquinolone antibacterials and their targeting of topoisomerase IV from Streptococcus pneumoniae, we have solved the X-ray structure of the complexes of two new 7,8-bridged fluoroquinolones (with restricted C7 group rotation favouring tight binding) in complex with the topoisomerase IV from S. pneumoniae and an 18-base-pair DNA binding site-the E-site-found by our DNA mapping studies to bind drug strongly in the presence of topoisomerase IV (Leo et al. 2005 J. Biol. Chem. 280, 14 252-14 263, doi:10.1074/jbc.M500156200). Although the degree of antibiotic resistance towards fluoroquinolones is much lower than that of β-lactams and a range of ribosome-bound antibiotics, there is a pressing need to increase the diversity of members of this successful clinically used class of drugs. The quinolone moiety of the new 7,8-bridged agents ACHN-245 and ACHN-454 binds similarly to that of clinafloxocin, levofloxacin, moxifloxacin and trovofloxacin but the cyclic scaffold offers the possibility of chemical modification to produce interactions with other topoisomerase residues at the active site.

  2. Nonpolar nucleobase analogs illuminate requirements for site-specific DNA cleavage by vaccinia topoisomerase.

    PubMed

    Yakovleva, Lyudmila; Lai, Jacob; Kool, Eric T; Shuman, Stewart

    2006-11-24

    Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a specific target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p downward arrow N(-1) in duplex DNA. Here we study the effects of nonpolar pyrimidine isosteres difluorotoluene (F) and monofluorotoluene (D) and the nonpolar purine analog indole at individual positions of the scissile and nonscissile strands on the rate of single-turnover DNA transesterification and the cleavage-religation equilibrium. Comparison of the effects of nonpolar base substitution to the effects of abasic lesions reported previously allowed us to surmise the relative contributions of base-stacking and polar edge interactions to the DNA transesterification reactions. For example, the deleterious effects of eliminating the +2T base on the scissile strand were rectified by introducing the nonpolar F isostere, whereas the requirement for the +1T base was not elided by F substitution. We impute a role for +1T in recruiting the catalytic residue Lys-167 to the active site. Topoisomerase is especially sensitive to suppression of DNA cleavage upon elimination of the +4G and +3G bases of the nonscissile strand. Indole provided little or no gain of function relative to abasic lesions. Inosine substitutions for +4G and +3G had no effect on transesterification rate, implying that the guanine exocyclic amine is not a critical determinant of DNA cleavage. Prior studies of 2-aminopurine and 7-deazaguanine effects had shown that the O6 and N7 of guanine were also not critical. These findings suggest that either the topoisomerase makes functionally redundant contacts with polar atoms (likely via Tyr-136, a residue important for precleavage active site assembly) or that it relies on contacts to N1 or N3 of the purine ring. The cleavage-religation equilibrium is strongly skewed toward trapping of the covalent intermediate by elimination of the +1A base of the nonscissile strand; the reaction equilibrium is restored by +1 indole

  3. Potential-modulated DNA cleavage by (N-salicylideneglycinato)copper(II) complex.

    PubMed

    Yang, Zhou-Sheng; Wang, Yan-Ling; Liu, Yun-Chun; Zhao, Guang-Chao

    2005-11-01

    The interaction of aqua (N-salicylideneglycinato)copper(II) (Cu(salgly)2+) complex with calf thymus DNA has been investigated by cyclic voltammetry. Potential-modulated DNA cleavage in the presence of Cu(salgly)2+ complex was performed at a gold electrode in a thin layer cell. DNA can be efficiently cleaved by electrochemically reducing Cu(salgly)2+ complex to Cu(salgly)+ complex at -0.7 V (vs. Ag/AgCl). When the solution was aerated with a small flow of O2 during electrolysis, the extent of DNA cleavage was dramatically enhanced, and hydroxyl radical scavengers inhibited DNA cleavage. These results suggested that O2 and hydroxyl radical were involved in potential-modulated DNA cleavage reaction. The percentage of DNA cleavage was enhanced as the working potential was shifted to more negative values and the electrolysis time was increased. It was also dependent on the ratio of Cu(salgly)2+ complex to DNA concentration. The cleaved DNA fragments were separated by high performance liquid chromatography (HPLC). The experimental results indicated that the method for potential-modulated DNA cleavage by Cu(salgly)2+ complex was simple and efficient.

  4. EGFR induces DNA decomposition via phosphodiester bond cleavage

    PubMed Central

    Tong, Yongpeng; Li, Shuiming; Huang, Chunliu

    2017-01-01

    EGFR may induce DNA degradation. This activity had not been previously described as an EGRF function. To confirm this unexpected activity, testing of EGFR in the presence of ATP and either 5A, 5C, 5G, 5T, or 5U oligonucleotides was performed. HPLC-MS analysis demonstrated that 5A and 5U levels significantly decreased in the presence of EGFR. Furthermore, fragments 4A and 4U were produced in 5A+EGFR+ATP and in 5U+EGFR+ATP reaction mixtures, respectively, but not in EGFR-negative controls. Degradation of Poly(A), Poly(C), Poly(G), Poly(I), Poly(T), and Poly(U) oligomers in the presence of EGFR and ATP correlated with the lower ability of reaction products to pair with complementary oligonucleotides. Gel electrophoresis showed that breakdown products migrated more quickly than controls, especially after addition of paired (complementary) oligomers, Poly(A) and Poly(U). Furthermore, λ DNA reaction products also migrated more quickly after incubation with EGFR. The results suggest that EGFR can induce breakage of certain types of nucleotide phosphodiester bonds, especially within the A residues of DNA or U residues of RNA, to induce DNA or RNA decomposition, respectively. This activity may be important in EGRF signaling, DNA degradation, or repair in normal or cancer cell activities. PMID:28272528

  5. Synthesis of thiazolidine-2,4-dione derivatives: anticancer, antimicrobial and DNA cleavage studies.

    PubMed

    Laxmi, S Vijaya; Anil, P; Rajitha, G; Rao, Asha Jyothi; Crooks, Peter A; Rajitha, B

    2016-10-01

    In the search of efficient anticancer agents, here, new 5-(4-alkylbenzyledene)thiazolidine-2,4-dione derivatives (5a-g) have been successfully synthesized and characterized and are evaluated for anticancer and antimicrobial activities using DNA cleavage studies. In vitro studies on anticancer activity of compound 5d (NSC: 768619/1) was done against the full panel of 60 human tumor cell lines. The five-level dose activity results revealed that, the compound 5d was active against all the cell lines, it has shown potential activity against leukemia SR (GI50: 2.04 μM), non-small cell lung cancer NCI-H522 (GI50: 1.36 μM), colon cancer COLO 205 (GI50: 1.64 μM), CNS cancer SF-539 (GI50: 1.87 μM), melanoma SK-MEL-2 (GI50: 1.64 μM), ovarian cancer OVCAR-3 (GI50: 1.87 μM), renal cancer RXF 393 (GI50: 1.15 μM), prostate cancer PC-3 (GI50: 1.90 μM), and breast cancer MDA-MB-468(GI50: 1.11 μM). DNA cleavage studies revealed that at 50 μg/mL concentration, partial DNA digestion was observed and when the concentration is increasing to threefold (150 μg/mL), complete linear DNA digestion and partial supercoiled DNA digestion was observed. Further antimicrobial studies indicate that all the synthesized compounds except compound 5a possess prominent activity against all the screened microbial species. This study throws a ray of light in the field of anticancer drugs.

  6. 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

  7. Role for Caspase-Mediated Cleavage of Rad51 in Induction of Apoptosis by DNA Damage

    PubMed Central

    Huang, YinYin; Nakada, Shuji; Ishiko, Takatoshi; Utsugisawa, Taiju; Datta, Rakesh; Kharbanda, Surender; Yoshida, Kiyotsugu; Talanian, Robert V.; Weichselbaum, Ralph; Kufe, Donald; Yuan, Zhi-Min

    1999-01-01

    We report here that the Rad51 recombinase is cleaved in mammalian cells during the induction of apoptosis by ionizing radiation (IR) exposure. The results demonstrate that IR induces Rad51 cleavage by a caspase-dependent mechanism. Further support for involvement of caspases is provided by the finding that IR-induced proteolysis of Rad51 is inhibited by Ac-DEVD-CHO. In vitro studies show that Rad51 is cleaved by caspase 3 at a DVLD/N site. Stable expression of a Rad51 mutant in which the aspartic acid residues were mutated to alanines (AVLA/N) confirmed that the DVLD/N site is responsible for the cleavage of Rad51 in IR-induced apoptosis. The functional significance of Rad51 proteolysis is supported by the finding that, unlike intact Rad51, the N- and C-terminal cleavage products fail to exhibit recombinase activity. In cells, overexpression of the Rad51(D-A) mutant had no effect on activation of caspase 3 but did abrogate in part the apoptotic response to IR exposure. We conclude that proteolytic inactivation of Rad51 by a caspase-mediated mechanism contributes to the cell death response induced by DNA damage. PMID:10082566

  8. An unusual (H(2)O)(20) discrete water cluster in the supramolecular host of a charge transfer platinum(ii) complex: cytotoxicity and DNA cleavage activities.

    PubMed

    Mandal, Sutanuva; Castiñeiras, Alfonso; Mondal, Tapan K; Mondal, Arindam; Chattopadhyay, Dhrubajyoti; Goswami, Sreebrata

    2010-10-28

    The chemical reaction of Pt(II)(L(1))Cl(2) [L(1) = N-4-tolylpyridine-2-aldimine] with a bidentate N,S-donor atom ligand, 2-methylthioaniline, (HL(2)) in alkaline methanolic medium yielded a mixed ligand donor-acceptor complex, [Pt(II)(L(1))(L(2))]Cl, [1]Cl. The complex has been characterized by different spectroscopic and electrochemical techniques. The complex showed intense interligand charge transfer (ILCT) transition in the long wavelength region of UV-vis spectrum (>600 nm). The single-crystal X-ray structure of complex, [1]Cl·3.3H(2)O is reported. The cationic complex upon crystallization from aqueous methanol solvent produces an assembly of discrete, three dimensional (H(2)O)(20) guest moiety within the reference Pt-complex host lattice. The water assembly showed a unique type of aggregation of a distorted cube encapsulated by hydrogen bonded network of a twelve-water ring. The complex displayed one reversible cathodic response at -0.75 V and two irreversible anodic responses at 0.42 and 0.79 V versus Ag/AgCl reference electrode. The redox processes are characterized by EPR and spectroelectrochemistry. Density-functional theory calculations were employed to confirm the structural features and to support the spectral and redox properties of the complex. The square-planar complex has been found to intercalate DNA. Fluorescence spectroscopy, circular dichroism, cyclic voltammetry, viscosity measurements, together with DNA melting studies have been employed to characterize the binding of [1]Cl with calf thymus DNA. Agarose gel electrophoresis indicates that the complex cleaves supercoiled (SC) pUC19 plasmid DNA to its nicked circular (NC) form via singlet oxygen. As determined by a MTT assay, [1]Cl exhibits significant cytotoxicity with IC(50) value 58 μM.

  9. Mechanisms for enzymatic cleavage of the N-glycosidic bond in DNA

    PubMed Central

    Drohat, Alexander C.; Maiti, Atanu

    2014-01-01

    DNA glycosylases remove damaged or enzymatically modified nucleobases from DNA, thereby initiating the base excision repair (BER) pathway, which is found in all forms of life. These ubiquitous enzymes promote genomic integrity by initiating repair of mutagenic and/or cytotoxic lesions that arise continuously due to alkylation, deamination, or oxidation of the normal bases in DNA. Glycosylases also perform essential roles in epigenetic regulation of gene expression, by targeting enzymatically-modified forms of the canonical DNA bases. Monofunctional DNA glycosylases hydrolyze the N-glycosidic bond to liberate the target base, while bifunctional glycosylases mediate glycosyl transfer using an amine group of the enzyme, generating a Schiff base intermediate that facilitates their second activity, cleavage of the DNA backbone. Here we review recent advances in understanding the chemical mechanism of monofunctional DNA glycosylases, with an emphasis on how the reactions are influenced by properties of the nucleobase leaving-group, the moiety that varies across the vast range of substrates targeted by these enzymes. PMID:25181003

  10. 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

  11. Phosphodiester hydrolysis and specific DNA binding and cleavage promoted by guanidinium-functionalized zinc complexes.

    PubMed

    He, Juan; Sun, Jing; Mao, Zong-Wan; Ji, Liang-Nian; Sun, Hongzhe

    2009-05-01

    Two new Zn(II) complexes containing guanidinium groups, [Zn(L(1))Cl(2)](ClO(4))(2).H(2)O.CH(3)OH (1) and [Zn(L(2))Cl(2)](ClO(4))(2).0.5H(2)O (2), were synthesized and characterized (L(1)=5,5'-di[1-(guanidyl)methyl]-2,2'-bipyridyl bication and L(2)=6,6'-di[1-(guanidyl)methyl]-2,2'-bipyridyl bication). Both complexes are able to catalyze bis(p-nitrophenyl) phosphate (BNPP) hydrolysis efficiently. Obtained kinetic data reveal that both 1 and 2 show nearly 300- and 600-fold rate enhancement of BNPP hydrolysis, respectively, compared to their simple analogue without the guanidinium groups [Zn(bpy)Cl(2)] (bpy=2,2'-bipyridy) (3). Enhanced acceleration for cleavage of BNPP could be attributed to cooperative interaction between the Zn(II) ion and the guanidinium groups by electrostatic interaction and H-bonding. Studies on inhibition of sequence-specific endonucleases (DraI and SmaI) by complexes show that 1 and 2 are able to recognize nucleotide sequence, -TTT;AAA-, and highly effectively cleave the plasmid DNA in the presence of hydrogen peroxide, while 3 has no specific binding to the DNA target sequences and only shows low DNA cleavage activity.

  12. DNA translocation blockage, a general mechanism of cleavage site selection by type I restriction enzymes.

    PubMed Central

    Janscak, P; MacWilliams, M P; Sandmeier, U; Nagaraja, V; Bickle, T A

    1999-01-01

    Type I restriction enzymes bind to a specific DNA sequence and subsequently translocate DNA past the complex to reach a non-specific cleavage site. We have examined several potential blocks to DNA translocation, such as positive supercoiling or a Holliday junction, for their ability to trigger DNA cleavage by type I restriction enzymes. Introduction of positive supercoiling into plasmid DNA did not have a significant effect on the rate of DNA cleavage by EcoAI endonuclease nor on the enzyme's ability to select cleavage sites randomly throughout the DNA molecule. Thus, positive supercoiling does not prevent DNA translocation. EcoR124II endonuclease cleaved DNA at Holliday junctions present on both linear and negatively supercoiled substrates. The latter substrate was cleaved by a single enzyme molecule at two sites, one on either side of the junction, consistent with a bi-directional translocation model. Linear DNA molecules with two recognition sites for endonucleases from different type I families were cut between the sites when both enzymes were added simultaneously but not when a single enzyme was added. We propose that type I restriction enzymes can track along a DNA substrate irrespective of its topology and cleave DNA at any barrier that is able to halt the translocation process. PMID:10228175

  13. Cleavage pattern of DNA caused by endonuclease: Theoretical modeling and experimental verification

    NASA Astrophysics Data System (ADS)

    Inagaki, Shio; Liu, Li; Takinoue, Masahiro; Yoshikawa, Kenichi

    2010-02-01

    In apoptotic cells, genomic DNA molecules are fragmented into multiple fragments with lengths that are integer multiples of approximately 180-200 base pairs (bp), i.e., the size of a single nucleosome. Here we propose a simple mathematical model for interpreting this cleavage pattern of DNA. Under the condition of a purely stochastic cleavage process, we derive a time evolution of the probability distribution of the fragment length by a Poisson distribution. We examine the applicability of our model by analyzing experimental results with apoptotic cells. Our model enables us to satisfactorily interpret the experimental trends. Interestingly, this theoretical fitting of the experimental data provides kinetic information for the cleavage reaction.

  14. Synthesis, DNA binding, photo-induced DNA cleavage, cytotoxicity studies of a family of heavy rare earth complexes.

    PubMed

    Chen, Gong-Jun; Wang, Zhi-Gang; Qiao, Xin; Xu, Jing-Yuan; Tian, Jin-Lei; Yan, Shi-Ping

    2013-10-01

    As a continuing investigation of our previous studies about the influence of the different rare earth metal ions on the bioactivity, a family of heavy rare earth metal complexes, [RE(acac)3(dpq)] (RE=Tb (1), Dy (2), Ho (3), Er (4), Tm (5), Yb (6), Lu (7)) and [RE(acac)3(dppz)]·CH3OH (RE=Tb (8), Dy (9), Ho (10), Er (11), Tm (12), Yb (13), Lu (14) viz. acetylacetonate (acac), dipyrido[3,2-d:20,30-f]quinoxaline (dpq), dipyrido[3,2-a:20,30-c] phenazine (dppz)), has been synthesized and their biological activities were also investigated. On the irradiation with UV-A light of 365nm or ambient light, all complexes exhibit efficient DNA cleavage activity via the mechanistic pathway involving the formation of singlet oxygen and hydroxyl radical as the reactive species. In addition, the in vitro cytotoxicity of these complexes on HeLa cells has been examined by MTT assay, which indicate that these compounds have the potential to act as effective anticancer drugs. The results of the above biological experiments also reveal that the choice of different rare earth metal ions has little influence on the DNA binding, DNA cleavage and cytotoxicity.

  15. Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity.

    PubMed

    Guilinger, John P; Pattanayak, Vikram; Reyon, Deepak; Tsai, Shengdar Q; Sander, Jeffry D; Joung, J Keith; Liu, David R

    2014-04-01

    Although transcription activator-like effector nucleases (TALENs) can be designed to cleave chosen DNA sequences, TALENs have activity against related off-target sequences. To better understand TALEN specificity, we profiled 30 unique TALENs with different target sites, array length and domain sequences for their abilities to cleave any of 10(12) potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. Based on these findings, we engineered a TALEN variant that exhibits equal on-target cleavage activity but tenfold lower average off-target activity in human cells.

  16. The nuclease domain of the SPP1 packaging motor coordinates DNA cleavage and encapsidation

    PubMed Central

    Cornilleau, Charlène; Atmane, Noureddine; Jacquet, Eric; Smits, Callum; Alonso, Juan C.; Tavares, Paulo; Oliveira, Leonor

    2013-01-01

    The large terminase subunit is a central component of the genome packaging motor from tailed bacteriophages and herpes viruses. This two-domain enzyme has an N-terminal ATPase activity that fuels DNA translocation during packaging and a C-terminal nuclease activity required for initiation and termination of the packaging cycle. Here, we report that bacteriophage SPP1 large terminase (gp2) is a metal-dependent nuclease whose stability and activity are strongly and preferentially enhanced by Mn2+ ions. Mutation of conserved residues that coordinate Mn2+ ions in the nuclease catalytic site affect the metal-induced gp2 stabilization and impair both gp2-specific cleavage at the packaging initiation site pac and unspecific nuclease activity. Several of these mutations block also DNA encapsidation without affecting ATP hydrolysis or gp2 C-terminus binding to the procapsid portal vertex. The data are consistent with a mechanism in which the nuclease domain bound to the portal switches between nuclease activity and a coordinated action with the ATPase domain for DNA translocation. This switch of activities of the nuclease domain is critical to achieve the viral chromosome packaging cycle. PMID:23118480

  17. 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.

  18. 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)

  19. Cleavage enhancement of specific chemical bonds in DNA-Cisplatin complexes induced by X-rays

    NASA Astrophysics Data System (ADS)

    Zheng, Yi; Yao, Xiaobin; Luo, Xinglan; Fu, Xianzhi

    2014-04-01

    The chemical bond transformation of cisplatin-DNA complexes can be probed efficiently by XPS which provides a concomitant X-ray irradiation source as well. The presence to Pt could considerably increase formation of the SE induced by X-ray and that the further interaction of these LEE with DNA leads to the enhancement of bond cleavages.

  20. 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.

  1. 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.

  2. Enantiopure copper(II) complex of natural product rosin derivative: DNA binding, DNA cleavage and cytotoxicity.

    PubMed

    Fei, Bao-Li; Yin, Bin; Li, Dong-Dong; Xu, Wu-Shuang; Lu, Yang

    2016-12-01

    To develop chiral anticancer drug candidates for molecular target DNA, the synthesis and characterization of a novel enantiomerically pure copper(II) complex [Cu 1 Cl 2 ] (2) of an optically pure ligand N-(pyridin-2-ylmethylene) dehydroabietylamine (1) was carried out. The coordination geometry of the copper center is a distorted square-planar arrangement. The interactions of 1 and 2 with salmon sperm DNA were investigated by viscosity measurements, UV, fluorescence and circular dichroism (CD) spectroscopic techniques. All the results reveal that 1 and 2 interacted with DNA through intercalation and 2 exhibited a higher DNA binding ability. Further, 1 and 2 could cleave supercoiled pBR322 DNA by single strand and 2 displayed stronger cleavage ability in the presence of ascorbic acid. In vitro cytotoxicity of 1 and 2 against HeLa, SiHa, HepG-2 and A431 cancer cell lines was studied using CCK-8 assay. The results indicate that 2 had a superior cytotoxicity than 1 and the widely used drug cisplatin under identical conditions. Flow cytometry analysis demonstrates 2 produced death of HeLa cancer cells through an apoptotic pathway. Cell cycle analysis shows that 2 mainly arrested HeLa cells at the S phase. A novel enantiomerically pure copper(II) complex [Cu 1 Cl 2 ] (2) of an optically pure ligand N-(pyridin-2-ylmethylene) dehydroabietylamine (1), based on natural product rosin has been synthesized. 2 has the potential to act as effective anticancer drug.

  3. 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

  4. Internucleosomal DNA cleavage triggered by plasma membrane damage during necrotic cell death. Involvement of serine but not cysteine proteases.

    PubMed Central

    Dong, Z.; Saikumar, P.; Weinberg, J. M.; Venkatachalam, M. A.

    1997-01-01

    Autolytic DNA breakdown, detected as smears in electrophoretic gels, is a late event in necrosis. On the other hand, internucleosomal DNA cleavage, visualized as ladders, is thought to be a hallmark of apoptosis. We now report that this specific form of DNA fragmentation also occurs during necrosis and is an early event but appears to be triggered by proteolytic mechanisms significantly different from those documented in apoptosis. Treatment of MDCK cells with a mitochondrial uncoupler and a Ca2+ ionophore led to ATP depletion, necrotic morphology, and progressive fragmentation of DNA in an internucleosomal or ladder pattern. DNA breakdown was immediately preceded by increased permeability of the plasma membrane to macromolecules. Provision of glycine along with the noxious agents did not modify the extent of ATP depletion, but prevented plasma membrane damage. This was accompanied by complete inhibition of DNA fragmentation. Internucleosomal DNA cleavage was observed also during necrosis after rapid permeabilization of plasma membranes by detergents or streptolysin-O in hepatocytes, thymocytes, and P19, Jurkat, and MDCK cells. DNA fragmentation associated with necrosis was Ca2+/Mg2+ dependent, was suppressed by endonuclease inhibitors, and was abolished by serine protease inhibitors but not by inhibitors of interleukin-1 beta converting enzyme (ICE)-related proteases or caspases. Moreover, unlike apoptosis, it was not accompanied by caspase-mediated proteolysis. On the other hand, the cleavage-site-directed chymotryptic inhibitor N-tosyl-L-phenylalanyl-chloromethyl ketone (TPCK) suppressed DNA fragmentation not only in necrotic cells but also during Fas-mediated apoptosis, without inhibiting caspase-related proteolysis. The results suggest a novel pathway of endonuclease activation during necrosis not involving the participation of caspases. In addition, they indicate that techniques based on double-strand DNA breaks may not reliably differentiate between

  5. Activation of human prolegumain by cleavage at a C-terminal asparagine residue.

    PubMed Central

    Chen, J M; Fortunato, M; Barrett, A J

    2000-01-01

    The processing and activation of prolegumain were studied using the recombinant protein synthesized by cells that had been stably transfected with a human legumain cDNA construct. A cell line termed C13 was selected for the high-level expression of prolegumain. C13 cells produced primarily 56 kDa prolegumain. The 56 kDa form was enzymically inactive but stable at neutral pH, unlike the 35 kDa mature pig legumain; it could be converted into a 46 kDa active form by incubation at pH 4.5. The 56 kDa pro-form and the 46 kDa active form were found to have the same N-terminal amino acid sequence, indicating that cleavage at the N-terminus was not necessary for prolegumain activation, and that the decrease in molecular mass was due to a C-terminal cleavage. The C-terminal processing site was identified as Asn(323). Replacement of Asn(323) at the cleavage site with aspartate, serine, alanine or glutamate abolished the processing and activation of prolegumain. In contrast, mutation of other asparagine and aspartate residues near the cleavage site had no effect. These results demonstrate that Asn(323) is essential for prolegumain activation. PMID:11085925

  6. Activation of human prolegumain by cleavage at a C-terminal asparagine residue.

    PubMed

    Chen, J M; Fortunato, M; Barrett, A J

    2000-12-01

    The processing and activation of prolegumain were studied using the recombinant protein synthesized by cells that had been stably transfected with a human legumain cDNA construct. A cell line termed C13 was selected for the high-level expression of prolegumain. C13 cells produced primarily 56 kDa prolegumain. The 56 kDa form was enzymically inactive but stable at neutral pH, unlike the 35 kDa mature pig legumain; it could be converted into a 46 kDa active form by incubation at pH 4.5. The 56 kDa pro-form and the 46 kDa active form were found to have the same N-terminal amino acid sequence, indicating that cleavage at the N-terminus was not necessary for prolegumain activation, and that the decrease in molecular mass was due to a C-terminal cleavage. The C-terminal processing site was identified as Asn(323). Replacement of Asn(323) at the cleavage site with aspartate, serine, alanine or glutamate abolished the processing and activation of prolegumain. In contrast, mutation of other asparagine and aspartate residues near the cleavage site had no effect. These results demonstrate that Asn(323) is essential for prolegumain activation.

  7. Local sequence requirements for DNA cleavage by mammalian topoisomerase II in the presence of doxorubicin.

    PubMed Central

    Capranico, G; Kohn, K W; Pommier, Y

    1990-01-01

    Doxorubicin, a DNA-intercalator, is one of several anti-cancer drugs that have been found to stabilizes topoisomerase II cleavage complexes at drug-specific DNA sites. The distribution and DNA sequence environments of doxorubicin-stabilized sites were determined in the SV40 genome. The sites were found to be most concentrated in the major nuclear matrix-associated region and nearly absent in the vicinity of the replication origin including the enhancer sequences in the 21-bp and 72-bp tandem repeats. Among 97 doxorubicin-stabilized sites that were localized at the DNA sequence level, none coincided with any of the 90 topoisomerase II cleavage sites detected in the same regions in the absence of drug. Cleavage at the 90 enzyme-only sites was inhibited by doxorubicin and never stimulated even at low drug concentrations. All of the doxorubicin-stabilized sites had an A at the 3' terminus of at least one member of each pair of strand breaks that would constitute a topoisomerase II double-strand scission. Conversely, none of the enzyme-only sites had an A simultaneously at the corresponding positions on opposite strands. The 3'-A requirement for doxorubicin-stabilized cleavage is therefore incompatible with enzyme-only cleavage and explains the mutual exclusivity of the two classes of sites. Images PMID:2174543

  8. Effect of the multifunctional proteins RPA, YB-1, and XPC repair factor on AP site cleavage by DNA glycosylase NEIL1.

    PubMed

    Pestryakov, Pavel; Zharkov, Dmitry O; Grin, Inga; Fomina, Elizaveta E; Kim, Ekaterina R; Hamon, Loïc; Eliseeva, Irina A; Petruseva, Irina O; Curmi, Patrick A; Ovchinnikov, Lev P; Lavrik, Olga I

    2012-04-01

    DNA glycosylases are key enzymes in the first step of base excision DNA repair, recognizing DNA damage and catalyzing the release of damaged nucleobases. Bifunctional DNA glycosylases also possess associated apurinic/apyrimidinic (AP) lyase activity that nick the damaged DNA strand at an abasic (or AP) site, formed either spontaneously or at the first step of repair. NEIL1 is a bifunctional DNA glycosylase capable of processing lesions, including AP sites, not only in double-stranded but also in single-stranded DNA. Here, we show that proteins participating in DNA damage response, YB-1 and RPA, affect AP site cleavage by NEIL1. Stimulation of the AP lyase activity of NEIL1 was observed when an AP site was located in a 60 nt-long double-stranded DNA. Both RPA and YB-1 inhibited AP site cleavage by NEIL1 when the AP site was located in single-stranded DNA. Taking into account a direct interaction of YB-1 with the AP site, located in single-stranded DNA, and the high affinity of both YB-1 and RPA for single-stranded DNA, this behavior is presumably a consequence of a competition with NEIL1 for the DNA substrate. Xeroderma pigmentosum complementation group C protein (XPC), a key protein of another DNA repair pathway, was shown to interact directly with AP sites but had no effect on AP site cleavage by NEIL1.

  9. Metal-organic frameworks with phosphotungstate incorporated for hydrolytic cleavage of a DNA-model phosphodiester.

    PubMed

    Han, Qiuxia; Zhang, Lejie; He, Cheng; Niu, Jiangyang; Duan, Chunying

    2012-05-07

    Five phosphotungstate-incorporated metal-organic frameworks {[Eu(4)(dpdo)(9)(H(2)O)(16)PW(12)O(40)]}(PW(12)O(40))(2)·(dpdo)(3)·Cl(3) (1); {ZnNa(2)(μ-OH)(dpdo)(4)(H(2)O)(4)[PW(12)O(40)]}·3H(2)O (2); {Zn(3)(dpdo)(7)}[PW(12)O(40)](2)·3H(2)O (3); and [Ln(2)H(μ-O)(2)(dpdo)(4)(H(2)O)(2)][PW(12)O(40)]·3H(2)O (Ln = Ho for 4 and Yb for 5) (dpdo = 4,4'-bipyridine-N,N'-dioxide) have been synthesized through a one-step hydrothermal reaction and characterized by elemental analyses, infrared (IR) spectroscopy, photoluminescence, and single-crystal X-ray diffraction (XRD). The structural analyses indicate that 1-5 display diversity structure from one-dimensional (1D) to three-dimensional (3D) series of hybrids. Kinetic experiments for the hydrolytic cleavage of DNA-model phosphodiester BNPP (bis(p-nitrophenyl)phosphate) were followed spectrophotometrically for the absorbance increase at 400 nm in EPPS (4-(2-hydroxyethyl)piperazine-1-propane sulfonic acid) buffer solution, because of the formation of p-nitrophenoxide with 1-5 under conditions of pH 4.0 and 50 °C. Ultraviolet (UV) spectroscopy indicate that the cleavage of the phosphodiester bond proceeds with the pseudo-first-order rate constant in the range of 10(-7)-10(-6) s(-1), giving an inorganic phosphate and p-nitrophenol as the final products of hydrolysis. The results demonstrate that 1-5 have good catalytic activity and reusability for hydrolytic cleavage of BNPP.

  10. 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.

  11. 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

  12. Natural products as topoisomerase II poisons: effects of thymoquinone on DNA cleavage mediated by human topoisomerase IIα.

    PubMed

    Ashley, Rachel E; Osheroff, Neil

    2014-05-19

    The seeds of Nigella sativa (often referred to as black seed) have long been utilized as a medicinal herb in Middle Eastern, Northern African, and Indian cultures. Historically, black seed has been used to treat a variety of illnesses associated with inflammation. More recent studies have found that it induces apoptosis and displays anticancer activity in animal and cellular models. The major bioactive compound of black seed is thymoquinone, which shares structural features with 1,4-benzoquinone and other covalent topoisomerase II poisons. Because a number of anticancer drugs target type II topoisomerases, we determined the effects of thymoquinone and a series of related quinones on human topoisomerase IIα. Thymoquinone enhanced enzyme-mediated DNA cleavage ~5-fold, which is similar to the increase seen with the anticancer drug etoposide. In order to enhance cleavage, compounds had to have at least two positions available for acylation. Furthermore, activity was decreased by the inclusion of electron-donating groups or bulky substituents. As predicted for a covalent topoisomerase II poison, the activity of thymoquinone (and related compounds) was abrogated by the addition of a reducing agent. Also, thymoquinone inhibited topoisomerase IIα activity when incubated with the enzyme prior to the addition of DNA. Cleavage complexes formed in the presence of the compound were stable for at least 8 h. Lastly, black seed extract and black seed oil both increased levels of enzyme-mediated DNA cleavage, suggesting that thymoquinone is active even in more complex herbal formulations. These findings indicate that thymoquinone can be added to the growing list of dietary and medicinal natural products with activity against human type II topoisomerases.

  13. 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

  14. 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.

  15. DNA cleavage by homo- and heterotetranuclear Cu(II) and Mn(II) complexes with tetrathioether-tetrathiol moiety.

    PubMed

    Dülger, S; Saglam, N; Beldüz, A O; Güner, S; Karaböcek, S

    2000-09-01

    Novel homotetranuclear Cu(II) and heteronuclear Cu(II)-Mn(II) complexes with tetrathioether-tetrathiol moiety have been prepared and their DNA relaxation activities with plasmid pCYTEXP (5kb) were electrophoretically established. The cleavage products analyzed by neutral agarose gel electrophoresis indicated that the interaction of the metal complexes with supercoiled plasmid DNA yielded linear, nicked or degraded DNA. The relaxation activities of both homo- and heterotetranuclear (SK4) complexes are time- and concentration-dependent. The findings suggest that SK4 with potent nucleolytic activity is a good nuclease substitute in the presence ofcooxidant. Furthermore, the observation of induction of DNA into smaller fragments by SK4 is also significant.

  16. The DNA cleavage reaction of topoisomerase II: wolf in sheep's clothing.

    PubMed

    Deweese, Joseph E; Osheroff, Neil

    2009-02-01

    Topoisomerase II is an essential enzyme that is required for virtually every process that requires movement of DNA within the nucleus or the opening of the double helix. This enzyme helps to regulate DNA under- and overwinding and removes knots and tangles from the genetic material. In order to carry out its critical physiological functions, topoisomerase II generates transient double-stranded breaks in DNA. Consequently, while necessary for cell survival, the enzyme also has the capacity to fragment the genome. The DNA cleavage/ligation reaction of topoisomerase II is the target for some of the most successful anticancer drugs currently in clinical use. However, this same reaction also is believed to trigger chromosomal translocations that are associated with specific types of leukemia. This article will familiarize the reader with the DNA cleavage/ligation reaction of topoisomerase II and other aspects of its catalytic cycle. In addition, it will discuss the interaction of the enzyme with anticancer drugs and the mechanisms by which these agents increase levels of topoisomerase II-generated DNA strand breaks. Finally, it will describe dietary and environmental agents that enhance DNA cleavage mediated by the enzyme.

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

    PubMed

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

    2005-04-04

    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.

  18. 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

  19. Vanadium(IV) and copper(II) complexes of salicylaldimines and aromatic heterocycles: Cytotoxicity, DNA binding and DNA cleavage properties.

    PubMed

    Correia, Isabel; Roy, Somnath; Matos, Cristina P; Borovic, Sladjana; Butenko, Nataliya; Cavaco, Isabel; Marques, Fernanda; Lorenzo, Julia; Rodríguez, Alejandra; Moreno, Virtudes; Pessoa, João Costa

    2015-06-01

    Five copper(II) complexes, [Cu(sal-Gly)(bipy)](1), [Cu(sal-Gly)(phen)] (2), [Cu(sal-l-Ala)(phen)] (3), [Cu(sal-D-Ala)(phen)] (4), [Cu(sal-l-Phe)(phen)] (5) and five oxidovanadium(IV) complexes, [V(IV)O(sal-Gly)(bipy)] (6), [V(IV)O(sal-Gly)(phen)] (7), [V(IV)O(sal-l-Phe)(H2O)] (8), [V(IV)O(sal-l-Phe)(bipy)] (9), [V(IV)O(sal-l-Phe)(phen)] (10) (sal=salicylaldehyde, bipy=2,2'-bipyridine, phen=1,10-phenanthroline) were synthesized and characterized, and their interaction with DNA was evaluated by different techniques: gel electrophoresis, fluorescence, UV-visible and circular dichroism spectroscopy. The complexes interact with calf-thymus DNA and efficiently cleave plasmid DNA in the absence (only 2 and 5) and/or presence of additives. The cleavage ability is concentration-dependent as well as metal and ligand-dependent. Moreover, DNA binding experiments show that the phen-containing Cu(II) and V(IV)O compounds display stronger DNA interaction ability than the corresponding bipy analogues. The complexes present cytotoxic activity against human ovarian (A2780) and breast (MCF7) carcinoma cells. Cell-growth inhibition (IC50) of compounds 1, 2 and 5 in human promyelocytic leukemia (HL60) and human cervical cancer (HeLa) cells were also determined. The copper complexes show much higher cytotoxic activity than the corresponding vanadium complexes and the reference drug cisplatin (except for the sal-Gly complexes); namely, the phenanthroline copper complexes 2-5 are ca. 10-fold more cytotoxic than cisplatin and more cytotoxic than their bipyridine analogues.

  20. Endonuclease cleavage of blocked replication forks: An indirect pathway of DNA damage from antitumor drug-topoisomerase complexes

    NASA Astrophysics Data System (ADS)

    Hong, George; Kreuzer, Kenneth N.

    2003-04-01

    The cytotoxicity of several important antitumor drugs depends on formation of the covalent topoisomerase-DNA cleavage complex. However, cellular processes such as DNA replication are necessary to convert the cleavage complex into a cytotoxic lesion, but the molecular mechanism of this conversion and the precise nature of the cytotoxic lesion are unknown. Using a bacteriophage T4 model system, we have previously shown that antitumor drug-induced cleavage complexes block replication forks in vivo. In this report, we show that these blocked forks can be cleaved by T4 endonuclease VII to create overt DNA breaks. The accumulation of blocked forks increased in endonuclease VII-deficient infections, suggesting that endonuclease cleavage contributes to fork processing in vivo. Furthermore, purified endonuclease VII cleaved the blocked forks in vitro close to the branch points. These results suggest that an indirect pathway of branched-DNA cleavage contributes to the cytotoxicity of antitumor drugs that target DNA topoisomerases.

  1. Double-strand DNA cleavage by copper complexes of 2,2'-dipyridyl with guanidinium/ammonium pendants.

    PubMed

    He, Juan; Hu, Ping; Wang, Yu-Jia; Tong, Ming-Liang; Sun, Hongzhe; Mao, Zong-Wan; Ji, Liang-Nian

    2008-06-28

    Two ligands with guanidinium/ammonium groups were synthesized and their copper complexes, [Cu(L1)Cl2](ClO4)2.H2O (1) and [Cu(L2)Cl2](ClO4)2 (2) (L1 = 5,5'-di[1-(guanidyl)methyl]-2,2'-bipyridyl cation and L2 = 5,5'-di[1-(amino)methyl]-2,2'-bipyridyl cation), were prepared to serve as nuclease mimics. X-Ray analysis revealed that Cu(II) ion in 1 has a planar square CuN2Cl2-configuration. The shortest distance between the nitrogen of guanidinium and copper atoms is 6.5408(5) A, which is coincident with that of adjacent phosphodiesters in DNA (ca. 6 A). In the absence of reducing agent, supercoiled plasmid DNA cleavage by the complexes were performed and their hydrolytic mechanisms were demonstrated with radical scavengers and T4 ligase. The pseudo-Michaelis-Menten kinetic parameters (kcat, KM) were calculated to be 4.42 h(-1), 7.46 x 10(-5) M for 1, and 4.21 h(-1), 1.07 x 10(-4) M for 2, respectively. The result shows that their cleavage efficiency is about 10-fold higher than the simple analogue [Cu(bipy)Cl2] (3) (0.50 h(-1), 3.5 x 10(-4) M). The pH dependence of DNA cleavage by 1 and its hydroxide species in solution indicates that mononuclear [Cu(L1)(OH)(H2O)]3+ ion is the active species. Highly effective DNA cleavage ability of is attributed to the effective cooperation of the metal moiety and two guanidinium pendants with the phosphodiester backbone of nucleic acid.

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

    PubMed

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

    2015-11-01

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

  3. Self-cleavage of DNA in the presence of metal ions.

    PubMed

    Maeda, Hidekatsu; Wada, Shinya; Minoura, Norihiko

    2006-01-01

    DNA is well known to be aggregated by metal ions including Mn ions, however, analysis of the aggregation process from a chemical aspect, which means identification of the product yielded during the process, has not been performed yet. On determination of what kinds of degraded materials were in the supernatant obtained on centrifugation of a DNA mixture aggregated under the conditions of 10 mM Mn ions ([Mn]/[P]=46.3) at 70 degrees C for 1 h, dAMP, dCMP, dGMP, and TMP produced through self-cleavage of DNA were found in the water-soluble part. These mononucleotides were purified by HPLC using TSKgel ODS-80Ts, and identified by LC-TOF/MS. The self-cleavage was effectively occurred under the conditions of more than 5 mM Mn ions, a reaction temperature of more than 70 degrees C, a reaction time of more than 30 min, and the use of DNA with a molecular weight of more than 140 bp. The self-cleavage was affected by the molecular size of the DNA.

  4. Carotenoid-cleavage activities of crude enzymes from Pandanous amryllifolius.

    PubMed

    Ningrum, Andriati; Schreiner, Matthias

    2014-11-01

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

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

    PubMed

    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

    2015-06-02

    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.

  6. 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.

  7. 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

  8. Cleavage enhancement of specific chemical bonds in DNA by cisplatin radiosensitization.

    PubMed

    Xiao, Fangxing; Luo, Xinglan; Fu, Xianzhi; Zheng, Yi

    2013-05-02

    X-ray photoelectron spectroscopy (XPS) is harnessed as an in situ efficient characterization technique for monitoring chemical bond transformation in DNA and cisplatin-DNA complexes under synergic X-ray irradiation. By analyzing the variation of relative peak area of core elements of DNA as a function of irradiation time, we find that the most vulnerable scission sites in DNA are those containing phosphate and glycosidic bonds. Compared to DNA, the effective rate constants of the corresponding phosphodiester and glycosidic bond cleavages for cisplatin-DNA complexes are 1.8 and 1.9 folds larger. These damages and their enhancements are similar to those induced by low energy electrons (LEE). Consistently, the magnitude of the secondary electron distribution produced by the X-rays on the cisplatin-DNA complexes is considerably increased compared to that of pristine DNA. The data suggest that DNA radiosensization by cisplatin results not only from the sensitization of DNA to the action of LEE, but also from an increase the production of LEE at the site of binding of the cisplatin. The results provide new insights into the mechanisms of cisplatin-induced sensitization of DNA under X-ray irradiation, which could be helpful in the design of new cisplatin-based antitumor drugs.

  9. Rates of chemical cleavage of DNA and RNA oligomers containing guanine oxidation products.

    PubMed

    Fleming, Aaron M; Alshykhly, Omar; Zhu, Judy; Muller, James G; Burrows, Cynthia J

    2015-06-15

    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.

  10. DNA cleavage on photoexposure at the d-d band in ternary copper(II) complexes using red-light laser.

    PubMed

    Dhar, Shanta; Nethaji, Munirathinam; Chakravarty, Akhil R

    2006-12-25

    Ternary copper(II) complexes [Cu(L1)B](ClO4) (1, 2) and [Cu(L2)B](ClO4) (3, 4), where HL1 and HL2 are tridentate NSO- and ONO-donor Schiff bases and B is a heterocyclic base, viz. dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 1 and 3) or dipyrido[3,2-a:2',3'-c]phenazine (dppz, 2 and 4), were prepared and their DNA binding and photoinduced DNA cleavage activity studied. Complex 1, structurally characterized by single-crystal X-ray crystallography, shows an axially elongated square-pyramidal (4 + 1) coordination geometry in which the monoanionic L1 binds at the equatorial plane. The NN-donor dpq ligand exhibits an axial-equatorial binding mode. The complexes display good binding propensity to calf thymus DNA, giving a relative order 2 (NSO-dppz) > 4 (ONO-dppz) > 1 (NSO-dpq) > 3 (ONO-dpq). They cleave supercoiled pUC19 DNA to its nicked circular form when treated with 3-mercaptopropionic acid (MPA) by formation of hydroxyl radicals as the cleavage active species under dark reaction conditions. The photoinduced DNA cleavage activity of the complexes was investigated using UV radiation of 365 nm and red light of 633, 647.1, and 676.4 nm (CW He-Ne and Ar-Kr mixed gas ion laser sources) in the absence of MPA. Complexes 1 and 2, having photoactive NSO-donor Schiff base and dpq/dppz ligands, show dual photosensitizing effects involving both the photoactive ligands in the ternary structure with significantly better cleavage properties when compared to those of 3 and 4, having only photoactive dpq/dppz ligands. Involvement of singlet oxygen in the light-induced DNA cleavage reactions is proposed. A significant enhancement of the red-light-induced DNA cleavage activity is observed for the dpq and dppz complexes containing the sulfur ligand when compared to their earlier reported phen (1,10-phenanthroline) analogue. Enhancement of the cleavage activity on photoexposure at the d-d band indicates the occurrence of metal-assisted photosensitization processes involving the LMCT and d

  11. DNA cleavage site selection by Type III restriction enzymes provides evidence for head-on protein collisions following 1D bidirectional motion.

    PubMed

    Schwarz, Friedrich W; van Aelst, Kara; Tóth, Júlia; Seidel, Ralf; Szczelkun, Mark D

    2011-10-01

    DNA cleavage by the Type III Restriction-Modification enzymes requires communication in 1D between two distant indirectly-repeated recognitions sites, yet results in non-specific dsDNA cleavage close to only one of the two sites. To test a recently proposed ATP-triggered DNA sliding model, we addressed why one site is selected over another during cleavage. We examined the relative cleavage of a pair of identical sites on DNA substrates with different distances to a free or protein blocked end, and on a DNA substrate using different relative concentrations of protein. Under these conditions a bias can be induced in the cleavage of one site over the other. Monte-Carlo simulations based on the sliding model reproduce the experimentally observed behaviour. This suggests that cleavage site selection simply reflects the dynamics of the preceding stochastic enzyme events that are consistent with bidirectional motion in 1D and DNA cleavage following head-on protein collision.

  12. Triple helix-forming oligonucleotides conjugated to indolocarbazole poisons direct topoisomerase I-mediated DNA cleavage to a specific site.

    PubMed

    Arimondo, P B; Bailly, C; Boutorine, A S; Moreau, P; Prudhomme, M; Sun, J S; Garestier, T; Hélène, C

    2001-01-01

    Topoisomerase I is an ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins as well as for indolocarbazole antibiotics such as rebeccamycin. To achieve a sequence-specific cleavage of DNA by topoisomerase I, a triple helix-forming oligonucleotide was covalently linked to indolocarbazole-type topoisomerase I poisons. The three indolocarbazole-oligonucleotide conjugates investigated were able to direct topoisomerase I cleavage at a specific site based upon sequence recognition by triplex formation. The efficacy of topoisomerase I-mediated DNA cleavage depends markedly on the intrinsic potency of the drug. We show that DNA cleavage depends also upon the length of the linker arm between the triplex-forming oligonucleotide and the drug. Based on a known structure of the DNA-topoisomerase I complex, a molecular model of the oligonucleotide conjugates bound to the DNA-topoisomerase I complex was elaborated to facilitate the design of a potent topoisomerase I inhibitor-oligonucleotide conjugate with an optimized linker between the two moieties. The resulting oligonucleotide-indolocarbazole conjugate at 10 nM induced cleavage at the triple helix site 2-fold more efficiently than 5 microM of free indolocarbazole, while the other drug-sensitive sites were not cleaved. The rational design of drug-oligonucleotide conjugates carrying a DNA topoisomerase poison may be exploited to improve the efficacy and selectivity of chemotherapeutic cancer treatments by targeting specific genes and reducing drug toxicity.

  13. Variola virus topoisomerase: DNA cleavage specificity and distribution of sites in Poxvirus genomes.

    PubMed

    Minkah, Nana; Hwang, Young; Perry, Kay; Van Duyne, Gregory D; Hendrickson, Robert; Lefkowitz, Elliot J; Hannenhalli, Sridhar; Bushman, Frederic D

    2007-08-15

    Topoisomerase enzymes regulate superhelical tension in DNA resulting from transcription, replication, repair, and other molecular transactions. Poxviruses encode an unusual type IB topoisomerase that acts only at conserved DNA sequences containing the core pentanucleotide 5'-(T/C)CCTT-3'. In X-ray structures of the variola virus topoisomerase bound to DNA, protein-DNA contacts were found to extend beyond the core pentanucleotide, indicating that the full recognition site has not yet been fully defined in functional studies. Here we report quantitation of DNA cleavage rates for an optimized 13 bp site and for all possible single base substitutions (40 total sites), with the goals of understanding the molecular mechanism of recognition and mapping topoisomerase sites in poxvirus genome sequences. The data allow a precise definition of enzyme-DNA interactions and the energetic contributions of each. We then used the resulting "action matrix" to show that favorable topoisomerase sites are distributed all along the length of poxvirus DNA sequences, consistent with a requirement for local release of superhelical tension in constrained topological domains. In orthopox genomes, an additional central cluster of sites was also evident. A negative correlation of predicted topoisomerase sites was seen relative to early terminators, but no correlation was seen with early or late promoters. These data define the full variola virus topoisomerase recognition site and provide a new window on topoisomerase function in vivo.

  14. 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.

  15. 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

  16. 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.

  17. A Study on Spectro-Analytical Aspects, DNA - Interaction, Photo-Cleavage, Radical Scavenging, Cytotoxic Activities, Antibacterial and Docking Properties of 3 - (1 - (6 - methoxybenzo [d] thiazol - 2 - ylimino) ethyl) - 6 - methyl - 3H - pyran - 2, 4 - dione and its Metal Complexes.

    PubMed

    Ravi, Mudavath; Chennam, Kishan Prasad; Ushaiah, B; Eslavath, Ravi Kumar; Perugu, Shyam; Ajumeera, Rajanna; Devi, Ch Sarala

    2015-09-01

    The focus of the present work is on the design, synthesis, characterization, DNA-interaction, photo-cleavage, radical scavenging, in-vitro cytotoxicity, antimicrobial, docking and kinetic studies of Cu (II), Cd (II), Ce (IV) and Zr (IV) metal complexes of an imine derivative, 3 - (1 - (6 - methoxybenzo [d] thiazol - 2 - ylimino) ethyl) - 6 - methyl - 3H - pyran - 2, 4 - dione. The investigation of metal ligand interactions for the determination of composition of metal complexes, corresponding kinetic studies and antioxidant activity in solution was carried out by spectrophotometric methods. The synthesized metal complexes were characterized by EDX analysis, Mass, IR, (1)H-NMR, (13)C-NMR and UV-Visible spectra. DNA binding studies of metal complexes with Calf thymus (CT) DNA were carried out at room temperature by employing UV-Vis electron absorption, fluorescence emission and viscosity measurement techniques. The results revealed that these complexes interact with DNA through intercalation. The results of in vitro antibacterial studies showed the enhanced activity of chelating agent in metal chelated form and thus inferring scope for further development of new therapeutic drugs. Cell viability experiments indicated that all complexes showed significant dose dependent cytotoxicity in selected cell lines. The molecular modeling and docking studies were carried out with energy minimized structures of metal complexes to identify the receptor to metal interactions.

  18. 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.

  19. Synthetic prodigiosenes and the influence of C-ring substitution on DNA cleavage, transmembrane chloride transport and basicity.

    PubMed

    Rastogi, Soumya; Marchal, Estelle; Uddin, Imam; Groves, Brandon; Colpitts, Julie; McFarland, Sherri A; Davis, Jeffery T; Thompson, Alison

    2013-06-21

    Analogues of the tripyrrolic natural product prodigiosin bearing an additional methyl and a carbonyl group at the C-ring were synthesised and evaluated. In vitro anticancer activity screening (NCI) and the study of modes of action (copper-mediated cleavage of double-stranded DNA and transmembrane transport of chloride anions) showed that the presence of the methyl group is not detrimental to activity. Furthermore, although the presence of an ester conjugated to the prodigiosene C-ring seems to decrease both pK(a) and chloride transport efficiency compared to the natural product, these analogues still exhibit a high rate of chloride transport. All analogues exhibit good in vitro anticancer activity and reduced toxicity compared to the natural product: compare an acute systemic toxicity of 100 mg kg(-1) in mice vs. 4 mg kg(-1) for prodigiosin, pointing towards a larger therapeutic window than for the natural product.

  20. Malt1-dependent RelB cleavage promotes canonical NF-kappaB activation in lymphocytes and lymphoma cell lines.

    PubMed

    Hailfinger, Stephan; Nogai, Hendrik; Pelzer, Christiane; Jaworski, Maike; Cabalzar, Katrin; Charton, Jean-Enno; Guzzardi, Montserrat; Décaillet, Chantal; Grau, Michael; Dörken, Bernd; Lenz, Peter; Lenz, Georg; Thome, Margot

    2011-08-30

    The protease activity of the paracaspase Malt1 contributes to antigen receptor-mediated lymphocyte activation and lymphomagenesis. Malt1 activity is required for optimal NF-κB activation, but little is known about the responsible substrate(s). Here we report that Malt1 cleaved the NF-κB family member RelB after Arg-85. RelB cleavage induced its proteasomal degradation and specifically controlled DNA binding of RelA- or c-Rel-containing NF-κB complexes. Overexpression of RelB inhibited expression of canonical NF-κB target genes and led to impaired survival of diffuse large B-cell lymphoma cell lines characterized by constitutive Malt1 activity. These findings identify a central role for Malt1-dependent RelB cleavage in canonical NF-κB activation and thereby provide a rationale for the targeting of Malt1 in immunomodulation and cancer treatment.

  1. 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

  2. 8-Oxoguanine Affects DNA Backbone Conformation in the EcoRI Recognition Site and Inhibits Its Cleavage by the Enzyme

    PubMed Central

    Kiryutin, Alexey S.; Kasymov, Rustem D.; Petrova, Darya V.; Endutkin, Anton V.; Popov, Alexander V.; Yurkovskaya, Alexandra V.; Fedechkin, Stanislav O.; Brockerman, Jacob A.; Zharkov, Dmitry O.; Smirnov, Serge L.

    2016-01-01

    8-oxoguanine is one of the most abundant and impactful oxidative DNA lesions. However, the reasons underlying its effects, especially those not directly explained by the altered base pairing ability, are poorly understood. We report the effect of the lesion on the action of EcoRI, a widely used restriction endonuclease. Introduction of 8-oxoguanine inside, or adjacent to, the GAATTC recognition site embedded within the Drew—Dickerson dodecamer sequence notably reduced the EcoRI activity. Solution NMR revealed that 8-oxoguanine in the DNA duplex causes substantial alterations in the sugar—phosphate backbone conformation, inducing a BI→BII transition. Moreover, molecular dynamics of the complex suggested that 8-oxoguanine, although does not disrupt the sequence-specific contacts formed by the enzyme with DNA, shifts the distribution of BI/BII backbone conformers. Based on our data, we propose that the disruption of enzymatic cleavage can be linked with the altered backbone conformation and dynamics in the free oxidized DNA substrate and, possibly, at the protein—DNA interface. PMID:27749894

  3. Novel designed enediynes: molecular design, chemical synthesis, mode of cycloaromatization and guanine-specific DNA cleavage.

    PubMed

    Toshima, K; Ohta, K; Kano, T; Nakamura, T; Nakata, M; Kinoshita, M; Matsumura, S

    1996-01-01

    The molecular design and chemical synthesis of novel enediyne molecules related to the neocarzinostatin chromophore (1), and their chemical and DNA cleaving properties are described. The 10-membered enediyne triols 16-18 were effectively synthesized from xylitol (10) in a short step, and found to be quite stable when handled at room temperature. The representative and acylated enediyne 16 was cycloaromatized by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in cyclohexa-1,4-diene-benzene to give the benzenoid product 21 through a radical pathway. On the other hand, the enediyne 16 was cycloaromatized by diethylamine in dimethyl sulfoxide-Tris-HCl, pH 8.5 buffer to afford another benzenoid product 22 as a diethylamine adduct through a polar pathway. Furthermore, the enediynes 16-18 were found to exhibit guanine-specific DNA cleavage under weakly basic conditions with no additive.

  4. Novel reagents for chemical cleavage at abasic sites and UV photoproducts in DNA.

    PubMed Central

    McHugh, P J; Knowland, J

    1995-01-01

    Hot piperidine is often used to cleave abasic and UV-irradiated DNA at the sites of damage. It can inflict non-specific damage on DNA, probably because it is a strong base and creates significant concentrations of hydroxyl ions which can attack purines and pyrimidines. We show that several other amines can cleave abasic DNA at or near neutral pH without non-specific damage. One diamine, N,N'-dimethylethylenediamine, efficiently cleaves abasic DNA at pH 7.4 by either beta- or beta,delta-elimination, depending on temperature. Using end-labelled oligonucleotides we show that cleavage depends mainly on elimination reactions, but that 4',5'-cyclization is also significant. This reagent also cleaves at photoproducts induced by UVC and UVB, producing the same overall pattern as piperidine, but with no non-specific damage. It should prove valuable in locating low levels of photoproducts in DNA, such as those induced by natural sunlight. Images PMID:7784169

  5. Antimicrobial and DNA-cleavage studies of 22-membered N4 tetraaza macrocyclic triazoles: template synthesis and physicochemical characterization.

    PubMed

    Patil, Sangamesh A; Kamble, Udaykumar V; Badami, Prema S

    2010-09-01

    A novel series of 22-membered macrocyclic complexes of the type [MLCl(2)] (M=Co(2+), Ni(2+) and Cu(2+)) have been synthesized with newly derived biologically active ligands (L(1)-L(IV)). These ligands were synthesized by the condensation of ortho-phthalaldehyde and bis-(4-amino-5-mercapto-1, 2, 4-triazole-3-yl)alkanes. The mode of bonding and overall geometry of the complexes have been inferred through IR, EPR, electronic spectral studies, conductivity, magnetic, thermal, and electrochemical studies. All these complexes have been screened for their antibacterial (Escherichia coli, Staphylococus aureus, Salmonella typhi, Pseudomonas aeruginosa) and antifungal activities (Aspergillus niger, Aspergillus flavus, and Cladosporium) by the minimal inhibitory concentration (MIC) method. The DNA cleavage study was done by agarose gel electrophoresis technique.

  6. The novel endonuclease Ankle1 requires its LEM and GIY-YIG motifs for DNA cleavage in vivo

    PubMed Central

    Brachner, Andreas; Braun, Juliane; Ghodgaonkar, Medini; Castor, Dennis; Zlopaša, Livija; Ehrlich, Veronika; Jiricny, Josef; Gotzmann, Josef; Knasmüller, Siegfried; Foisner, Roland

    2015-01-01

    Summary The Lamina-associated polypeptide, Emerin, MAN1 - (LEM) domain defines a group of nuclear proteins, which bind chromatin through interaction of the LEM motif with the conserved DNA cross-linking protein, Barrier-to-Auto-Integration factor (BAF). Here, we describe a novel LEM protein, annotated in databases as “Ankyrin and LEM domain containing protein 1” (ANKLE1). We show that Ankle1 is conserved in metazoans and contains a unique C-terminal GIY-YIG motif that confers endonuclease activity in vitro and in vivo. In mammals, Ankle1 is predominantly expressed in hematopoietic tissues. While most characterized LEM proteins are components of the inner nuclear membrane, ectopic Ankle1 shuttles between cytoplasm and nucleus, and Ankle1 enriched in the nucleoplasm induces DNA cleavage and DNA damage response. This activity requires both the catalytic C-terminal GIY-YIG domain and the LEM motif, which binds chromatin via BAF. Hence, Ankle1 represents a novel LEM-protein with a GIY-YIG type endonuclease activity in higher eukaryotes. PMID:22399800

  7. Catalytic site-specific cleavage of a DNA-target by an oligonucleotide carrying bleomycin A5.

    PubMed Central

    Sergeyev, D S; Godovikova, T S; Zarytova, V F

    1995-01-01

    Oligonucleotide reagents have been created which are capable of catalytic site-specific cleavage of DNA-targets. The oligonucleotide reagent Blm-R-pd(CCAAACA) bearing the bleomycin A5 (Blm-RH) residue was used to degrade the DNA-target pd(TGTTTGGCGAAGGA). It has been shown that at equimolar reagent: target concentration the bleomycin oligonucleotide derivative can repeatedly cleave the target at G9, G7, T5, T4 and T3 in site-specific manner. This paper demonstrates that with a 10-fold excess of the DNA-target relative to the reagent 30% degradation of the target was observed primarily at a single position G7. The paper also shows that one reagent molecule containing bleomycin A5 residue was capable to degrade three molecules of the DNA-target. The catalytic activity of Blm-R-pd(CCAAACA) was the highest in the temperature range close to the melting temperature of the reagent-target complex, that is under conditions where the oligonucleotide reagent can form a complementary complex and easily dissociate to interact with the next molecule of the target. The number of target molecules degraded by the bleomycin reagent is limited by the degradation of the antibiotic residue itself. Images PMID:7501462

  8. Oxidation and glycolytic cleavage of etheno and propano DNA base adducts.

    PubMed

    Knutson, Charles G; Rubinson, Emily H; Akingbade, Dapo; Anderson, Carolyn S; Stec, Donald F; Petrova, Katya V; Kozekov, Ivan D; Guengerich, F Peter; Rizzo, Carmelo J; Marnett, Lawrence J

    2009-02-03

    Non-invasive strategies for the analysis of endogenous DNA damage are of interest for the purpose of monitoring genomic exposure to biologically produced chemicals. We have focused our research on the biological processing of DNA adducts and how this may impact the observed products in biological matrixes. Preliminary research has revealed that pyrimidopurinone DNA adducts are subject to enzymatic oxidation in vitro and in vivo and that base adducts are better substrates for oxidation than the corresponding 2'-deoxynucleosides. We tested the possibility that structurally similar exocyclic base adducts may be good candidates for enzymatic oxidation in vitro. We investigated the in vitro oxidation of several endogenously occurring etheno adducts [1,N(2)-epsilon-guanine (1,N(2)-epsilon-Gua), N(2),3-epsilon-Gua, heptanone-1,N(2)-epsilon-Gua, 1,N(6)-epsilon-adenine (1,N(6)-epsilon-Ade), and 3,N(4)-epsilon-cytosine (3,N(4)-epsilon-Cyt)] and their corresponding 2'-deoxynucleosides. Both 1,N(2)-epsilon-Gua and heptanone-1,N(2)-epsilon-Gua were substrates for enzymatic oxidation in rat liver cytosol; heteronuclear NMR experiments revealed that oxidation occurred on the imidazole ring of each substrate. In contrast, the partially or fully saturated pyrimidopurinone analogues [i.e., 5,6-dihydro-M(1)G and 1,N(2)-propanoguanine (PGua)] and their 2'-deoxynucleoside derivatives were not oxidized. The 2'-deoxynucleoside adducts, 1,N(2)-epsilon-dG and 1,N(6)-epsilon-dA, underwent glycolytic cleavage in rat liver cytosol. Together, these data suggest that multiple exocyclic adducts undergo oxidation and glycolytic cleavage in vitro in rat liver cytosol, in some instances in succession. These multiple pathways of biotransformation produce an array of products. Thus, the biotransformation of exocyclic adducts may lead to an additional class of biomarkers suitable for use in animal and human studies.

  9. 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

  10. 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.

  11. 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

  12. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.

    PubMed

    Pattanayak, Vikram; Lin, Steven; Guilinger, John P; Ma, Enbo; Doudna, Jennifer A; Liu, David R

    2013-09-01

    The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.

  13. Structural, magnetic, electrochemical, catalytic, DNA binding and cleavage studies of new macrocyclic binuclear copper(II) complexes.

    PubMed

    Anbu, S; Kandaswamy, M; Suthakaran, P; Murugan, V; Varghese, Babu

    2009-03-01

    The macrocyclic symmetrical and a series of unsymmetrical binuclear copper(II) complexes have been synthesized by using mononuclear complex [CuL] [3,3'-((1E,7E)-3,6-dioxa-2,7-diazaocta-1,7-diene-1,8-diyl)bis(3-formyl-5-methyl-2-diolato)copper(II)]. Another compartment of the [CuL] have been condensed with various diamines like 1,2-bis(aminooxy)ethane (L(1)), 1,2-diamino ethane(L(2a)), 1,3-diamino propane(L(2b)), 1,4-diamino butane(L(2c)), 1,2-diamino benzene(L(2d)), 1,8-diamino naphthalene(L(2e)) and characterized by elemental, spectroscopic, and X-ray crystallographic methods. The influence of the coordination geometry and the ring size of the binucleating ligands on the electronic, redox, magnetic, catecholase activity, DNA binding and cleavage properties have been studied. The molecular structures of the symmetrical binuclear complex [Cu(2)L(1)(H(2)O)(2)](ClO(4))(2) (1) and unsymmetrical binuclear complex [Cu(2)L(2b)(ClO(4))(H(2)O)]ClO(4) (2b) were determined by X-ray crystallography. Both of them were discrete binuclear species in which each Cu(II) ions are in distorted square pyramid. The Cu...Cu distances vary from 3.0308 (2b) to 3.0361 A (1). Electrochemical studies evidenced that two quasi-reversible one electron-transfer reduction waves (E(pc)(1)) -0.91 to -1.01 V, (E(pc)(2)) -1.26 to -1.55 V) for binuclear complexes are obtained in the cathodic region. Cryomagnetic investigation of the binuclear complexes reveals a weak antiferromagnetic spin exchange interaction between the Cu(II) ions within the complexes (-2J=104.4-127.5 cm(-1)). The initial rate (V(in)) for the oxidation of 3,5-di-tert-butylcatechol to o-quinone by the binuclear Cu(II)complexes are in the range 3.6 x 10(-5) to 7.3 x 10(-5)Ms(-1). The binuclear Cu(II) complexes are avid binders to calf thymus DNA. The complexes display significant oxidative cleavage of circular plasmid pBR322 DNA in the presence of mercaptoethanol using the singlet oxygen as a reactive species. The aromatic diamine

  14. 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.

  15. Calcium influx-mediated translocation of m-calpain induces Ku80 cleavage and enhances the Ku80-related DNA repair pathway

    PubMed Central

    Baek, Kyung Hye; Yu, Han Vit; Kim, Eosu; Na, Younghwa; Kwon, Youngjoo

    2016-01-01

    Proteomic analysis of ionomycin-treated and untreated mammary epithelial MCF10A cells elucidated differences in Ku80 cleavage. Ku80, a subunit of the Ku protein complex, is an initiator of the non-homologous, end-joining (NHEJ), double-strand breaks (DSBs) repair pathway. The nuclear Ku80 was cleaved in a calcium concentration-dependent manner by m-calpain but not by m-calpain. The cleavage of nuclear Ku80 at its α/β domain was validated by Western blotting analysis using flag-tagged expression vectors of truncated versions of Ku80 and a flag antibody and was confirmed in m-calpain knock-down cells and in vitro cell-free evaluation with recombinant proteins of calpains, Ku70, and Ku80. In addition, the cleaved Ku80 still formed a Ku heterodimer and promoted DNA DSB repair activity. Taken together, these findings indicate that translocated m-calpain enhances the NHEJ pathway through the cleavage of Ku80. Based on the present study, m-calpain in DNA repair pathways might be a novel anticancer drug target, or its mechanism might be a possible route for resistance acquisition of DNA damage-inducing chemotherapeutics. PMID:27121057

  16. The mechanism of human tyrosyl-DNA phosphodiesterase 1 in the cleavage of AP site and its synthetic analogs.

    PubMed

    Lebedeva, Natalia A; Rechkunova, Nadejda I; Ishchenko, Alexander A; Saparbaev, Murat; Lavrik, Olga I

    2013-12-01

    The mechanism of hydrolysis of the apurinic/apyrimidinic (AP) site and its synthetic analogs by using tyrosyl-DNA phosphodiesterase 1 (Tdp1) was analyzed. Tdp1 catalyzes the cleavage of AP site and the synthetic analog of the AP site, 3-hydroxy-2(hydroxymethyl)-tetrahydrofuran (THF), in DNA by hydrolysis of the phosphodiester bond between the substituent and 5' adjacent phosphate. The product of Tdp1 cleavage in the case of the AP site is unstable and is hydrolyzed with the formation of 3'- and 5'-margin phosphates. The following repair demands the ordered action of polynucleotide kinase phosphorylase, with XRCC1, DNA polymerase β, and DNA ligase. In the case of THF, Tdp1 generates break with the 5'-THF and the 3'-phosphate termini. Tdp1 is also able to effectively cleave non-nucleotide insertions in DNA, decanediol and diethyleneglycol moieties by the same mechanism as in the case of THF cleavage. The efficiency of Tdp1 catalyzed hydrolysis of AP-site analog correlates with the DNA helix distortion induced by the substituent. The following repair of 5'-THF and other AP-site analogs can be processed by the long-patch base excision repair pathway.

  17. Synthesis and properties of new DNA cleavage agents based on oxoruthenium(IV)

    SciTech Connect

    Gupta, N.; Grover, N.; Neyhart, G.A.; Singh, P.; Thorp, H.H. )

    1993-02-03

    New aquaruthenium(II) reagents that are capable of being oxidized to hydrooxoruthenium(III) and oxoruthenium(IV) have been prepared. Complexes based on Ru(tpy)(L)OH[sub 2][sup 2+] (L = [eta][sup 2]-tpt, phen, dppz, tmen; tpy = 2,2[prime]:6[prime],2[double prime]-terpyridine, tpt = 2,4,6-tripyridyltriazine, phen = 1,10-phenanthroline, dppz = dipyridophenazine, and tmen = N,N,N[prime],N[prime]-tetramethylethylenediamine) have been prepared and can all be reversibly oxidized to their Ru[sup IV]O forms, which are component DNA cleavage agents, as in Ru(phen)[sub 2](py)O[sup 2+]. In addition to Ru(tpy)([eta][sup 2]-tpt)OH[sub 2][sup 2+], the [eta][sup 3] complex of tpt, Ru(tpy)([eta][sup 3]-tpt)[sup 2+], can also be prepared under similar conditions. In the presence of Ag[sup +] ion, a novel Ru[sub 2]Ag complex can be isolated and has been crystallographically characterized. The complex [Ru(tpy)([eta][sup 3]-tpt)](ClO[sub 4])[sub 2][center dot]0.5AgClO[sub 4][center dot]0.5H[sub 2]O crystallizes in the monoclinic space group A2/A with a = 14.723 (5) [Angstrom], b = 26.061 (6) [Angstrom], c = 22.148 (6) [Angstrom], [beta] = 106.33 (3)[degrees], V = 8155 (5) [Angstrom][sup 3], Z = 4, R = 0.0807, and R[sub w] = 0.1156 for 2,923 reflections with I [ge] 2[sigma](I). The Ru(tpy)OH[sub 2][sup 2+] unit can also be attached to the tmen-AO[sup +] ligand, where a N,N[prime],N[prime]-trimethylethylenediamine function is appended via a (CH[sub 2])[sub 6] linker to the acridine orange intercalator. The Ru(tpy)(tmen-AO)OH[sub 2][sup 3+] complex is an effective cleavage agent, but only when oxidation is performed on the complex prebound to DNA. In homogeneous solution, electrochemically reversible access of only the Ru[sup III]OH form is possible, probably because of oxidation of the polymethylene linker. 25 refs., 11 figs., 9 tabs.

  18. Cleavage Factor I Links Transcription Termination to DNA Damage Response and Genome Integrity Maintenance in Saccharomyces cerevisiae

    PubMed Central

    Gaillard, Hélène; Aguilera, Andrés

    2014-01-01

    During transcription, the nascent pre-mRNA undergoes a series of processing steps before being exported to the cytoplasm. The 3′-end processing machinery involves different proteins, this function being crucial to cell growth and viability in eukaryotes. Here, we found that the rna14-1, rna15-1, and hrp1-5 alleles of the cleavage factor I (CFI) cause sensitivity to UV-light in the absence of global genome repair in Saccharomyces cerevisiae. Unexpectedly, CFI mutants were proficient in UV-lesion repair in a transcribed gene. DNA damage checkpoint activation and RNA polymerase II (RNAPII) degradation in response to UV were delayed in CFI-deficient cells, indicating that CFI participates in the DNA damage response (DDR). This is further sustained by the synthetic growth defects observed between rna14-1 and mutants of different repair pathways. Additionally, we found that rna14-1 suffers severe replication progression defects and that a functional G1/S checkpoint becomes essential in avoiding genetic instability in those cells. Thus, CFI function is required to maintain genome integrity and to prevent replication hindrance. These findings reveal a new function for CFI in the DDR and underscore the importance of coordinating transcription termination with replication in the maintenance of genomic stability. PMID:24603480

  19. 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

  20. DNA binding, DNA cleavage and HSA interaction of several metal complexes containing N-(2-hydroxyethyl)-N'-benzoylthiourea and 1,10-phenanthroline ligands.

    PubMed

    Peng, Bo; Gao, Zhuantao; Li, Xibo; Li, Tingting; Chen, Guorong; Zhou, Min; Zhang, Ji

    2016-10-01

    Four novel ternary metal complexes of the type [M(Phen)(L1)2)] [phen = 1,10-phenanthroline, L1 = N-(2-hydroxyethyl)-N'-benzoylthiourea, M = Ni(II)(1), Co(II) (2), Cu(II) (3), Pd(II) (4)] were synthesized. The organic ligands and their corresponding organometallic complexes have been characterized using UV-vis absorption spectroscopy, element analysis, infrared radiation spectroscopy and fluorescence spectra. DNA binding and cleavage studies of these complexes were conducted in detail. In vitro DNA-binding properties were studied by electronic absorption spectra and fluorescence spectra methods. The results indicate that all of the ternary metal complexes can efficiently bind to DNA via intercalation mode. The DNA-binding constants for all ternary compounds are around 4 × 10(6) M(-1). The binding propensity of the complexes to human serum albumin (HSA) was also investigated. Agarose gel electrophoresis study revealed that the metal complexes could cleave super-coiled pBR322 DNA to a nicked form in the absence of external agents. In vitro anti bacterial studies show that copper complex has weak antibacterial activities. Copper complex exhibits a better biological activity among all complexes. This study provides a new perspective and evaluation on the role and importance of the effect factors on the medicinal properties of benzoylthiourea compounds. Synchronous fluorescence spectra of HSA (10 μM) as a function of concentration of the complexes 1-4.

  1. Efficient single-strand cleavage of DNA mediated by a MnIIIMnIV-based artificial nuclease.

    PubMed

    Qian, Jing; Yu, Shasha; Wang, Wenjun; Wang, Liping; Tian, Jinlei; Yan, Shiping

    2014-02-14

    A water-soluble Mn(IV) 1,4,7-triazacyclononane complex, [Mn(IV)2L2(μ-O)2](ClO4)2·2H2O (1), was prepared to serve as a nuclease mimic (L = 1,4,7-triazacyclononane-N-acetate). Complex 1 was readily synthesized from the highly water soluble ligand (L), with Mn(III) salt, [Mn3O(MeCO2)7]·3H2O in basic condition, and characterized by X-ray, IR, electronic spectroscopy, cyclic voltammetry and magnetic susceptibility as well as ESI-MS. The bond valence sum (BVS) analysis and magnetic data suggest that 1 is a Mn(IV)-μ-O2-Mn(IV) species. The electrospray mass spectrum and electronic spectrum of 1 in aqueous solution indicates that dinuclear Mn complex [Mn(III)Mn(IV)L2(μ-O)2](+) (2) is the active species. A predominantly hydrolytic cleavage mechanism was confirmed through experiments performed in the presence of various radical scavengers, T4 ligase and under anaerobic conditions. The kinetic aspects of DNA cleavage under pseudo- or true-Michaelis-Menten conditions were also detailed, kinetic parameters (kcat, KM, Vmax) were calculated to be 6.27 h(-1), 7.35 × 10(-5) M, 4.6 × 10(-4) M h(-1); 0.683 h(-1), 1.93 × 10(-5) M, 1.32 × 10(-5) M h(-1) for 2, respectively.

  2. Sterol Regulatory Element-binding Protein (SREBP) Cleavage Regulates Golgi-to-Endoplasmic Reticulum Recycling of SREBP Cleavage-activating Protein (SCAP)*

    PubMed Central

    Shao, Wei; Espenshade, Peter J.

    2014-01-01

    Sterol regulatory element-binding protein (SREBP) transcription factors are central regulators of cellular lipogenesis. Release of membrane-bound SREBP requires SREBP cleavage-activating protein (SCAP) to escort SREBP from the endoplasmic reticulum (ER) to the Golgi for cleavage by site-1 and site-2 proteases. SCAP then recycles to the ER for additional rounds of SREBP binding and transport. Mechanisms regulating ER-to-Golgi transport of SCAP-SREBP are understood in molecular detail, but little is known about SCAP recycling. Here, we have demonstrated that SCAP Golgi-to-ER transport requires cleavage of SREBP at site-1. Reductions in SREBP cleavage lead to SCAP degradation in lysosomes, providing additional negative feedback control to the SREBP pathway. Current models suggest that SREBP plays a passive role prior to cleavage. However, we show that SREBP actively prevents premature recycling of SCAP-SREBP until initiation of SREBP cleavage. SREBP regulates SCAP in human cells and yeast, indicating that this is an ancient regulatory mechanism. PMID:24478315

  3. Beetroot betalain inhibits peroxynitrite-mediated tyrosine nitration and DNA strand cleavage.

    PubMed

    Sakihama, Yasuko; Maeda, Makiko; Hashimoto, Makoto; Tahara, Satoshi; Hashidoko, Yasuyuki

    2012-01-01

    Two major betalains, red-purple betacyanins and yellow betaxanthins, were isolated from red beetroots (Beta vulgaris L.), and their peroxynitrite (ONOO(-)) scavenging capacity was investigated. Apparent colours of the betalains were bleached by the addition of ONOO(-), and the absorbance decreases were suppressed in the presence of glutathione, a ONOO(-) scavenger. After bleaching, a new absorption maximum was observed at 350 nm in the spectrum of the resulting reaction mixture. New peaks were detected from HPLC analysis of the reaction products of betanin, a representative constituent of red beetroot betacyanins, treated with ONOO(-) monitoring at 350 nm, and the intensity of the major peak was positively correlated with ONOO(-) concentration. Betanin inhibited the ONOO(-) (0.5 mM)-dependent nitration of tyrosine (0.1 mM). Additionally, the IC(50) value of betanin (19.2 μM) was lower than that of ascorbate (79.6 μM). The presence of betanin (0.05-1.0 mM) also inhibited ONOO(-) (0.5 mM)-dependent DNA strand cleavage in a concentration-dependent manner. These results suggest that betalains can protect cells from nitrosative stress in addition to protecting them from oxidative stresses.

  4. 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

  5. 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.

  6. Angiopoietin-like protein 3 inhibits lipoprotein lipase activity through enhancing its cleavage by proprotein convertases.

    PubMed

    Liu, Jun; Afroza, Huq; Rader, Daniel J; Jin, Weijun

    2010-09-03

    Lipoprotein lipase (LPL)-mediated lipolysis of triglycerides is the first and rate-limiting step in chylomicron/very low density lipoprotein clearance at the luminal surface of the capillaries. Angiopoietin-like protein 3 (ANGPTL3) is shown to inhibit LPL activity and plays important roles in modulating lipoprotein metabolism in vivo. However, the mechanism by which it inhibits LPL activity remains poorly understood. Using cell-based analysis of the interaction between ANGPTL3, furin, proprotein convertase subtilisin/kexin type 5 (PCSK5), paired amino acid converting enzyme-4 (PACE4), and LPL, we demonstrated that the cleavage of LPL by proprotein convertases is an inactivation process, similar to that seen for endothelial lipase cleavage. At physiological concentrations and in the presence of cells, ANGPTL3 is a potent inhibitor of LPL. This action is due to the fact that ANGPTL3 can enhance LPL cleavage by endogenous furin and PACE4 but not by PCSK5. This effect is specific to LPL but not endothelial lipase. Both N- and C-terminal domains of LPL are required for ANGPTL3-enhanced cleavage, and the N-terminal domain of ANGPTL3 is sufficient to exert its effect on LPL cleavage. Moreover, ANGPTL3 enhances LPL cleavage in the presence of either heparan sulfate proteoglycans or glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1). By enhancing LPL cleavage, ANGPTL3 dissociates LPL from the cell surface, inhibiting both the catalytic and noncatalytic functions of LPL. Taken together, our data provide a molecular connection between ANGPTL3, LPL, and proprotein convertases, which may represent a rapid signal communication among different metabolically active tissues to maintain energy homeostasis. These novel findings provide a new paradigm of specific protease-substrate interaction and further improve our knowledge of LPL biology.

  7. Human Holliday junction resolvase GEN1 uses a chromodomain for efficient DNA recognition and cleavage

    PubMed Central

    Lee, Shun-Hsiao; Princz, Lissa Nicola; Klügel, Maren Felizitas; Habermann, Bianca; Pfander, Boris; Biertümpfel, Christian

    2015-01-01

    Holliday junctions (HJs) are key DNA intermediates in homologous recombination. They link homologous DNA strands and have to be faithfully removed for proper DNA segregation and genome integrity. Here, we present the crystal structure of human HJ resolvase GEN1 complexed with DNA at 3.0 Å resolution. The GEN1 core is similar to other Rad2/XPG nucleases. However, unlike other members of the superfamily, GEN1 contains a chromodomain as an additional DNA interaction site. Chromodomains are known for their chromatin-targeting function in chromatin remodelers and histone(de)acetylases but they have not previously been found in nucleases. The GEN1 chromodomain directly contacts DNA and its truncation severely hampers GEN1’s catalytic activity. Structure-guided mutations in vitro and in vivo in yeast validated our mechanistic findings. Our study provides the missing structure in the Rad2/XPG family and insights how a well-conserved nuclease core acquires versatility in recognizing diverse substrates for DNA repair and maintenance. DOI: http://dx.doi.org/10.7554/eLife.12256.001 PMID:26682650

  8. p53 facilitates pRb cleavage in IL-3-deprived cells: novel pro-apoptotic activity of p53.

    PubMed Central

    Gottlieb, E; Oren, M

    1998-01-01

    In the interleukin-3 (IL-3)-dependent lymphoid cell line DA-1, functional p53 is required for efficient apoptosis in response to IL-3 withdrawal. Activation of p53 in these cells, by either DNA damage or p53 overexpression, results in a vital growth arrest in the presence of IL-3 and in accelerated apoptosis in its absence. Thus, IL-3 can control the choice between p53-dependent cell-cycle arrest and apoptosis. Here we report that the cross-talk between p53 and IL-3 involves joint control of pRb cleavage and degradation. Depletion of IL-3 results in caspase-mediated pRb cleavage, occurring preferentially within cells which express functional p53. Moreover, pRb can be cleaved efficiently by extracts prepared from DA-1 cells but not from their derivatives which lack p53 function. Inactivation of pRb through expression of the human papillomavirus (HPV) E7 oncogene overrides the effect of IL-3 in a p53-dependent manner. Our data suggest a novel role for p53 in the regulation of cell death and a novel mechanism for the cooperation between p53 and survival factor deprivation. Thus, p53 makes cells permissive to pRb cleavage, probably by controlling the potential activity of a pRb-cleaving caspase, whereas IL-3 withdrawal provides signals that turn on this potential activity and lead to the actual cleavage and subsequent degradation of pRb. Elimination of a presumptive anti-apoptotic effect of pRb may then facilitate conversion of p53-mediated growth arrest into apoptosis. PMID:9649429

  9. The carbohydrate domain of calicheamicin gamma I1 determines its sequence specificity for DNA cleavage.

    PubMed Central

    Drak, J; Iwasawa, N; Danishefsky, S; Crothers, D M

    1991-01-01

    We have investigated the DNA cleaving properties of calicheamicinone, the synthetic core aglycone of calicheamicin gamma I1, a natural product with extremely potent antitumor activity. Our experiments have shown that the synthetic analog binds and cleaves DNA, albeit without any sequence selectivity and with less efficiency than the natural compound. We propose that a key element in the sequence recognition process is the thiobenzoate ring present in the natural compound. We have demonstrated by one-dimensional NMR that there is direct hydrogen abstraction from DNA by calicheamicinone, with enhanced binding affinity contributed by the carbohydrate domain. The reduced efficiency of hydrogen abstraction from DNA by bound calicheamicinone, compared with the natural compound, implicates the carbohydrate moiety in positioning the drug for hydrogen abstraction. Images PMID:1881884

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

    PubMed

    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 (HL(1)), 2-((4-oxo-4H-chromen-3-yl)methylneamino) benzoicacid (HL(2)), 3-((3-hydroxypyridin-2-ylimino)methyl)-4H-chromen-4-one (HL(3)) and 3-((2-mercaptophenylimino)methyl)-4H-chromen-4-one (HL(4)). 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.

  11. Mechanism of the Glycosidic Bond Cleavage of Mismatched Thymine in Human Thymine DNA Glycosylase Revealed by Classical Molecular Dynamics and Quantum Mechanical/Molecular Mechanical Calculations.

    PubMed

    Kanaan, Natalia; Crehuet, Ramon; Imhof, Petra

    2015-09-24

    Base excision of mismatched or damaged nucleotides catalyzed by glycosylase enzymes is the first step of the base excision repair system, a machinery preserving the integrity of DNA. Thymine DNA glycosylase recognizes and removes mismatched thymine by cleaving the C1'-N1 bond between the base and the sugar ring. Our quantum mechanical/molecular mechanical calculations of this reaction in human thymine DNA glycosylase reveal a requirement for a positive charge in the active site to facilitate C1'-N1 bond scission: protonation of His151 significantly lowers the free energy barrier for C1'-N1 bond dissociation compared to the situation with neutral His151. Shuttling a proton from His151 to the thymine base further reduces the activation free energy for glycosidic bond cleavage. Classical molecular dynamics simulations of the H151A mutant suggest that the mutation to the smaller, neutral, residue increases the water accessibility of the thymine base, rendering direct proton transfer from the bulk feasible. Quantum mechanical/molecular mechanical calculations of the glycosidic bond cleavage reaction in the H151A mutant show that the activation free energy is slightly lower than in the wild-type enzyme, explaining the experimentally observed higher reaction rates in this mutant.

  12. Surface invasive cleavage assay on a maskless light-directed diamond DNA microarray for genome-wide human SNP mapping.

    PubMed

    Nie, Bei; Yang, Min; Fu, Weiling; Liang, Zhiqing

    2015-07-07

    The surface invasive cleavage assay, because of its innate accuracy and ability for self-signal amplification, provides a potential route for the mapping of hundreds of thousands of human SNP sites. However, its performance on a high density DNA array has not yet been established, due to the unusual "hairpin" probe design on the microarray and the lack of chemical stability of commercially available substrates. Here we present an applicable method to implement a nanocrystalline diamond thin film as an alternative substrate for fabricating an addressable DNA array using maskless light-directed photochemistry, producing the most chemically stable and biocompatible system for genetic analysis and enzymatic reactions. The surface invasive cleavage reaction, followed by degenerated primer ligation and post-rolling circle amplification is consecutively performed on the addressable diamond DNA array, accurately mapping SNP sites from PCR-amplified human genomic target DNA. Furthermore, a specially-designed DNA array containing dual probes in the same pixel is fabricated by following a reverse light-directed DNA synthesis protocol. This essentially enables us to decipher thousands of SNP alleles in a single-pot reaction by the simple addition of enzyme, target and reaction buffers.

  13. Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells.

    PubMed

    Zhang, Yilan; Ge, Xianglian; Yang, Fayu; Zhang, Liping; Zheng, Jiayong; Tan, Xuefang; Jin, Zi-Bing; Qu, Jia; Gu, Feng

    2014-06-23

    CRISPR/Cas9-mediated DNA cleavage (CCMDC) is becoming increasingly used for efficient genome engineering. Proto-spacer adjacent motif (PAM) adjacent to target sequence is one of the key components in the design of CCMDC strategies. It has been reported that NAG sequences are the predominant non-canonical PAM for CCMDC at the human EMX locus, but it is not clear whether it is universal at other loci. In the present study, we attempted to use a GFP-reporter system to comprehensively and quantitatively test the efficiency of CCMDC with non-canonical PAMs in human cells. The initial results indicated that the effectiveness of NGA PAM for CCMDC is much higher than that of other 14 PAMs including NAG. Then we further designed another three pairs of NGG, NGA and NAG PAMs at different locations in the GFP gene and investigated the corresponding DNA cleavage efficiency. We observed that one group of NGA PAMs have a relatively higher DNA cleavage efficiency, while the other groups have lower efficiency, compared with the corresponding NAG PAMs. Our study clearly demonstrates that NAG may not be the universally predominant non-canonical PAM for CCMDC in human cells. These findings raise more concerns over off-target effects in CRISPR/Cas9-mediated genome engineering.

  14. 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

  15. A cleavage-resistant urokinase plasminogen activator receptor exhibits dysregulated cell-surface clearance.

    PubMed

    Nieves, Evelyn C; Manchanda, Naveen

    2010-04-23

    Urokinase plasminogen activator receptor (u-PAR) binds urokinase plasminogen activator (u-PA) and participates in plasminogen activation in addition to modulating several cellular processes such as adhesion, proliferation, and migration. u-PAR is susceptible to proteolysis by its cognate ligand and several other proteases. To elucidate the biological significance of receptor cleavage by u-PA, we engineered and expressed a two-chain urokinase plasminogen activator (tcu-PA) cleavage-resistant u-PAR (cr-u-PAR). This mutated receptor was similar to wild-type u-PAR in binding u-PA and initiating plasminogen activation. However, cr-u-PAR exhibited accelerated internalization and resurfacing due to direct association with the endocytic receptor alpha(2)-macroglobulin receptor/low density lipoprotein receptor-related protein in the absence of the enzyme x inhibitor complex of tcu-PA and plasminogen activator inhibitor-1 (tcu-PA.PAI-1). cr-u-PAR-expressing cells had enhanced migration compared with wild-type u-PAR-expressing cells, and cr-u-PAR was less sensitive to chymotrypsin cleavage as compared with wt u-PAR. Our studies suggest that these mutations in the linker region result in a rearrangement within the cr-u-PAR structure that makes it resemble its ligand-bound form. This constitutively active variant may mimic highly glycosylated cleavage-resistant u-PAR expressed in certain highly malignant cancer-cells.

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

    PubMed

    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, (1)H NMR, (13)C 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.

  17. Regulation of Dpp activity by tissue-specific cleavage of an upstream site within the prodomain

    PubMed Central

    Sopory, Shailaja; Kwon, Sunjong; Wehrli, Marcel; Christian, Jan L.

    2010-01-01

    BMP4 is synthesized as an inactive precursor that is cleaved at two sites during maturation: initially at a site (S1) adjacent to the ligand domain, and then at an upstream site (S2) within the prodomain. Cleavage at the second site regulates the stability of mature BMP4 and this in turn influences its signaling intensity and range of action. The Drosophila ortholog of BMP4, Dpp, functions as a long- or short-range signaling molecule in the wing disc or embryonic midgut, respectively but mechanisms that differentially regulate its bioactivity in these tissues have not been explored. In the current studies we demonstrate, by dpp mutant rescue, that cleavage at the S2 site of proDpp is required for development of the wing and leg imaginal discs, whereas cleavage at the S1 site is sufficient to rescue Dpp function in the midgut. Both the S1 and S2 site of proDpp are cleaved in the wing disc, and S2-cleavage is essential to generate sufficient ligand to exceed the threshold for pMAD activation at both short- and long-range in most cells. By contrast, proDpp is cleaved at the S1 site alone in the embryonic mesoderm and this generates sufficient ligand to activate physiological target genes in neighboring cells. These studies provide the first biochemical and genetic evidence that that selective cleavage of the S2 site of proDPP provides a tissue-specific mechanism for regulating Dpp activity, and that differential cleavage can contribute to, but is not an absolute determinant of signaling range. PMID:20659445

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

    PubMed Central

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

    2008-01-01

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

  19. [Structural regularities in activated cleavage sites of thrombin receptors].

    PubMed

    Mikhaĭlik, I V; Verevka, S V

    1999-01-01

    Comparison of thrombin receptors activation splitting sites sequences testifies to their similarity both in activation splitting sites of protein precursors and protein proteinase inhibitors reactive sites. In all these sites corresponded to effectory sites P2'-positions are placed by hydrophobic amino-acids only. The regularity defined conforms with previous thesis about the role of effectory S2'-site in regulation of the processes mediated by serine proteinases.

  20. Glycine cleavage system in ketotic hyperglycinemia: a reduction of H-protein activity.

    PubMed

    Hayasaka, K; Narisawa, K; Satoh, T; Tateda, H; Metoki, K; Tada, K; Hiraga, K; Aoki, T; Kawakami, T; Akamatsu, H; Matsuo, N

    1982-01-01

    Glycine cleavage activity was compared in the livers from three cases of ketotic hyperglycinemia (two cases of propionic acidemia and one case of methylmalonic acidemia) and three controls. In one case of propionic acidemia, glycine cleavage activity (5.2 nmole/mg protein/hr) was normal in the liver obtained at biopsy when the patient was well controlled by the treatment with low protein diet (0.8 g/kg/day) and the level of serum glycine was lowered to normal. In the two other cases of ketotic hyperglycinemia, glycine cleavage activity was significantly reduced in the liver obtained at autopsy when the patients died in the state of metabolic acidosis. Its activity in the liver of one case of propionic acidemia (0.7 nmole/mg protein/hr) was 6-26% of that in controls (2.7-10.8 nmole/mg protein/hr), and 2-7% in the case of methylmalonic acidemia (0.2 nmole/mg protein/hr). Analysing of the individual components of the glycine cleavage system, a marked decrease in the activity of H-protein was revealed in the livers of the both patients; it (0.2 nmole/mg protein/hr) was only 3-4% of that in controls (4.9-6.3 nmole/mg Protein/hr). These findings suggest that the reduction of the glycine cleavage system in the liver of ketotic hyperglycinemia occurs secondarily as speculated previously and is caused mainly by a decrease of H-protein activity.

  1. The N-terminal Domain Allosterically Regulates Cleavage and Activation of the Epithelial Sodium Channel*

    PubMed Central

    Kota, Pradeep; Buchner, Ginka; Chakraborty, Hirak; Dang, Yan L.; He, Hong; Garcia, Guilherme J. M.; Kubelka, Jan; Gentzsch, Martina; Stutts, M. Jackson; Dokholyan, Nikolay V.

    2014-01-01

    The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr370 in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation. PMID:24973914

  2. 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.

  3. Roles of DNA helicases and Exo1 in the avoidance of mutations induced by Top1-mediated cleavage at ribonucleotides in DNA

    PubMed Central

    Niu, Hengyao; Potenski, Catherine J.; Epshtein, Anastasiya; Sung, Patrick; Klein, Hannah L.

    2016-01-01

    ABSTRACT The replicative DNA polymerases insert ribonucleotides into DNA at a frequency of approximately 1/6500 nucleotides replicated. The rNMP residues make the DNA backbone more susceptible to hydrolysis and can also distort the helix, impeding the transcription and replication machineries. rNMPs in DNA are efficiently removed by RNaseH2 by a process called ribonucleotides excision repair (RER). In the absence of functional RNaseH2, rNMPs are subject to cleavage by Topoisomerase I, followed by further processing to result in deletion mutations due to slippage in simple DNA repeats. The topoisomerase I-mediated cleavage at rNMPs results in DNA ends that cannot be ligated by DNA ligase I, a 5′OH end and a 2′–3′ cyclic phosphate end. In the budding yeast, the mutation level in RNaseH2 deficient cells is kept low via the action of the Srs2 helicase and the Exo1 nuclease, which collaborate to process the Top1-induced nick with subsequent non-mutagenic gap filling. We have surveyed other helicases and nucleases for a possible role in reducing mutagenesis at Top1 nicks at rNMPs and have uncovered a novel role for the RecQ family helicase Sgs1 in this process. PMID:26716562

  4. Roles of DNA helicases and Exo1 in the avoidance of mutations induced by Top1-mediated cleavage at ribonucleotides in DNA.

    PubMed

    Niu, Hengyao; Potenski, Catherine J; Epshtein, Anastasiya; Sung, Patrick; Klein, Hannah L

    2016-01-01

    The replicative DNA polymerases insert ribonucleotides into DNA at a frequency of approximately 1/6500 nucleotides replicated. The rNMP residues make the DNA backbone more susceptible to hydrolysis and can also distort the helix, impeding the transcription and replication machineries. rNMPs in DNA are efficiently removed by RNaseH2 by a process called ribonucleotides excision repair (RER). In the absence of functional RNaseH2, rNMPs are subject to cleavage by Topoisomerase I, followed by further processing to result in deletion mutations due to slippage in simple DNA repeats. The topoisomerase I-mediated cleavage at rNMPs results in DNA ends that cannot be ligated by DNA ligase I, a 5'OH end and a 2'-3' cyclic phosphate end. In the budding yeast, the mutation level in RNaseH2 deficient cells is kept low via the action of the Srs2 helicase and the Exo1 nuclease, which collaborate to process the Top1-induced nick with subsequent non-mutagenic gap filling. We have surveyed other helicases and nucleases for a possible role in reducing mutagenesis at Top1 nicks at rNMPs and have uncovered a novel role for the RecQ family helicase Sgs1 in this process.

  5. 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

  6. Ku-mediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia.

    PubMed

    Marmignon, Antoine; Bischerour, Julien; Silve, Aude; Fojcik, Clémentine; Dubois, Emeline; Arnaiz, Olivier; Kapusta, Aurélie; Malinsky, Sophie; Bétermier, Mireille

    2014-08-01

    During somatic differentiation, physiological DNA double-strand breaks (DSB) can drive programmed genome rearrangements (PGR), during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES). IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ) pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium DNA cleavage

  7. Ku-Mediated Coupling of DNA Cleavage and Repair during Programmed Genome Rearrangements in the Ciliate Paramecium tetraurelia

    PubMed Central

    Marmignon, Antoine; Bischerour, Julien; Silve, Aude; Fojcik, Clémentine; Dubois, Emeline; Arnaiz, Olivier; Kapusta, Aurélie; Malinsky, Sophie; Bétermier, Mireille

    2014-01-01

    During somatic differentiation, physiological DNA double-strand breaks (DSB) can drive programmed genome rearrangements (PGR), during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES). IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ) pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium DNA cleavage

  8. 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.

  9. Engineering of TM1459 from Thermotoga maritima for Increased Oxidative Alkene Cleavage Activity

    PubMed Central

    Fink, Matthias; Trunk, Sarah; Hall, Mélanie; Schwab, Helmut; Steiner, Kerstin

    2016-01-01

    Oxidative cleavage of alkenes is a widely employed process allowing oxyfunctionalization to corresponding carbonyl compounds. Recently, a novel biocatalytic oxidative alkene cleavage activity on styrene derivatives was identified in TM1459 from Thermotoga maritima. In this work we engineered the enzyme by site-saturation mutagenesis of active site amino acids to increase its activity and to broaden its substrate scope. A high-throughput assay for the detection of the ketone products was successfully developed. Several variants with up to twofold improved conversion level of styrene derivatives were successfully identified. Especially, changes in or removal of the C-terminus of TM1459 increased the activity most significantly. These best variants also displayed a slightly enlarged substrate scope. PMID:27713741

  10. Development of a high-throughput fluorescence polarization DNA cleavage assay for the identification of FEN1 inhibitors.

    PubMed

    McWhirter, Claire; Tonge, Michael; Plant, Helen; Hardern, Ian; Nissink, Willem; Durant, Stephen T

    2013-06-01

    Flap endonuclease-1 (FEN1) is a highly conserved metallonuclease and is the main human flap endonuclease involved in the recognition and cleavage of single-stranded 5' overhangs from DNA flap structures. The involvement of FEN1 in multiple DNA metabolism pathways and the identification of FEN1 overexpression in a variety of cancers has led to interest in FEN1 as an oncology target. In this article, we describe the development of a 1536-well high-throughput screening assay based on the change in fluorescence polarization of a FEN1 DNA substrate labeled with Atto495 dye. The assay was subsequently used to screen 850 000 compounds from the AstraZeneca compound collection, with a Z' factor of 0.66 ± 0.06. Hits were followed up by IC50 determination in both a concentration-response assay and a technology artifact assay.

  11. Optical mapping of site-directed cleavages on single DNA molecules by the RecA-assisted restriction endonuclease technique.

    PubMed Central

    Wang, Y K; Huff, E J; Schwartz, D C

    1995-01-01

    Fluorescence in situ hybridization (FISH) resolution has advanced because newer techniques use increasingly decondensed chromatin. FISH cannot analyze restriction enzyme cutting sites due to limitations of the hybridization and detection technologies. The RecA-assisted restriction endonuclease (RARE) technique cleaves chromosomal DNA at a single EcoRI site within a given gene or selected sequence. We recently described a mapping technique, optical mapping, which uses fluorescence microscopy to produce high-resolution restriction maps rapidly by directly imaging restriction digestion cleavage events occurring on single deproteinized DNA molecules. Ordered maps are then constructed by noting fragment order and size, using several optically based techniques. Since we also wanted to map arbitrary sequences and gene locations, we combined RARE with optical mapping to produce site-specific visible EcoRI restriction cleavage sites on single DNA molecules. Here we describe this combined method, named optical RARE, and its initial application to mapping gene locations on yeast chromosomes. Images Fig. 2 Fig. 3 PMID:7816810

  12. 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

  13. The position of DNA cleavage by TALENs and cell synchronization influences the frequency of gene editing directed by single-stranded oligonucleotides.

    PubMed

    Rivera-Torres, Natalia; Strouse, Bryan; Bialk, Pawel; Niamat, Rohina A; Kmiec, Eric B

    2014-01-01

    With recent technological advances that enable DNA cleavage at specific sites in the human genome, it may now be possible to reverse inborn errors, thereby correcting a mutation, at levels that could have an impact in a clinical setting. We have been developing gene editing, using single-stranded DNA oligonucleotides (ssODNs), as a tool to direct site specific single base changes. Successful application of this technique has been demonstrated in many systems ranging from bacteria to human (ES and somatic) cells. While the frequency of gene editing can vary widely, it is often at a level that does not enable clinical application. As such, a number of stimulatory factors such as double-stranded breaks are known to elevate the frequency significantly. The majority of these results have been discovered using a validated HCT116 mammalian cell model system where credible genetic and biochemical readouts are available. Here, we couple TAL-Effector Nucleases (TALENs) that execute specific ds DNA breaks with ssODNs, designed specifically to repair a missense mutation, in an integrated single copy eGFP gene. We find that proximal cleavage, relative to the mutant base, is key for enabling high frequencies of editing. A directionality of correction is also observed with TALEN activity upstream from the target base being more effective in promoting gene editing than activity downstream. We also find that cells progressing through S phase are more amenable to combinatorial gene editing activity. Thus, we identify novel aspects of gene editing that will help in the design of more effective protocols for genome modification and gene therapy in natural genes.

  14. Alpha-momorcharin: a ribosome-inactivating protein from Momordica charantia, possessing DNA cleavage properties.

    PubMed

    Wang, Shuzhen; Zheng, Yinzhen; Yan, Junjie; Zhu, Zhixuan; Wu, Zhihua; Ding, Yi

    2013-11-01

    Ribosome-inactivating proteins (RIPs) function to inhibit protein synthesis through the removal of specific adenine residues from eukaryotic ribosomal RNA and rending the 60S subunit unable to bind elongation factor 2. They have received much attention in biological and biomedical research due to their unique activities toward tumor cells, as well as the important roles in plant defense. Alpha-momorcharin (α-MC), a member of the type I family of RIPs, is rich in the seeds of Momordica charantia L. Previous studies demonstrated that α-MC is an effective antifungal and antibacterial protein. In this study, a detailed analysis of the DNase-like activity of α-MC was conducted. Results showed that the DNase-like activity toward plasmid DNA was time-dependent, temperature-related, and pH-stable. Moreover, a requirement for divalent metal ions in the catalytic domain of α-MC was confirmed. Additionally, Tyr(93) was found to be a critical residue for the DNase-like activity, while Tyr(134), Glu(183), Arg(186), and Trp(215) were activity-related residues. This study on the chemico-physical properties and mechanism of action of α-MC will improve its utilization in scientific research, as well as its potential industrial uses. These results may also assist in the characterization and elucidation of the DNase-like enzymatic properties of other RIPs.

  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. Enantiomeric in vitro DNA binding, pBR322 DNA cleavage and molecular docking studies of chiral L- and D-ternary copper(II) complexes of histidine and picolinic acid.

    PubMed

    Parveen, Shazia; Arjmand, Farukh; Ahmad, Iqbal

    2014-01-05

    Novel chiral ternary Cu(II) and Ni(II) complexes of l/d-histidine and picolinic acid, 1 and 2(a and b) were synthesized and characterized by elemental analysis, molar conductance and spectroscopic data (IR, NMR, EPR, UV-vis). In vitro DNA binding profile of both Cu(II) and Ni(II) complexes have been investigated by UV-vis titrations, while fluorescence spectroscopy, circular dichroism and viscosity measurements were carried out for Cu(II) complexes 1(a and b). Both the enantiomers of 1 and 2(a and b) bind to CT DNA via electrostatic interactions and the intrinsic binding constant, Kb values for complexes 1 and 2(a and b) were found to be 5.6×10(4), 9.8×10(3), 8.2×10(3) and 6.7×10(3)M(-1), respectively suggesting greater binding propensity of l-form of Cu(II) complex 1a. The DNA cleavage activity of complexes 1(a and b), investigated by agarose gel electrophoresis suggested an oxidative pathway for DNA cleavage. Further, the molecular docking studies of complexes 1(a and b) were carried out with B-DNA revealing that the complexes bind to the adenine-thymine residues in the minor groove of the DNA. The resulting binding energies of docked metal complexes 1(a and b) were found to be -265.1 and -218.9KJmol(-1), respectively. Furthermore, enantiomeric complexes 1 and 2(a and b) were screened for in vitro antimicrobial activity.

  17. DNA hydrolytic cleavage by the diiron(III) complex Fe(2)(DTPB)(mu-O)(mu-Ac)Cl(BF(4))(2): comparison with other binuclear transition metal complexes.

    PubMed

    Liu, Changlin; Yu, Siwang; Li, Dongfeng; Liao, Zhanru; Sun, Xiaohui; Xu, Huibi

    2002-02-25

    The binuclear structure of Fe(2)(DTPB)(mu-O)(mu-Ac)Cl(BF(4))(2) (DTPB = 1,1,4,7,7-penta (2'-benzimidazol-2-ylmethyl)-triazaheptane, Ac = acetate) was characterized by UV-visible absorption and infrared spectra and NMR and ESR. The binding interaction of DNA with the diiron complex was examined spectroscopically. Supercoiled and linear DNA hydrolytic cleavage by the diiron complex is supported by the evidence from anaerobic reactions, free radical quenching, high performance liquid chromatography experiments, and enzymatic manipulation such as T4 ligase ligation, 5'-(32)P end-labeling, and footprinting analysis. The estimation of rate for the supercoiled DNA double strand cleavage shows one of the largest known rate enhancement factors, approximately 10(10) against DNA. Moreover, the DNA hydrolysis chemistry needs no coreactant such as hydrogen peroxide. The poor sequence-specific DNA cleavage indicated by the restriction analysis of the pBR322 DNA linearized by the diiron complex might be due to the diiron complex bound to DNA by a coordination of its two ferric ions to the DNA phosphate oxygens, as suggested by spectral characterizations. The hydrolysis chemistry for a variety of binuclear metal complexes including Fe(2)(DTPB)(mu-O)(mu-Ac)Cl(BF(4))(2) is compared. It is established that the dominant factors for the DNA hydrolysis activities of the binuclear metal complexes are the mu-oxo bridge, labile and anionic ligands, and open coordination site(s). Concerning the hydrolytic mechanisms, the diiron complex Fe(2)(DTPB)(mu-O)(mu-Ac)Cl(BF(4))(2) might share many points in common with the native purple acid phosphatases.

  18. The recognition of DNA cleavage sites by porcine spleen topoisomerase II.

    PubMed

    Huang, H W; Juang, J K; Liu, H J

    1992-02-11

    The cutting sites specificity of topoisomerase II from porcine spleen were determined by a modified Sanger's DNA sequencing method. The topoisomerase II prefers to cut DNA at the 3' side of A and leave 5' protruding end with two staggering bases. Through the free energy analysis for DNA duplex, we also found that the topoisomerase II seemed cut DNA preferably at energetically unstable regions. So it is concluded that the specific DNA cutting by porcine spleen topoisomerase II has two structural recognition factors: one is to localize around the energetically unstable region and another is to act at the 3' side of A base.

  19. Structural and functional analyses of an archaeal XPF/Rad1/Mus81 nuclease: asymmetric DNA binding and cleavage mechanisms.

    PubMed

    Nishino, Tatsuya; Komori, Kayoko; Ishino, Yoshizumi; Morikawa, Kosuke

    2005-08-01

    XPF/Rad1/Mus81/Hef proteins recognize and cleave branched DNA structures. XPF and Rad1 proteins cleave the 5' side of nucleotide excision repair bubble, while Mus81 and Hef cleave similar sites of the nicked Holliday junction, fork, or flap structure. These proteins all function as dimers and consist of catalytic and helix-hairpin-helix DNA binding (HhH) domains. We have determined the crystal structure of the HhH domain of Pyrococcus furiosus Hef nuclease (HefHhH), which revealed the distinct mode of protein dimerization. Our structural and biochemical analyses also showed that each of the catalytic and HhH domains binds to distinct regions within the fork-structured DNA: each HhH domain from two separate subunits asymmetrically binds to the arm region, while the catalytic domain binds near the junction center. Upon binding to DNA, Hef nuclease disrupts base pairs near the cleavage site. It is most likely that this bipartite binding mode is conserved in the XPF/Rad1/Mus81 nuclease family.

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

    SciTech Connect

    Yang, Hui; Gao, Pu; Rajashankar, Kanagalaghatta R.; Patel, Dinshaw J.

    2016-12-01

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

  1. DNA cleavage at the AP site via β-elimination mediated by the AP site-binding ligands.

    PubMed

    Abe, Yukiko S; Sasaki, Shigeki

    2016-02-15

    DNA is continuously damaged by endogenous and exogenous factors such as oxidation and alkylation. In the base excision repair pathway, the damaged nucleobases are removed by DNA N-glycosylase to form the abasic sites (AP sites). The alkylating antitumor agent exhibits cytotoxicity through the formation of the AP site. Therefore blockage or modulation of the AP site repair pathway may enhance the antitumor efficacy of DNA alkylating agents. In this study, we have examined the effects of the nucleobase-polyamine conjugated ligands (G-, A-, C- and T-ligands) on the cleavage of the AP site. The G- and A-ligands cleaved DNA at the AP site by promoting β-elimination in a non-selective manner by the G-ligand, and in a selective manner for the opposing dT by the A-ligand. These results suggest that the nucleobase-polyamine conjugate ligands may have the potential for enhancement of the cytotoxicities of the AP site.

  2. Complementary addressed modification and cleavage of a single stranded DNA fragment with alkylating oligonucleotide derivatives.

    PubMed Central

    Vlassov, V V; Zarytova, V F; Kutiavin, I V; Mamaev, S V; Podyminogin, M A

    1986-01-01

    A single stranded DNA fragment was modified with alkylating derivatives of oligonucleotides complementary to a certain nucleotide sequences in the fragment. The derivatives carried aromatic 2-chloroethylamino groups at their 3'- or 5'-terminal nucleotide residues. Some of the derivatives carried both alkylating group and intercalating phenazine group which stabilized complementary complexes. It was found that these oligonucleotide derivatives modify the DNA fragment in a specific way near the target complementary nucleotide sequences, and the DNA fragment can be cleaved at the alkylated nucleotides positions. Alkylating derivatives carrying phenazine groups were found to be the most efficient in reaction with the DNA fragment. Images PMID:3714471

  3. Bio-important antipyrine derived Schiff bases and their transition metal complexes: Synthesis, spectroscopic characterization, antimicrobial, anthelmintic and DNA cleavage investigation

    NASA Astrophysics Data System (ADS)

    Manjunath, M.; Kulkarni, Ajaykumar D.; Bagihalli, Gangadhar B.; Malladi, Shridhar; Patil, Sangamesh A.

    2017-01-01

    Spectroscopic (IR, NMR, UV-vis, ESR, ESI-mass), magnetic and TGA studies suggests octahedral geometry for all the CoII, NiII and CuII complexes of the Schiff bases, derived from 4-aminoantipyrine and 8-formyl-7-Hydroxy-4-methylcoumarin/5-formyl-6-hydroxycoumarin, coordinated through ONO donor sites. Antibacterial (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi), antifungal (Aspergillus niger, Aspergillus flavus and Cladosporium) and DNA cleavage properties of the metal complexes are investigated. The results suggested that some of the synthesized compounds are potential antimicrobials. The synthesized compounds tested for their anthelmintic activities and it was found that CoII and NiII complexes exhibited good anthelmintic properties.

  4. 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.

  5. 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

  6. Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.

    PubMed

    Zay, Agnes; Choy, Francis Y M; Patrick, Chelsea; Sinclair, Graham

    2011-06-01

    The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to

  7. Escherichia coli DNA helicase I catalyzes a sequence-specific cleavage/ligation reaction at the F plasmid origin of transfer.

    PubMed

    Sherman, J A; Matson, S W

    1994-10-21

    Recent studies have shown that the Escherichia coli F plasmid-encoded traI gene product (TraIp), also known as DNA helicase I, catalyzes the formation of the site- and strand-specific nick that initiates F plasmid DNA transfer. Scission of the phosphodiester bond at the nic site within the origin of transfer (oriT) is accompanied by the covalent attachment of TraIp to the 5'-phosphate of the nicked DNA strand. This mechanism suggests that TraIp may also be capable of catalyzing a DNA ligation reaction using the energy stored in the protein-DNA intermediate. To test this possibility, an in vitro assay was designed that utilized short single-stranded DNA oligonucleotides of different lengths derived from the region within oriT that spanned the nic site. Purified TraIp was capable of efficiently cleaving single-stranded DNA that contained a nic site, and upon cleavage, the protein became covalently linked to the 5'-end of the nic site. When TraIp was incubated with two oligonucleotides of different length that contained the nic site, there was formation of novel recombinant products resulting from a TraIp-catalyzed cleavage/ligation reaction. Furthermore, the cleavage and ligation reactions were both sequence-specific. These data suggest that TraIp plays an important role in the initiation and termination of conjugative DNA transfer.

  8. 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

  9. DNA cleavage in red light promoted by copper(II) complexes of alpha-amino acids and photoactive phenanthroline bases.

    PubMed

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

    2008-12-28

    Ternary copper(II) complexes [Cu(L-trp)(B)(H(2)O)](NO(3)) (1-3) and [Cu(L-phe)(B)(H(2)O)](NO(3)) (4-6) of L-tryptophan (L-trp) and L-phenylalanine (L-phe) having phenanthroline bases (B), viz. 1,10-phenanthroline (phen, 1 and 4), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 2 and 5) and dipyrido[3,2-a:2',3'-c]phenazine (dppz, 3 and 6), were prepared and characterized by physico-chemical techniques. Complexes 3 and 6 were structurally characterized by X-ray crystallography and show the presence of a square pyramidal (4 + 1) CuN(3)O(2) coordination geometry in which the N,O-donor amino acid (L-trp or L-phe) and N,N-donor phenanthroline base bind at the equatorial plane with an aqua ligand coordinated at the elongated axial site. Complex 3 shows significant distortion from the square pyramidal geometry and a strong intramolecular pi-pi stacking interaction between the pendant indole ring of L-trp and the planar dppz aromatic moiety. All the complexes display good binding propensity to the calf thymus DNA giving an order: 3,6 (dppz) > 2,5 (dpq) > 1,4 (phen). The binding constant (K(b)) values are in the range of 2.1 x 10(4)-1.1 x 10(6) mol(-1) with the binding site size (s) values of 0.17-0.63. The phen and dpq complexes are minor groove binders while the dppz analogues bind at the DNA major groove. Theoretical DNA docking studies on 2 and 3 show the close proximity of two photosensitizers, viz. the indole moiety of L-trp and the quinoxaline/phenazine of the dpq/dppz bases, to the complementary DNA strands. Complexes 2 and 3 show oxidative DNA double strand breaks (dsb) of supercoiled (SC) DNA forming a significant quantity of linear DNA along with the nicked circular (NC) form on photoexposure to UV-A light of 365 nm and red light of 647.1 nm (Ar-Kr laser). Complexes 1,5 and 6 show only single strand breaks (ssb) forming NC DNA. The red light induced DNA cleavage involves metal-assisted photosensitization of L-trp and dpq/dppz base resulting in the formation of a reactive

  10. 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

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

    PubMed

    Rodrigo, María J; Alquézar, Berta; Alós, Enriqueta; Medina, Víctor; Carmona, Lourdes; Bruno, Mark; Al-Babili, Salim; Zacarías, Lorenzo

    2013-11-01

    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.

  12. Synthesis, structure, and DNA cleavage properties of copper(II) complexes of 1,4,7-triazacyclononane ligands featuring pairs of guanidine pendants.

    PubMed

    Tjioe, Linda; Joshi, Tanmaya; Brugger, Joël; Graham, Bim; Spiccia, Leone

    2011-01-17

    Two new ligands, L(1) and L(2), have been prepared via N-functionalization of 1,4,7-triazacyclononane (tacn) with pairs of ethyl- or propyl-guanidine pendants, respectively. The X-ray crystal structure of [CuL(1)](ClO4)2 (C1) isolated from basic solution (pH 9) indicates that a secondary amine nitrogen from each guanidine pendants coordinates to the copper(II) center in addition to the nitrogen atoms in the tacn macrocycle, resulting in a five-coordinate complex with intermediate square-pyramidal/trigonal bipyramidal geometry. The guanidines adopt an unusual coordination mode in that their amine nitrogen nearest to the tacn macrocycle binds to the copper(II) center, forming very stable five-membered chelate rings. A spectrophotometric pH titration established the pK(app) for the deprotonation and coordination of each guanidine group to be 3.98 and 5.72, and revealed that [CuL(1)](2+) is the only detectable species present in solution above pH ∼ 8. The solution speciation of the CuL(2) complex (C2) is more complex, with at least 5 deprotonation steps over the pH range 4-12.5, and mononuclear and binuclear complexes coexisting. Analysis of the spectrophotometric data provided apparent deprotonation constants, and suggests that solutions at pH ∼ 7.5 contain the maximum proportion of polynuclear complexes. Complex C1 exhibits virtually no cleavage activity toward the model phosphate diesters, bis(p-nitrophenyl)phosphate (BNPP) and 2-hydroxypropyl-p-nitrophenyl phosphate (HPNPP), while C2 exhibits moderate activity. For C2, the respective kobs values measured at pH 7.0 (7.24 (± 0.08) × 10(-5) s(-1) (BNPP at 50 °C) and 3.2 (± 0.3) × 10(-5) s(-1) (HPNPP at 25 °C)) are 40- and 10-times faster than [Cu(tacn)(OH2)2](2+) complex. Both complexes cleave supercoiled pBR 322 plasmid DNA, indicating that the guanidine pendants of [CuL(1)](2+) may have been displaced from the copper coordination sphere to allow for DNA binding and subsequent cleavage. The rate of DNA

  13. BMP1 controls TGFbeta1 activation via cleavage of latent TGFbeta-binding protein.

    PubMed

    Ge, Gaoxiang; Greenspan, Daniel S

    2006-10-09

    Transforming growth factor beta1 (TGFbeta1), an important regulator of cell behavior, is secreted as a large latent complex (LLC) in which it is bound to its cleaved prodomain (latency-associated peptide [LAP]) and, via LAP, to latent TGFbeta-binding proteins (LTBPs). The latter target LLCs to the extracellular matrix (ECM). Bone morphogenetic protein 1 (BMP1)-like metalloproteinases play key roles in ECM formation, by converting precursors into mature functional proteins, and in morphogenetic patterning, by cleaving the antagonist Chordin to activate BMP2/4. We provide in vitro and in vivo evidence that BMP1 cleaves LTBP1 at two specific sites, thus liberating LLC from ECM and resulting in consequent activation of TGFbeta1 via cleavage of LAP by non-BMP1-like proteinases. In mouse embryo fibroblasts, LAP cleavage is shown to be predominantly matrix metalloproteinase 2 dependent. TGFbeta1 is a potent inducer of ECM formation and of BMP1 expression. Thus, a role for BMP1-like proteinases in TGFbeta1 activation completes a novel fast-forward loop in vertebrate tissue remodeling.

  14. 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

  15. Structural, electrochemical, phosphate-hydrolysis, DNA binding and cleavage studies of new macrocyclic binuclear nickel(II) complexes.

    PubMed

    Anbu, Sellamuthu; Kandaswamy, Muthusamy; Varghese, Babu

    2010-04-28

    New macrocyclic binuclear nickel(ii) complexes have been synthesized by using the bicompartmental mononuclear complex [NiL] [3,30-((1E,7E)-3,6-dioxa-2,7-diazaocta-1,7-diene-1,8-diyl)bis(3-formyl-5-methyl-2-diolato)nickel(II)] with various diamines like 1,2-bis(aminooxy)ethane (L(1)), 1,2-diamino ethane (L(2)), 1,3-diamino propane (L(3)), 1,4-diamino butane (L(4)), 1,2-diamino benzene (L(5)), and 1,8-diamino naphthalene (L(6)). The complexes were characterized by elemental analysis and spectroscopic methods. The molecular structures of the symmetrical binuclear complex [Ni(2)L(1)(H(2)O)(4)](ClO(4))(2) (1) and unsymmetrical binuclear complex [Ni(2)L(3)(H(2)O)(4)](ClO(4))(2).(H(2)O)(4) (3) were determined by single-crystal X-ray diffraction. The geometry around both the nickel(II) ions in each molecule is a slightly distorted octahedral. The distance between the Ni...Ni centers for complex 1 is 3.039 A and for complex 3 is 3.059 A. The influence of the coordination geometry and the ring size of the binucleating ligands on the electronic, redox, phosphate hydrolysis, DNA binding and cleavage properties have been studied. Electrochemical studies of the complexes show two quasi-reversible one electron reduction processes between -0.49 to -1.69 V. The reduction potential of the binuclear Ni(II) complexes shifts towards anodically upon increasing the macrocyclic ring size. The observed first order rate constant values for the hydrolysis of 4-nitrophenyl phosphate reaction are in the range from 8.69 x 10(-3) to 1.85 x 10(-2) s(-1). The complexes show good binding propensity to calf thymus DNA giving binding constant values in the range from 1.4 x 10(4) to 17.5 x 10(4) M(-1). The absorption, fluorescence and CD spectral data suggests that the complexes are strongly interacting with DNA. These complexes display hydrolytic cleavage of supercoiled pBR322DNA in the presence of H(2)O(2) at pH 7.2 and 37 degrees C. The hydrolytic cleavage of DNA by the complexes is supported by

  16. 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

  17. Mec1/Tel1–dependent phosphorylation of Slx4 stimulates Rad1–Rad10 dependent cleavage of non–homologous DNA tails

    PubMed Central

    Toh, Geraldine W.-L.; Sugawara, Neal; Dong, Junchao; Toth, Rachel; Lee, Sang Eun; Haber, James E.; Rouse, John

    2015-01-01

    Budding yeast Slx4 interacts with the Rad1–Rad10 endonuclease that is involved in nucleotide excision repair (NER), homologous recombination (HR) and single–strand annealing (SSA). We previously showed that Slx4 is dispensable for NER but is essential for SSA. Slx4 is phosphorylated by the Mec1 and Tel1 kinases after DNA damage on at least six Ser/Thr residues, and mutation of all six residues to Ala reduces the efficiency of SSA. In this study, we further investigated the role of Slx4 phosphorylation in SSA, specifically in regulating cleavage of 3′ non–homologous (NH) DNA tails by Rad1-Rad10 during SSA and HR. Slx4 became phosphorylated after induction of a single double–strand break (DSB) during SSA and dephosphorylation coincided approximately with completion of repair. Slx4 is recruited to 3′ NH tails during DSB repair, but this does not require phosphorylation of Slx4. However, we identified specific damage-dependent Mec1/Tel1 site of Slx4 phosphorylation, Thr 113, that is required for efficient cleavage of NH tails by Rad1–Rad10. Consistent with these data, deletion of both Mec1 and Tel1 severely reduces the efficiency of NH DNA tail cleavage during HR. These data show that phosphorylation of Slx4 by Mec1 and Tel1 plays an important role in facilitating NH DNA tail cleavage during HR. PMID:20382573

  18. 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.

  19. 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.

  20. Co(II), Ni(II) and Cu(II) complexes with coumarin-8-yl Schiff-bases: Spectroscopic, in vitro antimicrobial, DNA cleavage and fluorescence studies

    NASA Astrophysics Data System (ADS)

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

    2011-09-01

    A new series of Co(II), Ni(II) and Cu(II) complexes of the type ML·2H 2O of Schiff-bases derived from m-substituted thiosemicarbazides and 8-acetyl-7-hydroxy-4-methylcoumarin have been synthesized and characterized by spectroscopic studies. Schiff-bases exhibit thiol-thione tautomerism wherein sulphur 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 analyses, spectral (IR, UV-vis, FAB-mass, ESR and fluorescence), magnetic and thermal studies. The low molar conductance values in DMF indicate that, the metal complexes are non-electrolytes. The cyclic voltammetric studies suggested that, the Cu(II) and Ni(II) complexes are of single electron transfer quasi-reversible nature. The Schiff-bases and its metal complexes have been evaluated for their in vitro antibacterial ( Escherichia coli, Staphilococcus aureus, Bascillus subtilis and Salmonella typhi) and antifungal activities ( Candida albicans, Cladosporium and Aspergillus niger) by MIC method. The Schiff-base I and its metal complexes exhibited DNA cleavage activity on isolated DNA of A. niger.

  1. Co(II), Ni(II) and Cu(II) complexes with coumarin-8-yl Schiff-bases: spectroscopic, in vitro antimicrobial, DNA cleavage and fluorescence studies.

    PubMed

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

    2011-09-01

    A new series of Co(II), Ni(II) and Cu(II) complexes of the type ML·2H2O of Schiff-bases derived from m-substituted thiosemicarbazides and 8-acetyl-7-hydroxy-4-methylcoumarin have been synthesized and characterized by spectroscopic studies. Schiff-bases exhibit thiol-thione tautomerism wherein sulphur 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 analyses, spectral (IR, UV-vis, FAB-mass, ESR and fluorescence), magnetic and thermal studies. The low molar conductance values in DMF indicate that, the metal complexes are non-electrolytes. The cyclic voltammetric studies suggested that, the Cu(II) and Ni(II) complexes are of single electron transfer quasi-reversible nature. The Schiff-bases and its metal complexes have been evaluated for their in vitro antibacterial (Escherichia coli, Staphilococcus aureus, Bascillus subtilis and Salmonella typhi) and antifungal activities (Candida albicans, Cladosporium and Aspergillus niger) by MIC method. The Schiff-base I and its metal complexes exhibited DNA cleavage activity on isolated DNA of A. niger.

  2. 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

  3. 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.

  4. Hpy188I–DNA pre- and post-cleavage complexes—snapshots of the GIY-YIG nuclease mediated catalysis

    PubMed Central

    Sokolowska, Monika; Czapinska, Honorata; Bochtler, Matthias

    2011-01-01

    The GIY-YIG nuclease domain is present in all kingdoms of life and has diverse functions. It is found in the eukaryotic flap endonuclease and Holliday junction resolvase Slx1–Slx4, the prokaryotic nucleotide excision repair proteins UvrC and Cho, and in proteins of ‘selfish’ genetic elements. Here we present the structures of the ternary pre- and post-cleavage complexes of the type II GIY-YIG restriction endonuclease Hpy188I with DNA and a surrogate or catalytic metal ion, respectively. Our structures suggest that GIY-YIG nucleases catalyze DNA hydrolysis by a single substitution reaction. They are consistent with a previous proposal that a tyrosine residue (which we expect to occur in its phenolate form) acts as a general base for the attacking water molecule. In contrast to the earlier proposal, our data identify the general base with the GIY and not the YIG tyrosine. A conserved glutamate residue (Glu149 provided in trans in Hpy188I) anchors a single metal cation in the active site. This metal ion contacts the phosphate proS oxygen atom and the leaving group 3′-oxygen atom, presumably to facilitate its departure. Taken together, our data reveal striking analogy in the absence of homology between GIY-YIG and ββα-Me nucleases. PMID:20935048

  5. 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.

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

    PubMed

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

    2015-02-25

    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, (1)H NMR, (13)C 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.

  7. Hydrolytic cleavage of double-strand DNA by the water-soluble dicobalt(III) complexes of 1,4,7-triazacyclononane-N-acetate.

    PubMed

    Qian, Jing; Ma, Xiaofang; Tian, Jinlei; Gu, Wen; Shang, Jing; Liu, Xin; Yan, Shiping

    2010-09-01

    Three water-soluble dicobalt(III) complexes, [Co(2)L(2)(micro-OH)(2)](ClO(4))(2).5H(2)O (1), [Co(2)L(2)(micro-OH)(2)](ClO(4))(2).CH(3)OH.H(2)O(2); [Co(2)L(2)(micro-OH)(2)](ClO(4))(2).4H(2)O(3) (L=1,4,7-triazacyclononane-N-acetate monoanion), were prepared to serve as nuclease mimics. The complexes were characterized by X-ray, IR and UV-vis spectroscopy as well as ESI-MS. Three complexes exhibit similar structures, just with different solvent molecules. The electrospray mass spectrum of 1 in solution indicates that dinuclear ion [Co(2)L(2)(micro-OH)(2)-H(+)] (+) (4) is the active species. In the absence of any reducing agent, the complexes cleave plasmid pBR322 DNA was performed and its hydrolytic mechanism was demonstrated with radical scavengers, anaerobic reaction and T4 ligase. The kinetic aspects of DNA cleavage under pseudo- or true-Michaelis-Menten conditions are also detailed, kinetic parameters (k(cat), K(M)) were calculated to be 3.57 h(-1), 6.92 x 10(-4)M; 0.28 h(-1), 1.9 x 10(-5)M for 4, respectively.

  8. The herpes simplex virus 1 UL15 gene encodes two proteins and is required for cleavage of genomic viral DNA.

    PubMed Central

    Baines, J D; Poon, A P; Rovnak, J; Roizman, B

    1994-01-01

    Previous studies have shown that a ts mutant [herpes simplex virus 1 (mP)ts66.4] in the UL15 gene fails to package viral DNA into capsids (A. P. W. Poon and B. Roizman, J. Virol. 67:4497-4503, 1993) and that although the intron separating the first and second exons of the UL15 gene contains UL16 and UL17 open reading frames, replacement of the first exon with a cDNA copy of the entire gene does not affect viral replication (J.D. Baines, and B. Roizman, J. Virol. 66:5621-5626, 1992). We report that (i) a polyclonal rabbit antiserum generated against a chimeric protein consisting of the bacterial maltose-binding protein fused in frame to the majority of sequences contained in the second exon of the UL15 gene reacted with two proteins with M(r) of 35,000 and 75,000, respectively, in cells infected with a virus containing the authentic gene yielding a spliced mRNA or with a virus in which the authentic UL15 gene was replaced with a cDNA copy. (ii) Insertion of 20 additional codons into the C terminus of UL15 exon II caused a reduction in the electrophoretic mobility of both the apparently 35,000- and 75,000-M(r) proteins, unambiguously demonstrating that both share the carboxyl terminus of the UL15 exon II. (iii) Accumulation of the 35,000-M(r) protein was reduced in cells infected and maintained in the presence of phosphonoacetate, an inhibitor of viral DNA synthesis. (iv) The UL15 proteins were localized in the perinuclear space at 6 h after infection and largely in the nucleus at 12 h after infection. (v) Viral DNA accumulating in cells infected with herpes simplex virus 1(mP)ts66.4 and maintained at the nonpermissive temperature was in an endless (concatemeric) form, and therefore UL15 is required for the cleavage of mature, unit-length molecules for packaging into capsids. Images PMID:7966602

  9. Revealing and Resolving the Restrained Enzymatic Cleavage of DNA Self-Assembled Monolayers on Gold: Electrochemical Quantitation and ESI-MS Confirmation.

    PubMed

    Gao, Xiaoyi; Geng, Mingxi; Li, Yunchao; Wang, Xinglin; Yu, Hua-Zhong

    2017-02-21

    Herein, we report a combined electrochemical and ESI-MS study of the enzymatic hydrolysis efficiency of DNA self-assembled monolayers (SAMs) on gold, platform systems for understanding nucleic acid surface chemistry, and for constructing DNA-based biosensors. Our electrochemical approach is based on the comparison of the amounts of surface-tethered DNA nucleotides before and after exonuclease I (Exo I) incubation using electrostatically bound [Ru(NH3)6](3+) as redox indicators. It is surprising to reveal that the hydrolysis efficiency of ssDNA SAMs does not depend on the packing density and base sequence, and that the cleavage ends with surface-bound shorter strands (9-13 mers). The ex-situ ESI-MS observations confirmed that the hydrolysis products for ssDNA SAMs (from 24 to 56 mers) are dominated with 10-15 mer fragments, in contrast to the complete digestion in solution. Such surface-restrained hydrolysis behavior is due to the steric hindrance of the underneath electrode to the Exo I/DNA binding, which is essential for the occurrence of Exo I-catalyzed processive cleavage. More importantly, we have shown that the hydrolysis efficiency of ssDNA SAMs can be remarkably improved by adopting long alkyl linkers (locating DNA strands further away from the substrates).

  10. 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.

  11. 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

  12. 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

  13. Topoisomerase inhibition, nucleolytic and electrolytic contribution on DNA binding activity exerted by biological active analogue of coordination compounds.

    PubMed

    Patel, Mohan N; Bhatt, Bhupesh S; Dosi, Promise A

    2012-04-01

    The neutral mononuclear copper complexes with the quinolone antibacterial drug ciprofloxacin and bipyridine derivatives have been synthesized and characterized. Complexes were screened for their antibacterial activity against three Gram((-)) and two Gram((+)) bacteria, and study suggests inhibition of gyrase activity by metal complexes as the possible mechanism. The nucleolytic activity of adducts was carried out on double stranded pUC19 DNA using gel electrophoresis in the presence of radical scavenging agents that suggest hydrolytic cleavage mechanism for plasmid DNA.

  14. Applying and testing the conveniently optimized enzyme mismatch cleavage method to clinical DNA diagnosis.

    PubMed

    Niida, Yo; Kuroda, Mondo; Mitani, Yusuke; Okumura, Akiko; Yokoi, Ayano

    2012-11-01

    Establishing a simple and effective mutation screening method is one of the most compelling problems with applying genetic diagnosis to clinical use. Because there is no reliable and inexpensive screening system, amplifying by PCR and performing direct sequencing of every coding exon is the gold standard strategy even today. However, this approach is expensive and time consuming, especially when gene size or sample number is large. Previously, we developed CEL nuclease mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining (CHIPS) as an ideal simple mutation screening system constructed with only conventional apparatuses and commercially available reagents. In this study, we evaluated the utility of CHIPS technology for genetic diagnosis in clinical practice by applying this system to screening for the COL2A1, WRN and RPS6KA3 mutations in newly diagnosed patients with Stickler syndrome (autosomal dominant inheritance), Werner syndrome (autosomal recessive inheritance) and Coffin-Lowry syndrome (X-linked inheritance), respectively. In all three genes, CHIPS detected all DNA variations including disease causative mutations within a day. Direct sequencing of all coding exons of these genes confirmed 100% sensitivity and specificity. We demonstrate high sensitivity, high cost performance and reliability of this simple system, with compatibility to all inheritance modes. Because of its low technology, CHIPS is ready to use and potentially disseminate to any laboratories in the world.

  15. Synthesis, structural characterisation, bio-potential efficiency and DNA cleavage applications of nicotinamide metal complexes

    NASA Astrophysics Data System (ADS)

    Surendra Dilip, C.; Siva Kumar, V.; John Venison, S.; Vetha potheher, I.; Rajalaxmi (a) Subahashini, D.

    2013-05-01

    Mixed ligand complexes were synthesised using nicotinamide as the primary ligand and nitrite as the secondary ligand were characterised by FT-IR, UV-Vis, 1H NMR, TG-DTA-DTG, X-ray powder diffraction and physical analytical studies. From the molar conductance, magnetic moment and electronic spectral data of the synthesised complexes a general formula of [M(ONO)2(NA)2] where M = Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Hg(II) and [Cr2(ONO)6(NA)2] with a distorted octahedral structure were proposed. Thermal analyses show that the complexes lose molecules of hydration initially and subsequently expel anionic and organic ligands in continuous steps. The kinetic parameter values, such as, E*, ΔH*, ΔS* and ΔG* illustrate the spontaneous association of the metal and ligands in the formation of the complexes. The antimicrobial efficacy of the ligand and its complexes were examined by in vitro method against various pathogenic bacterial and fungal strains. The metal complexes were found to posses efficient antimicrobial properties compared to nicotinamide and a few of these complexes could turn out to be excellent models for the design of effective antibiotic drug substances. The intercalating interaction of Cu(II) complex with CT-DNA was inspected by absorption spectral and viscosity studies, thermal denaturation and electro-analytical experiments.

  16. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain.

    PubMed

    Li, Ting; Huang, Sheng; Jiang, Wen Zhi; Wright, David; Spalding, Martin H; Weeks, Donald P; Yang, Bing

    2011-01-01

    DNA double-strand breaks enhance homologous recombination in cells and have been exploited for targeted genome editing through use of engineered endonucleases. Here we report the creation and initial characterization of a group of rare-cutting, site-specific DNA nucleases produced by fusion of the restriction enzyme FokI endonuclease domain (FN) with the high-specificity DNA-binding domains of AvrXa7 and PthXo1. AvrXa7 and PthXo1 are members of the transcription activator-like (TAL) effector family whose central repeat units dictate target DNA recognition and can be modularly constructed to create novel DNA specificity. The hybrid FN-AvrXa7, AvrXa7-FN and PthXo1-FN proteins retain both recognition specificity for their target DNA (a 26 bp sequence for AvrXa7 and 24 bp for PthXo1) and the double-stranded DNA cleaving activity of FokI and, thus, are called TAL nucleases (TALNs). With all three TALNs, DNA is cleaved adjacent to the TAL-binding site under optimal conditions in vitro. When expressed in yeast, the TALNs promote DNA homologous recombination of a LacZ gene containing paired AvrXa7 or asymmetric AvrXa7/PthXo1 target sequences. Our results demonstrate the feasibility of creating a tool box of novel TALNs with potential for targeted genome modification in organisms lacking facile mechanisms for targeted gene knockout and homologous recombination.

  17. 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

  18. Signal transduction activated by the cancer chemopreventive isothiocyanates: cleavage of BID protein, tyrosine phosphorylation and activation of JNK

    PubMed Central

    Xu, K; Thornalley, P J

    2001-01-01

    Phenethyl isothiocyanate and allyl isothiocyanate induce apoptosis of human leukaemia HL60 cells in vitro. Apoptosis was associated with cleavage of p22 BID protein to p15, p13 and p11 fragments and activation of JNK and tyrosine phosphorylation (18 kDa and 45 kDa proteins). All these effects and apoptosis were prevented by exogenous glutathione (15 mM). Protein tyrosine phosphatase activity was unchanged. The general caspase inhibitor Z-VAD-fmk prevented apoptosis but not JNK activation – excluding a role for caspases in JNK activation, whereas curcumin prevented JNK activation but only delayed apoptosis. This suggests that in isothiocyanate-induced apoptosis, the caspase pathway has an essential role, the JNK pathway a supporting role, and inhibition of protein tyrosine phosphatases is not involved. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11237388

  19. Synthesis, physico-chemical investigations of Co(II), Ni(II) and Cu(II) complexes and their in vitro microbial, cytotoxic, DNA cleavage studies.

    PubMed

    Bagihalli, Gangadhar B; Patil, Sangamesh A

    2010-06-01

    A series of metal complexes of cobalt(II), nickel(II), and copper(II) have been synthesized with newly derived biologically active ligands. These ligands were synthesized by the condensation of 2-amino-4-phenyl-1,3-thiazole with 8-formyl-7-hydroxy- 4-methylcoumarin. The probable structure of the complexes has been proposed on the basis of analytical and spectroscopic data (IR, UV-Vis, ESR, FAB-mass, and thermoanalytical). Electrochemical study of the complexes is also reported. Elemental analysis of the complexes confined them to stoichiometry of the type ML(2).2H(2)O [M = Co(II), Ni(II), and Cu(II)]. The Schiff base and its metal(II) complexes have been screened for their antibacterial (Escherichia coli, Staphylococcus aureus, Staphylococcus pyogenes, and Pseudomonas aeruginosa) and antifungal activities (Aspergillus niger, Aspergillus flavus, and Cladosporium) by the MIC method. The brine shrimp bioassay was carried out to study their in vitro cytotoxic properties, and also the Schiff base and its metal(II) complexes have been studied for DNA cleavage.

  20. 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.

  1. Reactive oxygen species-dependent HSP90 protein cleavage participates in arsenical As(+3)- and MMA(+3)-induced apoptosis through inhibition of telomerase activity via JNK activation.

    PubMed

    Shen, Shing-Chuan; Yang, Liang-Yo; Lin, Hui-Yi; Wu, Chin-Yen; Su, Tsung-Hsien; Chen, Yen-Chou

    2008-06-01

    The effects of six arsenic compounds including As(+3), MMA(+3), DMA(+3), As(+5), MMA(+5), and DMA(+5) on the viability of NIH3T3 cells were examined. As(+3) and MMA(+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(+3) and MMA(+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(+3) and MMA(+3) treatments. An increase in the intracellular peroxide level was examined in As(+3)- and MMA(+3)-treated NIH3T3 cells, and As(+3)- and MMA(+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(+3)- and MMA(+3)-induced cytotoxicity. Suppression of JNKs significantly inhibited As(+3)- and MMA(+3)-induced apoptosis by blocking HSP90 protein cleavage and telomerase reduction in NIH3T3 cells. Furthermore, Hb, SnPP, and dexferosamine showed no effect against As(+3)- and MMA(+3)-induced apoptosis, and overexpression of HO-1 protein or inhibition of HO-1 protein expression did not affect the apoptosis induced by As(+3) or MMA(+3). These data provide the first evidence to indicate that apoptosis induced by As(+3) and MMA(+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. Cae I: an endonuclease isolated from the African green monkey with properties indicating site-specific cleavage of homologous and heterologous mammalian DNA.

    PubMed Central

    Brown, F L; Musich, P R; Maio, J J

    1978-01-01

    Component alpha DNA is a highly repetitive sequence that comprises nearly a quarter of the African green monkey (Cercopithecus aethiops) genome. A previous microbial restriction enzyme analysis showed that the repeat structure of component alpha DNA is based upon a monomeric unit of 176 +/- 4 base-pairs. An endonuclease, provisionally termed Case I, has been isolated from African green monkey testes that cleaves component alpha DNA into multimeric segments based upon the same repeat periodicity as that revealed by microbial restriction enzymes. The primary sites of Cae I cleavage in the component alpha sequence appear to be 120 +/- 6 base-pairs distant from the Hind III sites and 73 +/- 6 base-pairs distant from the Eco RI* sites. Cae I has been partially characterized with special reference to the effects of ATP and S-adenosylmethionine on the cleavage of component alpha DNA. Cae I may be a member of a class of similar site-specific nucleases present in mammalian cells. Cae I also cleaves mouse satellite DNA into a multimeric series of discrete segments: the periodicity of this series is shorter than that revealed by Eco RII retriction analysis of mouse satellite DNA. Images PMID:206873

  3. Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation.

    PubMed

    Ginster, Stefanie; Bardet, Maureen; Unterreiner, Adeline; Malinverni, Claire; Renner, Florian; Lam, Stephen; Freuler, Felix; Gerrits, Bertran; Voshol, Johannes; Calzascia, Thomas; Régnier, Catherine H; Renatus, Martin; Nikolay, Rainer; Israël, Laura; Bornancin, Frédéric

    2017-01-01

    The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed that TRAF6 can activate MALT1 independently of the CBM. Intramolecular cleavage at R781/R770 removes a C-terminal TRAF6-binding site in both MALT1 isoforms, leaving MALT1B devoid of the two key interaction sites with TRAF6. A previously identified auto-proteolysis site of MALT1 at R149 leads to deletion of the death-domain, thereby abolishing interaction with BCL10. By using MALT1 isoforms and cleaved fragments thereof, as well as TRAF6 WT and mutant forms, this work shows that TRAF6 induces N-terminal auto-proteolytic cleavage of MALT1 at R149 and accelerates MALT1 protein turnover. The MALT1 fragment generated by N-terminal self-cleavage at R149 was labile and displayed enhanced signaling properties that required an intact K644 residue, previously shown to be a site for mono-ubiquitination of MALT1. Conversely, C-terminal self-cleavage at R781/R770 hampered the ability for self-cleavage at R149 and stabilized MALT1 by hindering interaction with TRAF6. C-terminal self-cleavage had limited impact on MALT1A but severely reduced MALT1B proteolytic and signaling functions. It also abrogated NF-κB activation by N-terminally cleaved MALT1A. Altogether, this study provides further insights into mechanisms that regulate the scaffolding and activation cycle of MALT1. It also emphasizes the reduced functional capacity of MALT1B as compared to MALT1A.

  4. Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation

    PubMed Central

    Renner, Florian; Lam, Stephen; Freuler, Felix; Gerrits, Bertran; Voshol, Johannes; Calzascia, Thomas; Régnier, Catherine H.; Renatus, Martin; Nikolay, Rainer; Israël, Laura; Bornancin, Frédéric

    2017-01-01

    The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed that TRAF6 can activate MALT1 independently of the CBM. Intramolecular cleavage at R781/R770 removes a C-terminal TRAF6-binding site in both MALT1 isoforms, leaving MALT1B devoid of the two key interaction sites with TRAF6. A previously identified auto-proteolysis site of MALT1 at R149 leads to deletion of the death-domain, thereby abolishing interaction with BCL10. By using MALT1 isoforms and cleaved fragments thereof, as well as TRAF6 WT and mutant forms, this work shows that TRAF6 induces N-terminal auto-proteolytic cleavage of MALT1 at R149 and accelerates MALT1 protein turnover. The MALT1 fragment generated by N-terminal self-cleavage at R149 was labile and displayed enhanced signaling properties that required an intact K644 residue, previously shown to be a site for mono-ubiquitination of MALT1. Conversely, C-terminal self-cleavage at R781/R770 hampered the ability for self-cleavage at R149 and stabilized MALT1 by hindering interaction with TRAF6. C-terminal self-cleavage had limited impact on MALT1A but severely reduced MALT1B proteolytic and signaling functions. It also abrogated NF-κB activation by N-terminally cleaved MALT1A. Altogether, this study provides further insights into mechanisms that regulate the scaffolding and activation cycle of MALT1. It also emphasizes the reduced functional capacity of MALT1B as compared to MALT1A. PMID:28052131

  5. Synthesis, characterization, biological studies (DNA binding, cleavage, antibacterial and topoisomerase I) and molecular docking of copper(II) benzimidazole complexes.

    PubMed

    Arjmand, Farukh; Parveen, Shazia; Afzal, Mohd; Shahid, Mohd

    2012-09-03

    To explore the therapeutic potential of copper-based benzimidazole complexes, tetranuclear Cu(II) complex 1 and dinuclear ternary amino acid complexes 2 and 3 {L-trp and L-val, respectively} were synthesized and thoroughly characterized. In vitro DNA binding studies of complexes 1-3 were carried out employing UV-vis titrations, fluorescence, circular dichroic and viscosity measurements which revealed that the complexes 1-3 bind to CT DNA preferably via groove binding. Complex 1 cleaved pBR322 DNA via hydrolytic pathway (validated by T4 DNA ligase assay), accessible to major groove while 2 followed oxidative mechanism, binding to minor groove of DNA double helix; binding events were further validated by molecular docking studies. Additionally, the complexes 1 and 2 exhibit high Topo-I inhibitory activity at different concentrations. The complexes 1-3 were evaluated for antibacterial activity against Escherichia coli and Staphylococcus aureus, and 2 was found to be most effective against Gram-positive bacteria.

  6. BIOLOGICAL ACTIVITY OF THE CLEAVAGE PRODUCT OF HUMAN IMMUNOGLOBULIN G WITH CYANOGEN BROMIDE

    PubMed Central

    Lahav, Mira; Arnon, Ruth; Sela, Michael

    1967-01-01

    Treatment of human IgG with cyanogen bromide in 0.05 M HCl under specified conditions resulted in the cleavage of about half of its methionyl peptide bonds. A major fragment of about 5S was isolated from the reaction mixture by gel filtration in quantitative yield. The CNBr fragment reacted fully with goat antiserum against human light chain, but its reaction with anti-heavy chain was markedly decreased. The treatment with CNBr caused a drastic decrease in the following biological activities of IgG: complement fixing, skin binding, reaction with antiglobulin factors, and reaction with specific anti-Gm(12) serum. On the other hand, the reaction with serum of anti-Gm(1) or anti-Gm(4) specificity was not impaired and antibody activity, namely antistreptolysin and isohemagglutinin, was retained after the treatment with CNBr. It is concluded that the CNBr cleaves preferentially the methionyl bonds in the Fc portion of IgG, and that the major fragment obtained, denoted F(ab'')2, has still the combining properties of a divalent antibody. The possible therapeutic uses of F(ab'')2 are discussed. PMID:4164692

  7. A camel-derived MERS-CoV with a variant spike protein cleavage site and distinct fusion activation properties

    PubMed Central

    Millet, Jean Kaoru; Goldstein, Monty E; Labitt, Rachael N; Hsu, Hung-Lun; Daniel, Susan; Whittaker, Gary R

    2016-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) continues to circulate in both humans and camels, and the origin and evolution of the virus remain unclear. Here we characterize the spike protein of a camel-derived MERS-CoV (NRCE-HKU205) identified in 2013, early in the MERS outbreak. NRCE-HKU205 spike protein has a variant cleavage motif with regard to the S2′ fusion activation site—notably, a novel substitution of isoleucine for the otherwise invariant serine at the critical P1′ cleavage site position. The substitutions resulted in a loss of furin-mediated cleavage, as shown by fluorogenic peptide cleavage and western blot assays. Cell–cell fusion and pseudotyped virus infectivity assays demonstrated that the S2′ substitutions decreased spike-mediated fusion and viral entry. However, cathepsin and trypsin-like protease activation were retained, albeit with much reduced efficiency compared with the prototypical EMC/2012 human strain. We show that NRCE-HKU205 has more limited fusion activation properties possibly resulting in more restricted viral tropism and may represent an intermediate in the complex pattern of MERS-CoV ecology and evolution. PMID:27999426

  8. 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.

  9. DNA endonuclease activities on psoralen plus ultraviolet light treated DNA

    SciTech Connect

    Lambert, M.W.; Clark, M.

    1986-03-01

    Activities of nuclear DNA endonucleases (Endos) from normal human lymphoblastoid cells on DNA treated with the DNA interstrand cross-linking agents 4,5'8-trimethyl psoralen (TMP) or 8-methoxypsoralen (MOP) plus long-wavelength (320-400 nm) ultraviolet light (UVA) were examined. Chromatin-associated DNA Endos were isolated from both cell lines and subjected to isoelectric focusing (IF). Each IF fraction was assayed for DNA Endo activity. Peaks of activity were pooled and assayed for activity on undamaged PM2 bacteriophage DNA and on PM2 DNA that had been treated with 15 ..mu..g/ml TMP or MOP in the dark and then exposed to UVA light. Unbound psoralen was removed by dialysis and a second dose of UVA light was given in order to increase the number of DNA cross-links. Two Endo activities were found which were active on TMP- and MOP-DNA: a major one, pI 4.6, which is also active on intercalated DNA, and a second, lesser one, pI 7.6, which is active on UVC (254 nm) light irradiated DNA. These results indicate that there are two different DNA Endos which act on both TMP- and MOP-treated DNA and that the major activity recognizes the intercalation of, and/or the cross-link produced by interaction of, psoralen with DNA.

  10. New strategy for specific activation of recombinant microbial pro-transglutaminase by introducing an enterokinase cleavage site.

    PubMed

    Wang, Kun; Wang, Bin; Yang, Hui-Lin; Pan, Li

    2013-03-01

    Recombinant microbial transglutaminase (rMTG) is usually expressed as a soluble zymogen (pro-rMTG) in heterologous expression systems but proteolytic activation of the inactive pro-rMTG is essential. Instead of screening proteases for activating pro-rMTG, we examined an alternative method by introducing a specific cleavage site of enterokinase between the pro-peptide and mature rMTG, generating three pro-rMTG variants (Pro-mrMTG, Pro-m-rMTG and mPro-rMTG). Pro-mrMTG and Pro-m-rMTG were activated by enterokinase without degrading mature rMTG. The activation productivity of Pro-m-rMTG by enterokinase reached 92 % after 22 h activation, while the activation productivity of Pro-rMTG activated by trypsin was 47 %. MALDI-MS analysis revealed that the pro-peptide including the cleavage site was specifically removed from Pro-m-rMTG after activation. This methodology has the potential to be applied in rMTG production by incorporating highly specific cleavage sites of other proteases.

  11. Inhibition of endonuclease cleavage and DNA replication of E. coli plasmid by the antitumor rhodium(II) complex.

    PubMed

    Rahman, Md Masudur; Yasuda, Hachiro; Katsura, Shinji; Mizuno, Akira

    2007-08-01

    Binding effect of the antitumor complex rhodium(II) acetate [Rh(2)(O(2)CCH(3))(4)] (Rh1) to the plasmid pUC19 DNA has been studied under different molar ratio of Rh1 compound to base pair of pUC19 DNA (R(f)) and reaction time. The Rh1 binding inhibited the activity of restriction enzyme. The binding effect was monitored using gel electrophoresis. The results indicate that at least one Rh1 binds with the recognition sequence and the binding has no preference between A-T and G-C pairs. At high value of R(f)=100, ICP-MS (Inductively Coupled Plasma Mass Spectrometry) measurement confirmed that 46% of Rh1 binds to DNA. PCR amplification of the DNA was also inhibited by the Rh1 binding. The transformation experiment using Escherichia coli suggested that the cell growth was inhibited after binding the Rh1 to the plasmid. These results indicated that DNA synthesis could be inhibited both in vitro and in vivo by the Rh(2)(O(2)CCH(3))(4) binding.

  12. DNA nuclease activity of Rev-coupled transition metal chelates.

    PubMed

    Joyner, Jeff C; Keuper, Kevin D; Cowan, J A

    2012-06-07

    paints a clearer picture of the factors governing DNA nuclease activity by redox active M-chelates than was previously possible. The results demonstrate enhancement of DNA cleavage by use of a targeting sequence, but also clearly underscore that significant orientational factors are required for optimal reactivity at the metal center. Moreover, the studies confirm high selectivity for the target HIV RRE RNA at the most likely dosage concentrations, lending further support to the feasibility of designing and applying targeted catalytic metallodrugs.

  13. A superstructure-based electrochemical assay for signal-amplified detection of DNA methyltransferase activity.

    PubMed

    Zhang, Hui; Yang, Yin; Dong, Huilei; Cai, Chenxin

    2016-12-15

    DNA methyltransferase (MTase) activity is highly correlated with the occurrence and development of cancer. This work reports a superstructure-based electrochemical assay for signal-amplified detection of DNA MTase activity using M.SssI as an example. First, low-density coverage of DNA duplexes on the surface of the gold electrode was achieved by immobilized mercaptohexanol, followed by immobilization of DNA duplexes. The duplex can be cleaved by BstUI endonuclease in the absence of DNA superstructures. However, the cleavage is blocked after the DNA is methylated by M.SssI. The DNA superstructures are formed with the addition of helper DNA. By using an electroactive complex, RuHex, which can bind to DNA double strands, the activity of M.SssI can be quantitatively detected by differential pulse voltammetry. Due to the high site-specific cleavage by BstUI and signal amplification by the DNA superstructure, the biosensor can achieve ultrasensitive detection of DNA MTase activity down to 0.025U/mL. The method can be used for evaluation and screening of the inhibitors of MTase, and thus has potential in the discovery of methylation-related anticancer drugs.

  14. Schistosome satellite DNA encodes active hammerhead ribozymes.

    PubMed

    Ferbeyre, G; Smith, J M; Cedergren, R

    1998-07-01

    Using a computer program designed to search for RNA structural motifs in sequence databases, we have found a hammerhead ribozyme domain encoded in the Smalpha repetitive DNA of Schistosoma mansoni. Transcripts of these repeats are expressed as long multimeric precursor RNAs that cleave in vitro and in vivo into unit-length fragments. This RNA domain is able to engage in both cis and trans cleavage typical of the hammerhead ribozyme. Further computer analysis of S. mansoni DNA identified a potential trans cleavage site in the gene coding for a synaptobrevin-like protein, and RNA transcribed from this gene was efficiently cleaved by the Smalpha ribozyme in vitro. Similar families of repeats containing the hammerhead domain were found in the closely related Schistosoma haematobium and Schistosomatium douthitti species but were not present in Schistosoma japonicum or Heterobilharzia americana, suggesting that the hammerhead domain was not acquired from a common schistosome ancestor.

  15. A novel L-asparaginase from Aquabacterium sp. A7-Y with self-cleavage activation.

    PubMed

    Sun, Zhibin; Li, Ding; Liu, Pingping; Wang, Wenhui; Ji, Kai; Huang, Yan; Cui, Zhongli

    2016-01-01

    We have identified a novel L-asparaginase, abASNase3, from Aquabacterium sp. A7-Y. abASNase3 is composed of 306 amino acids and exhibits 34 % sequence homology to human asparaginase (hASNase3). Further analysis revealed that abASNase3 belongs to the N-terminal nucleophile (Ntn) family of hydrolases. Previous reports about the Ntn hydrolase family and the results of our study suggest that abASNase3 must form two subunits by self-cleavage between Gly189 and Thr190 to attain catalytic activity. The two subunits remained tightly associated to build a single functional unit. The optimum pH for abASNase3 was found to be 8.0 in Tris-HCl buffer and the enzyme was found to be stable over a broad pH range from pH 6.0 to 12.0. The optimum temperature for abASNase3 was found to be approximately 40 °C, and the enzyme was stable below 65 °C. abASNase3 showed high substrate specificity toward L-asparagine and had no or only slight activity toward D-asparagine, L-glutamine and D-glutamine. abASNase3 was significantly activated by Mg(2+) and was substantially inhibited by Ni(2+), Cu(2+), Mn(2+) and Co(2+). The Michaelis-Menten constant and turnover number of abASNase3 for L-asparagine were estimated to be 3.37 × 10(-2) M and 8.72 × 10(-3) s(-1), respectively. Our results indicate that abASNase3 is a novel L-asparaginase in the Ntn family of hydrolases.

  16. Synthesis, spectroscopic and electrochemical studies of N,N-bis[(E)-2-thienylmethylidene]-1,8-naphthalenediamine and its Cu(II) complex: DNA cleavage and generation of superoxide anion.

    PubMed

    Shakir, Mohammad; Azam, Mohammad; Ullah, M F; Hadi, S M

    2011-09-02

    A novel tetradentate Cu(II) complex of the type, [CuL](NO(3))(2) was synthesized by the interaction of Schiff base ligand, N,N-bis[(E)-2-thienylmethylidene]-1,8-naphthalenediamine, L obtained by the condensation of thiophene-2-carboxaldehyde and 1,8-diaminonaphthalene. The formation of Schiff base ligand, L and its Cu(II) complex was confirmed on the basis of results of elemental analyses, mass, FT-IR, (1)H and (13)C{(1)H} NMR spectral studies. UV-Vis, EPR and magnetic susceptibility data support a square planar environment around Cu(II) ion. However, molar conductance values confirmed 1:2 electrolytic nature for the Cu(II) complex. The electrochemical studies of Cu(II) complex was carried out by using cyclic voltammetry which revealed the complex to exhibit quasi reversible process. The biological activity of Cu(II) complex such as ability to bind DNA and DNA cleavage were studied where the Cu(II) complex was shown to cause considerable DNA cleavage and also generated reactive oxygen species such as superoxide anion. Since it is known that various anticancer drugs act through induction of oxidative stress that is mediated by reactive oxygen species, our results suggest a putative role of Cu(II) complex similar to various anticancer drugs.

  17. 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.

  18. 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

  19. Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.

    PubMed

    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

    2012-12-07

    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.

  20. Cleavage of nucleic acids

    DOEpatents

    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.

  1. 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.

  2. 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.

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

    PubMed

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

    2006-11-01

    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(1), diffract to a resolution of 2.9 A and possess unit-cell parameters a = 83.6, b = 171.5, c = 87.8 A, beta = 90.1 degrees, while crystals of GrlA59 belong to space group P2(1)2(1)2, with unit-cell parameters a = 41.5, b = 171.89, c = 87.9 A. These crystals diffract to a resolution of 2.8 A. 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.

  4. The caspase-3 cleavage product of the plasma membrane Ca2+-ATPase 4b is activated and appropriately targeted.

    PubMed

    Pászty, Katalin; Antalffy, Géza; Penheiter, Alan R; Homolya, László; Padányi, Rita; Iliás, Attila; Filoteo, Adelaida G; Penniston, John T; Enyedi, Agnes

    2005-11-01

    The calmodulin-activated transporter hPMCA4 (human plasma membrane Ca2+-ATPase isoform 4) is a target for cleavage by caspase-3 during apoptosis. We have demonstrated that caspase-3 generates a 120 kDa fragment of this pump which lacks the complete autoinhibitory sequence [Paszty, Verma, Padanyi, Filoteo, Penniston and Enyedi (2002) J. Biol. Chem. 277, 6822-6829]. In the present study we analysed further the characteristics of the fragment of hPMCA4b produced by caspase-3. We did this by overexpressing the caspase-3 cleavage product of hPMCA4b in COS-7 and MDCKII (Madin-Darby canine kidney II) cells. This technique made it possible to clearly define the properties of this fragment, and we showed that it is constitutively active, as it forms a phosphoenzyme intermediate and has high Ca2+ transport activity in the absence of calmodulin. When this fragment of hPMCA4b was stably expressed in MDCKII cell clones, it was targeted without degradation to the basolateral plasma membrane. In summary, our studies emphasize that the caspase-3 cleavage product of hPMCA4b is constitutively active, and that the C-terminus is not required for proper targeting of hPMCA4b to the plasma membrane. Also, for the first time, we have generated cell clones that stably express a constitutively active PMCA.

  5. Aniline mustard analogues of the DNA-intercalating agent amsacrine: DNA interaction and biological activity.

    PubMed

    Fan, J Y; Valu, K K; Woodgate, P D; Baguley, B C; Denny, W A

    1997-04-01

    Two series of analogues of the clinical antileukemic drug and DNA-intercalating ligand amsacrine have been prepared, containing aniline mustard sidechains of varying reactivity, linked either at the 4-position of the intercalating acridine chromophore (type A) or at the 1'-position of the 9-anilino group (type B). DNase I footprinting assays showed that compounds of type B had stronger reversible binding to DNA than did compounds of type A. Compounds of each type showed similar patterns of alkylation-induced cleavage of DNA, and alkylate at the N7 of guanines in runs of guanines (similar to the pattern for untargeted mustards) as well as some adenines. Both classes of compounds crosslinked DNA, although those bearing relatively inactive mustards did so only at high drug/base pair ratios. However, while the patterns of DNA alkylation were broadly similar, the compounds were considerably more cytotoxic than analogous untargeted mustards. Comparison of their cytotoxicities in wild-type and DNA repair-deficient lines indicated this toxicity was due to DNA crosslinks (except for the least reactive SO2-linked mustards). The 4-linked analogues showed slightly higher in vivo antileukemic activity than the corresponding 1'-linked analogues.

  6. Modification of the Hemagglutinin Cleavage Site Allows Indirect Activation of Avian Influenza Virus H9N2 by Bacterial Staphylokinase

    PubMed Central

    Tse, Longping V.; Whittaker, Gary R.

    2015-01-01

    Influenza H9N2 is considered to be a low pathogenicity avian influenza (LPAI) virus that commonly infects avian species and can also infect humans. In 1996, the influenza virus, A/chicken/Korea/MS96-CE6/1996/H9N2 (MS96) was isolated from an outbreak in multiple farms in South Korea that resulted in upwards of 30% mortality in infected chickens, with the virus infecting a number of extrapulmonary tissues, indicating internal spread. However, in experimental infections, complete recovery of specific pathogen free (SPF) chickens occurred. Such a discrepancy indicated an alternative pathway for MS96 virus to gain virulence in farmed chickens. A key determinant of influenza pathogenesis is the susceptibility of the viral hemagglutinin (HA) to proteolytic cleavage/activation. Here, we identified that an amino acid substitution, Ser to Tyr found at the P2 position of the MS96 HA cleavage site optimizes cleavage by the protease plasmin (Pm). Importantly, we identified that certain Staphylococcus sp. are able to cleave and activate MS96 HA by activating plasminogen (Plg) to plasmin by use of a virulence factor, staphylokinase. Overall, these studies provide an in-vitro mechanism for bacterially mediated enhancement of influenza activation, and allow insight into the microbiological mechanisms underlying the avian influenza H9N2 outbreak in Korea in1996. PMID:25841078

  7. Active DNA unwinding dynamics during processive DNA replication.

    PubMed

    Morin, José A; Cao, Francisco J; Lázaro, José M; Arias-Gonzalez, J Ricardo; Valpuesta, José M; Carrascosa, José L; Salas, Margarita; Ibarra, Borja

    2012-05-22

    Duplication of double-stranded DNA (dsDNA) requires a fine-tuned coordination between the DNA replication and unwinding reactions. Using optical tweezers, we probed the coupling dynamics between these two activities when they are simultaneously carried out by individual Phi29 DNA polymerase molecules replicating a dsDNA hairpin. We used the wild-type and an unwinding deficient polymerase variant and found that mechanical tension applied on the DNA and the DNA sequence modulate in different ways the replication, unwinding rates, and pause kinetics of each polymerase. However, incorporation of pause kinetics in a model to quantify the unwinding reaction reveals that both polymerases destabilize the fork with the same active mechanism and offers insights into the topological strategies that could be used by the Phi29 DNA polymerase and other DNA replication systems to couple unwinding and replication reactions.

  8. A rapid synthesis of 2-substituted 1,2,3- triazole-1-oxide derivative starting from 4-(methyl)isonitrosoacetophenone and its Ni(II) complex: Characterization, DNA binding and cleavage properties

    NASA Astrophysics Data System (ADS)

    Gup, Ramazan; Erer, Oktay; Dilek, Nefise

    2017-02-01

    An efficient route, not including any metal salt as a catalyst, for the synthesis of a new 2-substituted 1,2,3- triazole-1-oxide is reported in this paper. The title compound has been synthesized via reacting 4-(methyl)isonitrosoacetophenone with hydrazine hydrate and dipyridyl ketone in high yield under mild reaction condition. The structure of the new 1,2,3-triazole-1-oxide has been characterized via single crystal X-ray and spectral studies. The 1:1 ratio reaction of the 1,2,3-triazole 1-oxide ligand with nickel(II) chloride gives the mononuclear complex [Ni(L)(DMF)(Cl)2] which is hexa-coordinated within an octahedral geometry. Characterization of the 1,2,3-triazole compound and its Ni(II) complex with FTIR, 1H and 13C NMR, UV-vis, TGA and elemental analysis also confirm the proposed structures for the compounds. The interactions of the compounds with Calf thymus DNA (CT-DNA) have been investigated via UV-visible spectra and viscosity measurements. The results suggested that both ligand and Ni(II) complex bind to DNA in electrostatic interaction and/or groove binding with a slight partial intercalation. DNA cleavage experiments have been also investigated by agarose gel electrophoresis in the presence and absence of an oxidative agent (H2O2). Both 1,2,3-triazole 1-oxide ligand and nickel(II) complex show nuclease activity, which significantly depends on concentrations of the compounds, both in the presence and absence of an oxidative agent. DNA binding and cleavage affinities of the Ni(II) complex is stronger than that of the 1,2,3-triazole 1-oxide ligand.

  9. Probing the structural dynamics of the CRISPR-Cas9 RNA-guided DNA-cleavage system by coarse-grained modeling.

    PubMed

    Zheng, Wenjun

    2017-02-01

    In the adaptive immune systems of many bacteria and archaea, the Cas9 endonuclease forms a complex with specific guide/scaffold RNA to identify and cleave complementary target sequences in foreign DNA. This DNA targeting machinery has been exploited in numerous applications of genome editing and transcription control. However, the molecular mechanism of the Cas9 system is still obscure. Recently, high-resolution structures have been solved for Cas9 in different structural forms (e.g., unbound forms, RNA-bound binary complexes, and RNA-DNA-bound tertiary complexes, corresponding to an inactive state, a pre-target-bound state, and a cleavage-competent or product state), which offered key structural insights to the Cas9 mechanism. To further probe the structural dynamics of Cas9 interacting with RNA and DNA at the amino-acid level of details, we have performed systematic coarse-grained modeling using an elastic network model and related analyses. Our normal mode analysis predicted a few key modes of collective motions that capture the observed conformational changes featuring large domain motions triggered by binding of RNA and DNA. Our flexibility analysis identified specific regions with high or low flexibility that coincide with key functional sites (such as DNA/RNA-binding sites, nuclease cleavage sites, and key hinges). We also identified a small set of hotspot residues that control the energetics of functional motions, which overlap with known functional sites and offer promising targets for future mutagenesis efforts to improve the specificity of Cas9. Finally, we modeled the conformational transitions of Cas9 from the unbound form to the binary complex and then the tertiary complex, and predicted a distinct sequence of domain motions. In sum, our findings have offered rich structural and dynamic details relevant to the Cas9 machinery, and will guide future investigation and engineering of the Cas9 systems. Proteins 2017; 85:342-353. © 2016 Wiley Periodicals

  10. Design of a New Fluorescent Oligonucleotide-Based Assay for a Highly Specific Real-Time Detection of Apurinic/Apyrimidinic Site Cleavage by Tyrosyl-DNA Phosphodiesterase 1.

    PubMed

    Lebedeva, Natalia A; Anarbaev, Rashid O; Kupryushkin, Maxim S; Rechkunova, Nadejda I; Pyshnyi, Dmitrii V; Stetsenko, Dmitry A; Lavrik, Olga I

    2015-10-21

    Tyrosyl-DNA phosphodiesterase 1 (Tdp1) promotes catalytic scission of a phosphodiester bond between the 3'-end of DNA and the hydroxyl group of a tyrosine residue, as well as cleaving off a variety of other 3'-terminal phosphate-linked DNA substituents. We have shown recently that Tdp1 can initiate an apurinic/apyrimidinic (AP) site repair pathway that is independent from the one mediated by AP endonuclease 1 (APE1). Until recently, there was no method available of tracking the AP-site cleaving activity of Tdp1 by real-time fluorescence assay. In the present study we demonstrate a highly specific real-time detection of the AP-site cleaving activity of Tdp1 which allows one to distinguish it from the activity of APE1 by using a short hairpin oligonucleotide with a 1,12-dodecanediol loop, a 5'-fluorophore, and a 3'-quencher. Specific phosphodiesterase activity of Tdp1, which is usually able to remove quencher from the 3'-end of DNA, was suppressed in our approach by introducing a noncleavable phosphate group mimic between the 3'-end and the quencher. As a nondigestible 3'-phosphate analogue, we have used a new uncharged tetramethyl phosphoryl guanidine (Tmg) group, which is resistant to 3'-phosphodiesterase cleavage.

  11. Factors influencing the DNA nuclease activity of iron, cobalt, nickel, and copper chelates.

    PubMed

    Joyner, Jeff C; Reichfield, Jared; Cowan, J A

    2011-10-05

    A library of complexes that included iron, cobalt, nickel, and copper chelates of cyclam, cyclen, DOTA, DTPA, EDTA, tripeptide GGH, tetrapeptide KGHK, NTA, and TACN was evaluated for DNA nuclease activity, ascorbate consumption, superoxide and hydroxyl radical generation, and reduction potential under physiologically relevant conditions. Plasmid DNA cleavage rates demonstrated by combinations of each complex and biological co-reactants were quantified by gel electrophoresis, yielding second-order rate constants for DNA(supercoiled) to DNA(nicked) conversion up to 2.5 × 10(6) M(-1) min(-1), and for DNA(nicked) to DNA(linear) up to 7 × 10(5) M(-1) min(-1). Relative rates of radical generation and characterization of radical species were determined by reaction with the fluorescent radical probes TEMPO-9-AC and rhodamine B. Ascorbate turnover rate constants ranging from 3 × 10(-4) to 0.13 min(-1) were determined, although many complexes demonstrated no measurable activity. Inhibition and Freifelder-Trumbo analysis of DNA cleavage supported concerted cleavage of dsDNA by a metal-associated reactive oxygen species (ROS) in the case of Cu(2+)(aq), Cu-KGHK, Co-KGHK, and Cu-NTA and stepwise cleavage for Fe(2+)(aq), Cu-cyclam, Cu-cyclen, Co-cyclen, Cu-EDTA, Ni-EDTA, Co-EDTA, Cu-GGH, and Co-NTA. Reduction potentials varied over the range from -362 to +1111 mV versus NHE, and complexes demonstrated optimal catalytic activity in the range of the physiological redox co-reactants ascorbate and peroxide (-66 to +380 mV).

  12. 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.

  13. 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.

  14. Respiratory syncytial virus fusion glycoprotein: nucleotide sequence of mRNA, identification of cleavage activation site and amino acid sequence of N-terminus of F1 subunit.

    PubMed Central

    Elango, N; Satake, M; Coligan, J E; Norrby, E; Camargo, E; Venkatesan, S

    1985-01-01

    The amino acid sequence of respiratory syncytial virus fusion protein (Fo) was deduced from the sequence of a partial cDNA clone of mRNA and from the 5' mRNA sequence obtained by primer extension and dideoxysequencing. The encoded protein of 574 amino acids is extremely hydrophobic and has a molecular weight of 63371 daltons. The site of proteolytic cleavage within this protein was accurately mapped by determining a partial amino acid sequence of the N-terminus of the larger subunit (F1) purified by radioimmunoprecipitation using monoclonal antibodies. Alignment of the N-terminus of the F1 subunit within the deduced amino acid sequence of Fo permitted us to identify a sequence of lys-lys-arg-lys-arg-arg at the C-terminus of the smaller N-terminal F2 subunit that appears to represent the cleavage/activation domain. Five potential sites of glycosylation, four within the F2 subunit, were also identified. Three extremely hydrophobic domains are present in the protein; a) the N-terminal signal sequence, b) the N-terminus of the F1 subunit that is analogous to the N-terminus of the paramyxovirus F1 subunit and the HA2 subunit of influenza virus hemagglutinin, and c) the putative membrane anchorage domain near the C-terminus of F1. Images PMID:2987829

  15. Further insights into the mechanism of the reaction of activated bleomycin with DNA

    PubMed Central

    Chow, Marina S.; Liu, Lei V.; Solomon, Edward I.

    2008-01-01

    Bleomycin (BLM) is a glycopeptide anticancer drug that effectively carries out single- and double-stranded DNA cleavage. Activated BLM (ABLM), a low-spin ferric-hydroperoxide, BLM–FeIII–OOH, is the last intermediate detected before DNA cleavage. We have previously shown through experiments and DFT calculations that both ABLM decay and reaction with H atom donors proceed via direct H atom abstraction. However, the rate of ABLM decay had been previously found, based on indirect methods, to be independent of the presence of DNA. In this study, we use a circular dichroism (CD) feature unique to ABLM to directly monitor the kinetics of ABLM reaction with a DNA oligonucleotide. Our results show that the ABLM + DNA reaction is appreciably faster, has a different kinetic isotope effect, and has a lower Arrhenius activation energy than does ABLM decay. In the ABLM reaction with DNA, the small normal kH/kD ratio is attributed to a secondary solvent effect through DFT vibrational analysis of reactant and transition state (TS) frequencies, and the lower Ea is attributed to the weaker bond involved in the abstraction reaction (C–H for DNA and N–H for the decay in the absence of DNA). The DNA dependence of the ABLM reaction indicates that DNA is involved in the TS for ABLM decay and thus reacts directly with BLM–FeIII–OOH instead of its decay product. PMID:18757754

  16. Synthesis, characterization and DNA binding/cleavage, protein binding and cytotoxicity studies of Co(II), Ni(II), Cu(II) and Zn(II) complexes of aminonaphthoquinone.

    PubMed

    Kosiha, A; Parthiban, C; Elango, Kuppanagounder P

    2017-03-01

    The Co(II), Ni(II), Cu(II) and Zn(II) complexes of an aminonaphthoquinone ligand (L) have been prepared and characterized using analytical and spectral techniques. The structures of L and its Zn(II) complex are confirmed by single crystal X-ray diffraction study. The results indicate that Co(II), Ni(II) and Zn(II) complexes possess tetrahedral geometry while Cu(II) complex exhibits square planar structure. The interaction of L and its complexes with CT-DNA reveal that they could interact with CT-DNA through intercalation. The DNA cleavage studies of the L and its complexes indicate that the Cu(II) and Ni(II) complexes cleave the circular form of the DNA relatively to a greater extent than the other complexes. The results of the interaction of these compounds with bovine serum albumin (BSA) indicate that the complexes exhibit a strong binding to BSA over the L. The in vitro anticancer activities indicate that these compounds exhibit substantial activity against human breast (MCF7) and lung cancer (A549) cell lines. The characteristics of apoptosis in cell morphology have been observed using AO/EB and DAPI staining and the results suggest that an apoptotic mode of cell death with these compounds. The overall results and discussion indicate that coordination of metal ions with the ligand enhances the biological activity.

  17. Different cleavage sites are aligned differently in the active site of M1 RNA, the catalytic subunit of Escherichia coli RNase P.

    PubMed Central

    Kufel, J; Kirsebom, L A

    1996-01-01

    We have studied RNase P RNA (M1 RNA) cleavage of model tRNA precursors that are cleaved at two independent positions. Here we present data demonstrating that cleavage at both sites depends on the 2'-OH immediately 5' of the respective cleavage site. However, we show that the 2-amino group of a guanosine at the cleavage site plays a significant role in cleavage at one of these sites but not at the other. These data suggest that these two cleavage sites are handled differently by the ribozyme. This theory is supported by our finding that the cross-linking pattern between Ml RNA and tRNA precursors carrying 4-thioU showed distinct differences, depending on the location of the 4-thioU relative to the respective cleavage site. These findings lead us to suggest that different cleavage sites are aligned differently in the active site, possibly as a result of different binding modes of a substrate to M1 RNA. We discuss a model in which the interaction between the 3'-terminal "RCCA" motif (first three residues interact) of a tRNA precursor and M1 RNA plays a significant role in this process. Images Fig. 2 Fig. 3 Fig. 4 PMID:8650223

  18. 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.

  19. Citrate and the conversion of carbohydrate in fat. The activities of citrate-cleavage enzyme and acetate thiokinase in livers of starved and re-fed rats

    PubMed Central

    Kornacker, Melodee S.; Lowenstein, J. M.

    1965-01-01

    1. The activity of citrate-cleavage enzyme varies in accordance with the nutritional state of the animal. It is suppressed on starvation and restored on re-feeding after starvation. 2. The increase in enzyme activity that occurs on re-feeding starved animals depends on the diet. It is largest on diets high in carbohydrate and low in fat, and smallest on diets high in fat. Intermediate increases are obtained with balanced diets. 3. The ratio of activities of citrate-cleavage enzyme to acetate thiokinase varies from 2·5 for animals maintained on a balanced diet to 20 for animals re-fed with a diet high in carbohydrate. 4. The changes in activity of citrate-cleavage enzyme correlate with changes in the rate of fatty acid synthesis and provide evidence for the involvement of the citrate-cleavage reaction in fatty acid synthesis. PMID:14342232

  20. Binding of BAL 31 RNA polymerase to PM2 DNA as determined by electron microscopy and protection against restriction endonuclease cleavage.

    PubMed Central

    Bull, P; Susaeta, M; González, B; Yudelevich, A

    1988-01-01

    Specific binding sites of BAL 31 RNA polymerase on PM2 DNA have been mapped by protection against HincII and HindIII cleavage and by observation of enzyme-DNA complexes by electron microscopy. Nine specific binding sites were observed at map units 0.19, 0.20, 0.28, 0.54, 0.63, 0.65, 0.71, 0.72, and 0.75 by the first method. All these sites were confirmed by electron microscopy which, in addition, revealed another site at 0.05 map unit. Published nucleotide sequences of the region surrounding sites at 0.71 and 0.75 map units show the presence of consensus sequences for procaryotic promoters. Images PMID:2843687

  1. 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

  2. Cholesterol side-chain cleavage in the rat adrenal cortex: isolation of a cycloheximide-sensitive activator peptide.

    PubMed Central

    Pedersen, R C; Brownie, A C

    1983-01-01

    A cytosolic peptide activator (Mr approximately equal to 2,200) of cholesterol side-chain cleavage in the adrenal cortex has been isolated from normal corticotropin-treated rats and from rats implanted with the MtT/F4 corticotropin-secreting pituitary tumor. The isolation techniques were those common to peptide hormone purification, including tissue extraction into a highly acidic medium, gel filtration, and reverse-phase HPLC. The amino acid composition has been determined on acid hydrolysates. The activity of this adrenal peptide is acutely increased in hypophysectomized animals treated with corticotropin, and this increase is blocked by cycloheximide. The addition of activator peptide to adrenal mitochondrial preparations results in a rapid stimulation of pregnenolone formation that is dependent on activator concentration and a source of NADPH. In the absence of NADPH, addition of activator peptide to adrenal mitochondria increases the rate of cholesterol association with side-chain cleavage cytochrome P-450. The peptide therefore exhibits properties that are believed to characterize the hypothetical corticotropin-dependent labile activator of adrenal steroidogenesis. PMID:6300876

  3. 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.

  4. Cathepsin K cleavage of SDF-1α inhibits its chemotactic activity towards glioblastoma stem-like cells.

    PubMed

    Hira, Vashendriya V V; Verbovšek, Urška; Breznik, Barbara; Srdič, Matic; Novinec, Marko; Kakar, Hala; Wormer, Jill; der Swaan, Britt Van; Lenarčič, Brigita; Juliano, Luiz; Mehta, Shwetal; Van Noorden, Cornelis J F; Lah, Tamara T

    2017-03-01

    Glioblastoma (GBM) is the most aggressive primary brain tumor with poor patient survival that is at least partly caused by malignant and therapy-resistant glioma stem-like cells (GSLCs) that are protected in GSLC niches. Previously, we have shown that the chemo-attractant stromal-derived factor-1α (SDF-1α), its C-X-C receptor type 4 (CXCR4) and the cysteine protease cathepsin K (CatK) are localized in GSLC niches in glioblastoma. Here, we investigated whether SDF-1α is a niche factor that through its interactions with CXCR4 and/or its second receptor CXCR7 on GSLCs facilitates their homing to niches. Furthermore, we aimed to prove that SDF-1α cleavage by CatK inactivates SDF-1α and inhibits the invasion of GSLCs. We performed mass spectrometric analysis of cleavage products of SDF-1α after proteolysis by CatK. We demonstrated that CatK cleaves SDF-1α at 3 sites in the N-terminus, which is the region of SDF-1α that binds to its receptors. Confocal imaging of human GBM tissue sections confirmed co-localization of SDF-1α and CatK in GSLC niches. In accordance, 2D and 3D invasion experiments using CXCR4/CXCR7-expressing GSLCs and GBM cells showed that SDF-1α had chemotactic activity whereas CatK cleavage products of SDF-1α did not. Besides, CXCR4 inhibitor plerixafor inhibited invasion of CXCR4/CXCR7-expressing GSLCs. In conclusion, CatK can cleave and inactivate SDF-1α. This implies that CatK activity facilitates migration of GSLCs out of niches. We propose that activation of CatK may be a promising strategy to prevent homing of GSLCs in niches and thus render these cells sensitive to chemotherapy and radiation.

  5. Mixed ligand copper(II) complexes of 1,10-phenanthroline with tridentate phenolate/pyridyl/(benz)imidazolyl Schiff base ligands: covalent vs non-covalent DNA binding, DNA cleavage and cytotoxicity.

    PubMed

    Rajarajeswari, Chandrasekaran; Ganeshpandian, Mani; Palaniandavar, Mallayan; Riyasdeen, Anvarbatcha; Akbarsha, Mohammad Abdulkadher

    2014-11-01

    A series of copper(II) complexes of the types [Cu(L)(phen)](ClO4) 1-2, where HL is a tridentate ligand with two nitrogen and one oxygen donor atoms (2NO) such as 2-(2-(1H-benzimidazol-2-yl)ethyliminomethyl)phenol (HL1) and 2-(2-(1H-benzimidazol-2-yl)ethyl-imino)methyl)-4-methylphenol (HL2), phen is 1,10-phenanthroline and [Cu(L)(phen)](ClO4)23-6, where L is a tridentate ligand with three nitrogen donor atoms (3N) such as (2-pyridin-2-ylethyl)pyridin-2-ylmethyleneamine (L3), 2-(1H-benzimidazol-2-yl)ethyl)-pyridin-2-yl-methyleneamine (L4), 2-(1H-benzimidazol-2-yl)ethyl)(1H-imidazol-2-ylmethylene)-amine (L5) and 2-(1H-benzimidazol-2-yl)ethyl)(4,4a-dihydroquinolin-2-ylmethylene)amine (L6), has been isolated and characterized by different spectral techniques. In single crystal X-ray structures, 1 possesses square pyramidal distorted trigonal bipyramidal (SPDTBP), geometry whereas 3 and 4 possess trigonal bipyramidal distorted square pyramidal (TBDSP) geometry. UV-Vis and fluorescence spectral studies reveal that the complexes 1-6 bind non-covalently to calf thymus DNA more strongly than the corresponding covalently bound chlorido complexes [Cu(2NO)Cl] 1a-2a and [Cu(3N)Cl2] 3a-6a. On prolonged incubation, all the complexes 1-6 exhibit double strand cleavage of supercoiled (SC) plasmid DNA in the absence of an activator. Also, they exhibit cytotoxicity against human breast cancer cell lines (HBL-100) more potent than their corresponding chlorido complexes 1a-6a, and have the potential to act as efficient cytotoxic drugs.

  6. Reactivity of mononuclear alkylperoxo copper(II) complex. O-O bond cleavage and C-H bond activation.

    PubMed

    Kunishita, Atsushi; Ishimaru, Hirohito; Nakashima, Satoru; Ogura, Takashi; Itoh, Shinobu

    2008-04-02

    A detailed reactivity study has been carried out for the first time on a new mononuclear alkylperoxo copper(II) complex, which is generated by the reaction of copper(II) complex supported by the bis(pyridylmethyl)amine tridentate ligand containing a phenyl group at the 6-position of the pyridine donor groups and cumene hydroperoxide (CmOOH) in CH3CN. The cumylperoxo copper(II) complex thus obtained has been found to undergo homolytic cleavage of the O-O bond and induce C-H bond activation of exogenous substrates, providing important insights into the catalytic mechanism of copper monooxygenases.

  7. Activation and cleavage of SASH1 by caspase-3 mediates an apoptotic response.

    PubMed

    Burgess, Joshua T; Bolderson, Emma; Adams, Mark N; Baird, Anne-Marie; Zhang, Shu-Dong; Gately, Kathy A; Umezawa, Kazuo; O'Byrne, Kenneth J; Richard, Derek J

    2016-11-10

    Apoptosis is a highly regulated cellular process that functions to remove undesired cells from multicellular organisms. This pathway is often disrupted in cancer, providing tumours with a mechanism to avoid cell death and promote growth and survival. The putative tumour suppressor, SASH1 (SAM and SH3 domain containing protein 1), has been previously implicated in the regulation of apoptosis; however, the molecular role of SASH1 in this process is still unclear. In this study, we demonstrate that SASH1 is cleaved by caspase-3 following UVC-induced apoptosis. Proteolysis of SASH1 enables the C-terminal fragment to translocate from the cytoplasm to the nucleus where it associates with chromatin. The overexpression of wild-type SASH1 or a cleaved form of SASH1 representing amino acids 231-1247 leads to an increase in apoptosis. Conversely, mutation of the SASH1 cleavage site inhibits nuclear translocation and prevents the initiation of apoptosis. SASH1 cleavage is also required for the efficient translocation of the transcription factor nuclear factor-κB (NF-κB) to the nucleus. The use of the NF-κB inhibitor DHMEQ demonstrated that the effect of SASH1 on apoptosis was dependent on NF-κB, indicating a codependence between SASH1 and NF-κB for this process.

  8. Activation and cleavage of SASH1 by caspase-3 mediates an apoptotic response

    PubMed Central

    Burgess, Joshua T; Bolderson, Emma; Adams, Mark N; Baird, Anne-Marie; Zhang, Shu-Dong; Gately, Kathy A; Umezawa, Kazuo; O'Byrne, Kenneth J; Richard, Derek J

    2016-01-01

    Apoptosis is a highly regulated cellular process that functions to remove undesired cells from multicellular organisms. This pathway is often disrupted in cancer, providing tumours with a mechanism to avoid cell death and promote growth and survival. The putative tumour suppressor, SASH1 (SAM and SH3 domain containing protein 1), has been previously implicated in the regulation of apoptosis; however, the molecular role of SASH1 in this process is still unclear. In this study, we demonstrate that SASH1 is cleaved by caspase-3 following UVC-induced apoptosis. Proteolysis of SASH1 enables the C-terminal fragment to translocate from the cytoplasm to the nucleus where it associates with chromatin. The overexpression of wild-type SASH1 or a cleaved form of SASH1 representing amino acids 231–1247 leads to an increase in apoptosis. Conversely, mutation of the SASH1 cleavage site inhibits nuclear translocation and prevents the initiation of apoptosis. SASH1 cleavage is also required for the efficient translocation of the transcription factor nuclear factor-κB (NF-κB) to the nucleus. The use of the NF-κB inhibitor DHMEQ demonstrated that the effect of SASH1 on apoptosis was dependent on NF-κB, indicating a codependence between SASH1 and NF-κB for this process. PMID:27831555

  9. Synthesis, characterization, DNA interaction and cleavage, and in vitro cytotoxicity of copper(II) mixed-ligand complexes with 2-phenyl-3-hydroxy-4(1H)-quinolinone.

    PubMed

    Buchtík, Roman; Trávníček, Zdeněk; Vančo, Ján; Herchel, Radovan; Dvořák, Zdeněk

    2011-10-07

    A series of mixed-ligand complexes [Cu(qui)(L)]NO(3)·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), have been synthesized and fully characterized. The X-ray structures of [Cu(qui)(phen)]NO(3)·H(2)O (2) and [Cu(qui)(ambpy)]NO(3) (3a) show a slightly distorted square-planar geometry in the vicinity of the central copper(II) atom. An in vitro cytotoxicity study of the complexes found significant activity against human osteosarcoma (HOS) and human breast adenocarcinoma (MCF7) cell lines, with the best results for complex 6, where IC(50) equals to 2.1 ± 0.2 μM, and 2.2 ± 0.4 μM, respectively. The strong interactions of the complexes with calf thymus DNA (CT-DNA) and high ability to cleave pUC19 DNA plasmid were found. A correlation has been found between the in vitro cytotoxicity and DNA cleavage studies of the complexes.

  10. Isolation of cDNA clones for mRNAs transcribed zygotically during cleavage in the ascidian, Halocynthia roretzi.

    PubMed

    Miya, Takahito; Nishida, Hiroki

    2002-02-01

    The ascidian larva consists of a relatively small number of different cell types, and the cell lineages during embryogenesis have been well described. The clonal restriction of developmental fate takes place considerably early in development. The fates of most of the blastomeres become tissue-restricted by the 110-cell stage, just before the onset of gastrulation. To elucidate the molecular basis of the early events of fate determination in the ascidian Halocynthia roretzi, we isolated the genes for which zygotic expression is initiated during the early cleavage stages. Here we report 18 genes isolated by subtractive hybridization screening between 110-cell embryos and fertilized eggs. The expression of most (13) of the genes was initiated at the 32-cell stage. The genes were subdivided into three groups according to their spatial expression patterns. The first group included clones expressed throughout almost the entire embryo. The second and third groups represented clones expressed mainly in the animal hemisphere and in a subset of vegetal blastomeres, respectively. One of the genes, HrHesl1, encoded a polypeptide containing the bHLH domain that is similar to those of the Hairy/Enhancer of split/Deadpan family of transcriptional repressors. HrHesl1 was expressed exclusively in epidermal precursor cells during cleavage. Another gene named HrWnt-5 beta was expressed in muscle precursors.

  11. DNA-stacking interactions determine the sequence specificity of the deoxyribonuclease activity of 1,10-phenanthroline-copper ion.

    PubMed

    Schaeffer, F; Rimsky, S; Spassky, A

    1996-07-26

    Bis(1,10-phenanthroline)-copper(I) ion (OP2Cu+) binds reversibly to B-DNA and makes single-stranded cuts by oxidative attack on the deoxyribose moiety. The deoxyribonuclease activity is sequence-dependent yet not nucleotide-specific at the cutting site. OP2Cu+ sequence specificity was analysed in terms of local variations of DNA stability. Kinetic constants of strand cleavage were measured at sequence positions on the two strands and converted into activation free energies of the cleavage reaction. DNA unwinding free energies were calculated from the base sequence using B-DNA stacking parameters for calculations. The two free-energy variations were statistically compared for a series of DNA restriction fragments bearing the binding sites of regulatory proteins and representing a total of 345 DNA base positions. This study shows that the mean activation free energy of strand cleavage at a pair of opposing sugars across the DNA minor groove varies like the unwinding free energy of the DNA sequence delimited by opposing sugars (3 to 4 bp). A statistical equality between the two free-energy variations is demonstrated when considering the sum of the two cleavage events at the opposing sugars. Systematic deviations between the two free-energy distributions were observed at specific sequences, including polypurine-polypyrimidine tracts (AnTm/AmTn, CnTmCp/GpAmGn), alternating purine-pyrimidine tracts ((TA)n/(TA)n, (TG)n/(CA)n) and at certain G+C-rich triplets (GGC, GCC and CGC). The physical significance of these observations is discussed and a model of OP2Cu+ binding and cleavage specificity based on the free-energy equality is proposed.

  12. DNA double-strand breaks and ATM activation by transcription-blocking DNA lesions.

    PubMed

    Sordet, Olivier; Nakamura, Asako J; Redon, Christophe E; Pommier, Yves

    2010-01-15

    A taxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA double-strand break (DSB) response. We recently showed that transcription-blocking topoisomerase I cleavage complexes (TOP1cc) produce DSBs related to R-loop formation and activate ATM in post-mitotic neurons and lymphocytes. Here we discuss how TOP1cc can produce transcription arrest with R-loop formation and generate DSBs that activate ATM, as well as data suggesting that those transcription-dependent DSBs tend to form at the IgH locus and at specific genomic sites. We also address the potential roles of ATM in response to transcription-blocking TOP1cc.

  13. Functionalized Graphene Oxide with Chitosan for Protein Nanocarriers to Protect against Enzymatic Cleavage and Retain Collagenase Activity

    PubMed Central

    Emadi, Fatemeh; Amini, Abbas; Gholami, Ahmad; Ghasemi, Younes

    2017-01-01

    Proteins have short half-life because of enzymatic cleavage. Here, a new protein nanocarrier made of graphene oxide (GO) + Chitosan (CS) is proposed to successfully prevent proteolysis in protein and simultaneously retain its activity. Bovine serum albumin (BSA) and collagenase were loaded on GO and GO-CS to explore the stability and activity of proteins. SEM, AFM, TEM, DSC, UV-Vis, FT-IR, RBS, Raman, SDS-PAGE and zymography were utilized as characterization techniques. The protecting role of GO and GO-CS against enzymatic cleavage was probed by protease digestion analysis on BSA, where the protease solution was introduced to GO-BSA and GO-CS-BSA at 37 °C for 0.5-1-3-6 hours. Characterizations showed the successful synthesis of few layers of GO and the coverage by CS. According to gelatin zymographic analysis, the loaded collagenase on GO and GO-CS lysed the gelatin and created non-staining bands which confirmed the activity of loaded collagenase. SDS-PAGE analysis revealed no significant change in the intact protein in the GO-BSA and GO-CS-BSA solution after 30-minute and 1-hour exposure to protease; however, free BSA was completely digested after 1 hour. After 6 hours, intact proteins were detected in GO-BSA and GO-CS-BSA solutions, while no intact protein was detected in the free BSA solution. PMID:28186169

  14. Functionalized Graphene Oxide with Chitosan for Protein Nanocarriers to Protect against Enzymatic Cleavage and Retain Collagenase Activity.

    PubMed

    Emadi, Fatemeh; Amini, Abbas; Gholami, Ahmad; Ghasemi, Younes

    2017-02-10

    Proteins have short half-life because of enzymatic cleavage. Here, a new protein nanocarrier made of graphene oxide (GO) + Chitosan (CS) is proposed to successfully prevent proteolysis in protein and simultaneously retain its activity. Bovine serum albumin (BSA) and collagenase were loaded on GO and GO-CS to explore the stability and activity of proteins. SEM, AFM, TEM, DSC, UV-Vis, FT-IR, RBS, Raman, SDS-PAGE and zymography were utilized as characterization techniques. The protecting role of GO and GO-CS against enzymatic cleavage was probed by protease digestion analysis on BSA, where the protease solution was introduced to GO-BSA and GO-CS-BSA at 37 °C for 0.5-1-3-6 hours. Characterizations showed the successful synthesis of few layers of GO and the coverage by CS. According to gelatin zymographic analysis, the loaded collagenase on GO and GO-CS lysed the gelatin and created non-staining bands which confirmed the activity of loaded collagenase. SDS-PAGE analysis revealed no significant change in the intact protein in the GO-BSA and GO-CS-BSA solution after 30-minute and 1-hour exposure to protease; however, free BSA was completely digested after 1 hour. After 6 hours, intact proteins were detected in GO-BSA and GO-CS-BSA solutions, while no intact protein was detected in the free BSA solution.

  15. 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.

  16. Ribosomal Protein S12 and Aminoglycoside Antibiotics Modulate A-site mRNA Cleavage and Transfer-Messenger RNA Activity in Escherichia coli*

    PubMed Central

    Holberger, Laura E.; Hayes, Christopher S.

    2009-01-01

    Translational pausing in Escherichia coli can lead to mRNA cleavage within the ribosomal A-site. A-site mRNA cleavage is thought to facilitate transfer-messenger RNA (tmRNA)·SmpB- mediated recycling of stalled ribosome complexes. Here, we demonstrate that the aminoglycosides paromomycin and streptomycin inhibit A-site cleavage of stop codons during inefficient translation termination. Aminoglycosides also induced stop codon read-through, suggesting that these antibiotics alleviate ribosome pausing during termination. Streptomycin did not inhibit A-site cleavage in rpsL mutants, which express streptomycin-resistant variants of ribosomal protein S12. However, rpsL strains exhibited reduced A-site mRNA cleavage compared with rpsL+ cells. Additionally, tmRNA·SmpB-mediated SsrA peptide tagging was significantly reduced in several rpsL strains but could be fully restored in a subset of mutants when treated with streptomycin. The streptomycin-dependent rpsL(P90K) mutant also showed significantly lower levels of A-site cleavage and tmRNA·SmpB activity. Mutations in rpsD (encoding ribosomal protein S4), which suppressed streptomycin dependence, were able to partially restore A-site cleavage to rpsL(P90K) cells but failed to increase tmRNA·SmpB activity. Taken together, these results show that perturbations to A-site structure and function modulate A-site mRNA cleavage and tmRNA·SmpB activity. We propose that tmRNA·SmpB binds to streptomycin-resistant rpsL ribosomes less efficiently, leading to a partial loss of ribosome rescue function in these mutants. PMID:19776006

  17. 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.

  18. DNA-ligase IV and DNA-protein kinase play a critical role in deficient caspases activation in apoptosis-resistant cancer cells by using doxorubicin.

    PubMed

    Friesen, Claudia; Uhl, Miriam; Pannicke, Ulrich; Schwarz, Klaus; Miltner, Erich; Debatin, Klaus-Michael

    2008-08-01

    Resistance toward cytotoxic drugs is one of the primary causes for therapeutic failure in cancer therapy. DNA repair mechanisms as well as deficient caspases activation play a critical role in apoptosis resistance of tumor cells toward anticancer drug treatment. Here, we discovered that deficient caspases activation in apoptosis-resistant cancer cells depends on DNA-ligase IV and DNA-protein kinase (DNA-PK), playing crucial roles in the nonhomologous end joining (NHEJ) pathway, which is the predominant pathway for DNA double-strand break repair (DNA-DSB-repair) in mammalian cells. DNA-PK(+/+) as well as DNA-ligase IV (+/+) cancer cells were apoptosis resistant and deficient in activation of caspase-3, caspase-9, and caspase-8 and in cleavage of poly(ADP-ribose) polymerase after doxorubicin treatment. Inhibition of NHEJ by knocking out DNA-PK or DNA-ligase IV restored caspases activation and apoptosis sensitivity after doxorubicin treatment. In addition, inhibition of caspases activation prevented doxorubicin-induced apoptosis but could not prevent doxorubicin-induced DNA damage, indicating that induction of DNA damage is independent of caspases activation. However, caspases activation depends on induction of DNA damage left unrepaired by NHEJ-DNA-DSB-repair. We conclude that DNA damage left unrepaired by DNA-ligase IV or DNA-PK might be the initiator for caspases activation by doxorubicin in cancer cells. Failure in caspases activation using doxorubicin depends on loss of DNA damage and is due to higher rates of NHEJ-DNA-DBS-repair.

  19. 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

  20. 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.

  1. 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.

  2. Structural snapshots of Xer recombination reveal activation by synaptic complex remodeling and DNA bending

    PubMed Central

    Bebel, Aleksandra; Karaca, Ezgi; Kumar, Banushree; Stark, W Marshall; Barabas, Orsolya

    2016-01-01

    Bacterial Xer site-specific recombinases play an essential genome maintenance role by unlinking chromosome multimers, but their mechanism of action has remained structurally uncharacterized. Here, we present two high-resolution structures of Helicobacter pylori XerH with its recombination site DNA difH, representing pre-cleavage and post-cleavage synaptic intermediates in the recombination pathway. The structures reveal that activation of DNA strand cleavage and rejoining involves large conformational changes and DNA bending, suggesting how interaction with the cell division protein FtsK may license recombination at the septum. Together with biochemical and in vivo analysis, our structures also reveal how a small sequence asymmetry in difH defines protein conformation in the synaptic complex and orchestrates the order of DNA strand exchanges. Our results provide insights into the catalytic mechanism of Xer recombination and a model for regulation of recombination activity during cell division. DOI: http://dx.doi.org/10.7554/eLife.19706.001 PMID:28009253

  3. 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

  4. 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.

  5. 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.

  6. 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.

    PubMed

    Alizadeh, Rahman; Afzal, Mohd; Arjmand, Farukh

    2014-10-15

    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.

  7. 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…

  8. Efficient energy transfer between coronene-modified permethyl-β-cyclodextrins and porphyrin for light induced DNA cleavage.

    PubMed

    Yu, Jie; Zhang, Ying-Ming; Li, Pei-Yu; Liu, Yu

    2017-03-28

    A novel supramolecular assembly was constructed by the noncovalent complexation of hexa-cata-hexabenzocoronene modified permethyl-β-cyclodextrins with tetrasodium tetraphenylporphyrintetrasulfonate in water, exhibiting highly efficient excited energy transfer behaviors and a promising DNA photocleavage ability.

  9. Synthetic RNA-cleaving molecules mimicking ribonuclease A active center. Design and cleavage of tRNA transcripts.

    PubMed Central

    Podyminogin, M A; Vlassov, V V; Giegé, R

    1993-01-01

    RNA cleaving molecules were synthesized by conjugating imidazole residues imitating the essential imidazoles in the active center of pancreatic ribonuclease to an intercalating compound, derivative of phenazine capable of binding to the double stranded regions of polynucleotides. Action of the molecules on tRNA was investigated. It was found, that some of the compounds bearing two imidazole residues cleave tRNA under physiological conditions. The cleavage reaction shows a bell-shaped pH dependence with a maximum at pH 7.0 indicating participation of protonated and non-protonated imidazole residues in the process. Under the conditions stabilizing the tRNA structure, a tRNAAsp transcript was cleaved preferentially at the junctions of the stem and loop regions of the cloverleaf tRNA fold, at the five positions C56, C43, C20.1, U13, and U8, with a marked preference for C56. This cleavage pattern is consistent with a hydrolysis mechanism involving non-covalent binding of the compounds to the double-stranded regions of tRNA followed by an attack of the imidazole residues at the juxtaposed flexible single-stranded regions of the molecule. The compounds provide new probes for the investigation of RNA structure in solution and potential reactive groups for antisense oligonucleotide derivatives. Images PMID:7507235

  10. Searching for discrimination rules in protease proteolytic cleavage activity using genetic programming with a min-max scoring function.

    PubMed

    Yang, Zheng Rong; Thomson, Rebecca; Hodgman, T Charles; Dry, Jonathan; Doyle, Austin K; Narayanan, Ajit; Wu, XiKun

    2003-11-01

    This paper presents an algorithm which is able to extract discriminant rules from oligopeptides for protease proteolytic cleavage activity prediction. The algorithm is developed using genetic programming. Three important components in the algorithm are a min-max scoring function, the reverse Polish notation (RPN) and the use of minimum description length. The min-max scoring function is developed using amino acid similarity matrices for measuring the similarity between an oligopeptide and a rule, which is a complex algebraic equation of amino acids rather than a simple pattern sequence. The Fisher ratio is then calculated on the scoring values using the class label associated with the oligopeptides. The discriminant ability of each rule can therefore be evaluated. The use of RPN makes the evolutionary operations simpler and therefore reduces the computational cost. To prevent overfitting, the concept of minimum description length is used to penalize over-complicated rules. A fitness function is therefore composed of the Fisher ratio and the use of minimum description length for an efficient evolutionary process. In the application to four protease datasets (Trypsin, Factor Xa, Hepatitis C Virus and HIV protease cleavage site prediction), our algorithm is superior to C5, a conventional method for deriving decision trees.

  11. Sea urchin akt activity is Runx-dependent and required for post-cleavage stage cell division.

    PubMed

    Robertson, Anthony J; Coluccio, Alison; Jensen, Sarah; Rydlizky, Katarina; Coffman, James A

    2013-05-15

    In animal development following the initial cleavage stage of embryogenesis, the cell cycle becomes dependent on intercellular signaling and controlled by the genomically encoded ontogenetic program. Runx transcription factors are critical regulators of metazoan developmental signaling, and we have shown that the sea urchin Runx gene runt-1, which is globally expressed during early embryogenesis, functions in support of blastula stage cell proliferation and expression of the mitogenic genes pkc1, cyclinD, and several wnts. To obtain a more comprehensive list of early runt-1 regulatory targets, we screened a Strongylocentrotus purpuratus microarray to identify genes mis-expressed in mid-blastula stage runt-1 morphants. This analysis showed that loss of Runx function perturbs the expression of multiple genes involved in cell division, including the pro-growth and survival kinase Akt (PKB), which is significantly underexpressed in runt-1 morphants. Further genomic analysis revealed that Akt is encoded by two genes in the S. purpuratus genome, akt-1 and akt-2, both of which contain numerous canonical Runx target sequences. The transcripts of both genes accumulate several fold during blastula stage, contingent on runt-1 expression. Inhibiting Akt expression or activity causes blastula stage cell cycle arrest, whereas overexpression of akt-1 mRNA rescues cell proliferation in runt-1 morphants. These results indicate that post-cleavage stage cell division requires Runx-dependent expression of akt.

  12. Activity-dependent regulation of {beta}-catenin via {epsilon}-cleavage of N-cadherin

    SciTech Connect

    Uemura, Kengo; Kihara, Takeshi; Kuzuya, Akira; Okawa, Katsuya; Nishimoto, Takaaki; Bito, Haruhiko; Ninomiya, Haruaki; Sugimoto, Hachiro; Kinoshita, Ayae . E-mail: akinoshita@hs.med.kyoto-u.ac.jp; Shimohama, Shun

    2006-07-07

    N-cadherin is essential for excitatory synaptic contact in the hippocampus. Presenilin 1 (PS1) is located at sites of synaptic contact, forming a complex with N-cadherin and {beta}-catenin. Here, we report that human N-cadherin is cleaved by PS1/{gamma}-secretase in response to physiological concentration of glutamate (Glu) stimulation, yielding a fragment Ncad/CTF2. The expression of Ncad/CTF2 in neuronal cells led to its translocation to the nucleus, and caused a prominent enhancement of cytoplasmic and nuclear {beta}-catenin levels in a cell-cell contact dependent manner, via following mechanisms: 1, inhibition of {beta}-catenin phosphorylation; 2, transactivation of {beta}-catenin; and 3, inhibition of N-cadherin transcription, and finally enhanced {beta}-catenin nuclear signaling. Since the regulation of cellular {beta}-catenin level is essential for synaptic function, disruption in the cleavage of N-cadherin may be causally linked to the synaptic dysfunction associated with Alzheimer's disease (AD)

  13. Factors Influencing the DNA Nuclease Activity of Iron, Cobalt, Nickel, and Copper Chelates

    PubMed Central

    Joyner, Jeff C.; Reichfield, Jared; Cowan, J. A.

    2012-01-01

    A library of complexes that included iron, cobalt, nickel, and copper chelates of cyclam, cyclen, DOTA, DTPA, EDTA, tripeptide GGH, tetrapeptide KGHK, NTA, and TACN was evaluated for DNA nuclease activity, ascorbate consumption, superoxide and hydroxyl radical generation, and reduction potential under physiologically relevant conditions. Plasmid DNA cleavage rates demonstrated by combinations of each complex and biological coreactants were quantified by gel electrophoresis, yielding second-order rate constants for DNAsupercoiled to DNAnicked conversion up to 2.5 ×106 M-1min-1, and for DNAnicked to DNAlinear up to 7 ×105 M-1min-1. Relative rates of radical generation and characterization of radical species were determined by reaction with the fluorescent radical probe TEMPO-9-AC and rhodamine B. Ascorbate turnover rate constants ranging from 9.1×10-3 to 8.2 min-1 were determined, although many complexes demonstrated no measureable activity. Inhibition and Freifelder-Trumbo analysis of DNA cleavage supported concerted cleavage of dsDNA by a metal associated ROS in the case of Cu2+(aq), Cu-KGHK, Co-KGHK, and Cu-NTA and stepwise cleavage for Fe2+(aq), Cu-cyclam, Cu-cyclen, Co-cyclen, Cu-EDTA, Ni-EDTA, Co-EDTA, Cu-GGH, and Co-NTA. Reduction potentials varied over the range from -362 mV to +1111 mV versus NHE, and complexes demonstrated optimal catalytic activity in the range of the physiological redox coreactants ascorbate and peroxide (-66 to +380 mV). PMID:21815680

  14. Oligodendrocyte Precursor Cells Modulate the Neuronal Network by Activity-Dependent Ectodomain Cleavage of Glial NG2

    PubMed Central

    Singh, Jeet; Frischknecht, Renato; Marongiu, Daniele; Binamé, Fabien; Perera, Sumudhu S.; Endres, Kristina; Lutz, Beat; Radyushkin, Konstantin; Trotter, Jacqueline; Mittmann, Thomas

    2014-01-01

    The role of glia in modulating neuronal network activity is an important question. Oligodendrocyte precursor cells (OPC) characteristically express the transmembrane proteoglycan nerve-glia antigen 2 (NG2) and are unique glial cells receiving synaptic input from neurons. The development of NG2+ OPC into myelinating oligodendrocytes has been well studied, yet the retention of a large population of synapse-bearing OPC in the adult brain poses the question as to additional functional roles of OPC in the neuronal network. Here we report that activity-dependent processing of NG2 by OPC-expressed secretases functionally regulates the neuronal network. NG2 cleavage by the α-secretase ADAM10 yields an ectodomain present in the extracellular matrix and a C-terminal fragment that is subsequently further processed by the γ-secretase to release an intracellular domain. ADAM10-dependent NG2 ectodomain cleavage and release (shedding) in acute brain slices or isolated OPC is increased by distinct activity-increasing stimuli. Lack of NG2 expression in OPC (NG2-knockout mice), or pharmacological inhibition of NG2 ectodomain shedding in wild-type OPC, results in a striking reduction of N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) in pyramidal neurons of the somatosensory cortex and alterations in the subunit composition of their α-amino-3-hydroxy-5-methyl-4-isoxazolepr opionicacid (AMPA) receptors. In NG2-knockout mice these neurons exhibit diminished AMPA and NMDA receptor-dependent current amplitudes; strikingly AMPA receptor currents can be rescued by application of conserved LNS protein domains of the NG2 ectodomain. Furthermore, NG2-knockout mice exhibit altered behavior in tests measuring sensorimotor function. These results demonstrate for the first time a bidirectional cross-talk between OPC and the surrounding neuronal network and demonstrate a novel physiological role for OPC in regulating information processing at neuronal synapses. PMID

  15. Endoplasmic Reticulum Exit of Golgi-resident Defective for SREBP Cleavage (Dsc) E3 Ligase Complex Requires Its Activity.

    PubMed

    Raychaudhuri, Sumana; Espenshade, Peter J

    2015-06-05

    Layers of quality control ensure proper protein folding and complex formation prior to exit from the endoplasmic reticulum. The fission yeast Dsc E3 ligase is a Golgi-localized complex required for sterol regulatory element-binding protein (SREBP) transcription factor activation that shows architectural similarity to endoplasmic reticulum-associated degradation E3 ligases. The Dsc E3 ligase consists of five integral membrane proteins (Dsc1-Dsc5) and functionally interacts with the conserved AAA-ATPase Cdc48. Utilizing an in vitro ubiquitination assay, we demonstrated that Dsc1 has ubiquitin E3 ligase activity that requires the E2 ubiquitin-conjugating enzyme Ubc4. Mutations that specifically block Dsc1-Ubc4 interaction prevent SREBP cleavage, indicating that SREBP activation requires Dsc E3 ligase activity. Surprisingly, Golgi localization of the Dsc E3 ligase complex also requires Dsc1 E3 ligase activity. Analysis of Dsc E3 ligase complex formation, glycosylation, and localization indicated that Dsc1 E3 ligase activity is specifically required for endoplasmic reticulum exit of the complex. These results define enzyme activity-dependent sorting as an autoregulatory mechanism for protein trafficking.

  16. Heterogeneity of genetic loci in chickens: analysis of endogenous viral and nonviral genes by cleavage of DNA with restriction endonucleases.

    PubMed

    Hughes, S H; Payvar, F; Spector, D; Schimke, R T; Robinson, H L; Payne, G S; Bishop, J M; Varmus, H E

    1979-10-01

    Restriction endonucleases can be used to define the structure and position of genetic loci for which specific molecular hybridization reagents are available. We have used this approach to compare 18 chicken embryos with respect to several cellular genes; endogenous viral DNA related to the replicative genes of avian sarcoma virus (ASV) or to RAV-O, an endogenous virus of chickens; and sequences related to the transforming (src) gene of ASV. Each cellular gene eas remarkably homogeneous within our test population. We found little or no variation in globin and ovomucoid genes; ovalbumin and transferrin (with one exception) showed variation which is probably allelic in nature. The endogenous viral DNA which has homology with RAV-O was found at several different positions in host DNA and its structure resembled that of proviruses acquired by experimental infection, with sequences from both ends of viral RNA repeated near both ends of viral DNA. Within the population of 18 chickens, one endogenous provirus was always present, whereas the several other proviruses were each found in only a few members of this group. However, screening of additional chickens identified individuals lacking the provirus common to the initial 18 animals surveyed; in at least one embryo no RAV-O-related DNA was detected. These findings suggest that the endogenous RAV-O-related sequences have entered the germ line by relatively recent infection and are still segregating in several contemporary chicken flocks. The sequences in the chicken genome which have homology with the src gene of ASV are invariant from bird to bird and in this sense resemble a cellular gene rather than a viral sequence.

  17. 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

  18. 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.

  19. Cleavage of Type I Collagen by Fibroblast Activation Protein-α Enhances Class A Scavenger Receptor Mediated Macrophage Adhesion.

    PubMed

    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.

  20. Active DNA Demethylation in Plants and Animals

    PubMed Central

    Zhang, H.; Zhu, J.-K.

    2013-01-01

    Active DNA demethylation regulates many vital biological processes, including early development and locus-specific gene expression in plants and animals. In Arabidopsis, bifunctional DNA glycosylases directly excise the 5-methylcytosine base and then cleave the DNA backbone at the abasic site. Recent evidence suggests that mammals utilize DNA glycosylases after 5-methylcytosine is oxidized and/or deaminated. In both cases, the resultant single-nucleotide gap is subsequently filled with an unmodified cytosine through the DNA base excision repair pathway. The enzymatic removal of 5-methylcytosine is tightly integrated with histone modifications and possibly noncoding RNAs. Future research will increase our understanding of the mechanisms and critical roles of active DNA demethylation in various cellular processes as well as inspire novel genetic and chemical therapies for epigenetic disorders. PMID:23197304

  1. 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.

  2. A Cell-based Fluorescence Resonance Energy Transfer (FRET) Sensor Reveals Inter- and Intragenogroup Variations in Norovirus Protease Activity and Polyprotein Cleavage.

    PubMed

    Emmott, Edward; Sweeney, Trevor R; Goodfellow, Ian

    2015-11-13

    The viral protease represents a key drug target for the development of antiviral therapeutics. Because many protease inhibitors mimic protease substrates, differences in substrate recognition between proteases may affect their sensitivity to a given inhibitor. Here we use a cell-based FRET sensor to investigate the activity of different norovirus proteases upon cleavage of various norovirus cleavage sites inserted into a linker region separating cyan fluorescent protein and yellow fluorescent protein. Using this system, we demonstrate that differences in substrate processing exist between proteases from human noroviruses (genogroups I (GI) and II) and the commonly used murine norovirus (MNV, genogroup V) model. These altered the cleavage efficiency of specific cleavage sites both within and between genogroups. The differences observed between these proteases may affect sensitivity to protease inhibitors and the suitability of MNV as a model system for testing such molecules against the human norovirus protease. Finally, we demonstrate that replacement of MNV polyprotein cleavage sites with the GI or GII equivalents, with the exception of the NS6-7 junction, leads to the production of infectious virus when the MNV NS6 protease, but not the GI or GII proteases, are present.

  3. A methylation-stimulated DNA machine: an autonomous isothermal route to methyltransferase activity and inhibition analysis.

    PubMed

    Zhu, Changfeng; Wen, Yanqin; Peng, Hongzhen; Long, Yitao; He, Yao; Huang, Qing; Li, Di; Fan, Chunhai

    2011-04-01

    The operation of DNA nanomachines is generally triggered by either conformational changes of DNA nanostructure or external environmental stimuli. In the present study, we demonstrate an alternative driving force, DNA methylation, to stimulate DNA machine operation. DNA methylation changes neither DNA sequence and conformation nor external environment, however, blocks its cleavage by corresponding methylation-sensitive restriction endonuclease. We thus designed a strand displacement amplification DNA machine, which could be stimulated upon DNA methylation and then autonomously generates accumulated amounts of peroxidase-mimicking DNAzyme signaling machine products in an isothermal manner. The machine product DNAzyme could catalyze the H(2)O(2)-mediated oxidation of 2,2'-azino-bis(3-ethylbenzo thiazoline-6-sulfonic acid) (ABTS(2-)) to a colored product ABTS(·-). This methylation-stimulated DNA machine was further used as a colorimetric assay for analysis of methyltransferases activities and screening of methylation inhibitors. As compared with classical methylation assay, this facile isothermal DNA machine avoids the introduction of methylation-specific polymerase chain reaction and radioactive labels, which might be employed as an effective tool for DNA methylation analysis.

  4. Rapid cytochrome c release, activation of caspases 3, 6, 7 and 8 followed by Bap31 cleavage in HeLa cells treated with photodynamic therapy.

    PubMed

    Granville, D J; Carthy, C M; Jiang, H; Shore, G C; McManus, B M; Hunt, D W

    1998-10-16

    Photodynamic therapy (PDT) is a clinical approach that utilizes light-activated drugs for the treatment of a variety of pathologic conditions. The initiating events of PDT-induced apoptosis are poorly defined. It has been shown for other proapoptotic stimuli that the integral endoplasmic reticulum protein Bap31 is cleaved by caspases 1 and 8, but not by caspase-3. Further, a 20 kDa Bap31 cleavage fragment is generated which can induce apoptosis. In the current report, we sought to determine whether Bap31 cleavage and generation of p20 is an early event in PDT-induced apoptosis. The mitochondrial release of cytochrome c, involvement of caspases 1, 2, 3, 4, 6, 7, 8, and 10 and the status of several known caspase substrates, including Bap31, were evaluated in PDT-treated HeLa cells. Cytochrome c appeared in the cytosol immediately following light activation of the photosensitizer benzoporphyrin derivative monoacid ring A. Activation of caspases 3, 6, 7, and 8 was evident within 1-2 h post PDT. Processing of caspases 1, 2, 4, and 10 was not observed. Cleavage of Bap31 was observed at 2-3 h post PDT. The caspase-3 inhibitor DEVD-fmk blocked caspase-8 and Bap31 cleavage suggesting that caspase-8 and Bap31 processing occur downstream of caspase-3 activation in PDT-induced apoptosis. These results demonstrate that release of mitochondrial cytochrome c into the cytoplasm is a primary event following PDT, preceding caspase activation and cleavage of Bap31. To our knowledge, this is the first example of a chemotherapeutic agent inducing caspase-8 activation and demonstrates that caspase-8 activation can occur after cytochrome c release.

  5. Cleavage of the NR2B subunit amino terminus of N-methyl-D-aspartate (NMDA) receptor by tissue plasminogen activator: identification of the cleavage site and characterization of ifenprodil and glycine affinities on truncated NMDA receptor.

    PubMed

    Ng, Kay-Siong; Leung, How-Wing; Wong, Peter T-H; Low, Chian-Ming

    2012-07-20

    Thrombolysis using tissue plasminogen activator (tPA) has been the key treatment for patients with acute ischemic stroke for the past decade. Recent studies, however, suggest that this clot-busting protease also plays various roles in brain physiological and pathophysiological glutamatergic-dependent processes, such as synaptic plasticity and neurodegeneration. In addition, increasing evidence implicates tPA as an important neuromodulator of the N-methyl-d-aspartate (NMDA) receptors. Here, we demonstrate that recombinant human tPA cleaves the NR2B subunit of NMDA receptor. Analysis of NR2B in rat brain lysates and cortical neurons treated with tPA revealed concentration- and time-dependent degradation of NR2B proteins. Peptide sequencing studies performed on the cleaved-off products obtained from the tPA treatment on a recombinant fusion protein of the amino-terminal domain of NR2B revealed that tPA-mediated cleavage occurred at arginine 67 (Arg(67)). This cleavage is tPA-specific, plasmin-independent, and removes a predicted ~4-kDa fragment (Arg(27)-Arg(67)) from the amino-terminal domain of the NR2B protein. Site-directed mutagenesis of putative cleavage site Arg(67) to Ala(67) impeded tPA-mediated degradation of recombinant protein. This analysis revealed that NR2B is a novel substrate of tPA and suggested that an Arg(27)-Arg(67)-truncated NR2B-containing NMDA receptor could be formed. Heterologous expression of NR2B with Gln(29)-Arg(67) deleted is functional but exhibits reduced ifenprodil inhibition and increased glycine EC(50) with no change in glutamate EC(50). Our results confirmed NR2B as a novel proteolytic substrate of tPA, where tPA may directly interact with NR2B subunits leading to a change in pharmacological properties of NR2B-containing NMDA receptors.

  6. Finding and characterizing the complexes of drug like molecules with quadruplex DNA: combined use of an enhanced hydroxyl radical cleavage protocol and NMR.

    PubMed

    Ranpura, Harikrushan; Bialonska, Dobroslawa; Bolton, Philip H

    2014-01-01

    Structural information on the complexes of drug like molecules with quadruplex DNAs can aid the development of therapeutics and research tools that selectively target specific quadruplex DNAs. Screening can identify candidate molecules that require additional evaluation. An enhanced hydroxyl radical cleavage protocol is demonstrated that can efficiently provide structural information on the complexes of the candidate molecules with quadruplex DNA. NMR methods have been used to offer additional structural information about the complexes as well as validate the results of the hydroxyl radical approach. This multi-step protocol has been demonstrated on complexes of the chair type quadruplex formed by the thrombin binding aptamer, d(GGTTGGTGTGGTTGG). The hydroxyl radical results indicate that NSC 176319, Cain's quinolinium that was found by screening, exhibits selective binding to the two TT loops. The NMR results are consistent with selective disruption of the hydrogen bonding between T4 and T13 as well as unstacking of these residues from the bottom quartet. Thus, the combination of screening, hydroxyl radical footprinting and NMR can find new molecules that selectively bind to quadruplex DNAs as well as provide structural information about their complexes.

  7. Molecular Mechanisms of DNA Replication Checkpoint Activation

    PubMed Central

    Recolin, Bénédicte; van der Laan, Siem; Tsanov, Nikolay; Maiorano, Domenico

    2014-01-01

    The major challenge of the cell cycle is to deliver an intact, and fully duplicated, genetic material to the daughter cells. To this end, progression of DNA synthesis is monitored by a feedback mechanism known as replication checkpoint that is untimely linked to DNA replication. This signaling pathway ensures coordination of DNA synthesis with cell cycle progression. Failure to activate this checkpoint in response to perturbation of DNA synthesis (replication stress) results in forced cell division leading to chromosome fragmentation, aneuploidy, and genomic instability. In this review, we will describe current knowledge of the molecular determinants of the DNA replication checkpoint in eukaryotic cells and discuss a model of activation of this signaling pathway crucial for maintenance of genomic stability. PMID:24705291

  8. 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

  9. 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

  10. 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 ...

  11. Force and twist dependence of RepC nicking activity on torsionally-constrained DNA molecules

    PubMed Central

    Pastrana, Cesar L.; Carrasco, Carolina; Akhtar, Parvez; Leuba, Sanford H.; Khan, Saleem A.; Moreno-Herrero, Fernando

    2016-01-01

    Many bacterial plasmids replicate by an asymmetric rolling-circle mechanism that requires sequence-specific recognition for initiation, nicking of one of the template DNA strands and unwinding of the duplex prior to subsequent leading strand DNA synthesis. Nicking is performed by a replication-initiation protein (Rep) that directly binds to the plasmid double-stranded origin and remains covalently bound to its substrate 5′-end via a phosphotyrosine linkage. It has been proposed that the inverted DNA sequences at the nick site form a cruciform structure that facilitates DNA cleavage. However, the role of Rep proteins in the formation of this cruciform and the implication for its nicking and religation functions is unclear. Here, we have used magnetic tweezers to directly measure the DNA nicking and religation activities of RepC, the replication initiator protein of plasmid pT181, in plasmid sized and torsionally-constrained linear DNA molecules. Nicking by RepC occurred only in negatively supercoiled DNA and was force- and twist-dependent. Comparison with a type IB topoisomerase in similar experiments highlighted a relatively inefficient religation activity of RepC. Based on the structural modeling of RepC and on our experimental evidence, we propose a model where RepC nicking activity is passive and dependent upon the supercoiling degree of the DNA substrate. PMID:27488190

  12. A study on Zr-Ir multiple bonding active for C-H bond cleavage.

    PubMed

    Oishi, Masataka; Oshima, Masato; Suzuki, Hiroharu

    2014-07-07

    Zr-Ir hydrido complexes with ansa-(cyclopentadienyl)(amide) as the supporting ligand in the zirconium fragment, e.g., (L(1)ZrR)(Cp*Ir)(μ-H)3 [L(1) = Me2Si(η(5)-C5Me4)(N(t)Bu), R = Cl (5), Ph (7), Me (10), alkyl, and aryl] were designed, synthesized, and isolated as tractable early-late heterodinuclear complexes. Despite the presence of the three supporting hydride ligands, Zr-Ir distances in the crystal structures of 5, alkyl, and aryl complexes [2.74-2.76 Å] were slightly longer than the sum of the element radii of Zr and Ir [2.719 Å]. These hydrocarbyl complexes displayed the thermolytic C-H activation of a variety of aromatic compounds and several organometallic compounds. Also, the substrate scope and limitation in the Zr-Ir system were studied. The regiochemical outcomes during the C-H activation of pyridine derivatives and methoxyarenes suggested the in situ generation of a Lewis acidic active intermediate, i.e., (L(1)Zr)(Cp*IrH2) (III). The existence of III and relevant σ-complex intermediates {L(1)Zr(η(2)-R-H)}(Cp*IrH2) (IIR) (R = Me, Ph) in the ligand exchange was demonstrated by the direct isolation of a Et3PO-adduct of III (39b) from 7 and kinetic studies. The structure of the direct Zr-Ir bonds in IIPh, IIMe, III, and 39b were probed using computational studies. The unprecedented strong M-M' interactions in the early-late heterobimetallic (ELHB) complexes have been proposed herein.

  13. Rapid three-step cleavage of RNA and DNA model systems promoted by a dinuclear Cu(II) complex in methanol. energetic origins of the catalytic efficacy.

    PubMed

    Lu, Zhong-Lin; Liu, C Tony; Neverov, Alexei A; Brown, R Stan

    2007-09-19

    A dinuclear Cu(II) complex of 1,3-bis-N(1)-(1,5,9-triazacyclododecyl)propane with an associated methoxide (2-Cu(II)(2):(-OCH(3))) was prepared, and its kinetics of reaction with an RNA model (2-hydroxypropyl-p-nitrophenyl phosphate (1, HPNPP)) and two DNA models (methyl p-nitrophenyl phosphate (3) and iso-butyl p-chlorophenyl phosphate (4)) were studied in methanol solution at (s)(s)pH 7.2 +/- 0.2. X-ray diffraction structures of 2-Cu(II)(2):(-OH)(H(2)O)(CF(3)SO(3)-)(3):0.5CH(3)CH(2)OCH(2)CH(3) and 2-Cu(II)(2):(-OH)((C(6)H(5)CH(2)O)(2)PO(2)-)(CF(3)SO(3)-)2 show the mode of coordination of the bridging -OH and H(2)O between the two Cu(II) ions in the first complex and bridging -OH and phosphate groups in the second. The kinetic studies with 1 and 3 reveal some common preliminary steps prior to the chemical one of the catalyzed formation of p-nitrophenol. With 3, and also with the far less reactive substrate (4), two relatively fast events are cleanly observed via stopped-flow kinetics. The first of these is interpreted as a binding step which is linearly dependent on [catalyst] while the second is a unimolecular step independent of [catalyst] proposed to be a rearrangement that forms a doubly Cu(II)-coordinated phosphate. The catalysis of the cleavage of 1 and 3 is very strong, the first-order rate constants for formation of p-nitrophenol from the complex being approximately 0.7 s(-1) and 2.4 x 10(-3) s(-1), respectively. With substrate 3, 2-Cu(II)(2):(-OCH(3)) exhibits Michaelis-Mentin kinetics with a k(cat)/K(M) value of 30 M(-1) s(-1) which is 3.8 x 10(7)-fold greater than the methoxide promoted reaction of 3 (7.9 x 10(-7) M(-1) s(-1)). A free energy calculation indicates that the binding of 2-Cu(II)(2):(-OCH(3)) to the transition states for 1 and 3 cleavage stabilizes them by -21 and -24 kcal/mol, respectively, relative to that of the methoxide promoted reactions. The results are compared with a literature example where the cleavage of 1 in water is promoted by

  14. Characterization of trans- and cis-cleavage activity of the SARS coronavirus 3CLpro protease: basis for the in vitro screening of anti-SARS drugs.

    PubMed

    Lin, Cheng-Wen; Tsai, Chang-Hai; Tsai, Fuu-Jen; Chen, Pei-Jer; Lai, Chien-Chen; Wan, Lei; Chiu, Hua-Hao; Lin, Kuan-Hsun

    2004-09-10

    Severe acute respiratory syndrome (SARS) has been globally reported. A novel coronavirus (CoV), SARS-CoV, was identified as the etiological agent of the disease. SARS-CoV 3C-like protease (3CLpro) mediates the proteolytic processing of replicase polypeptides 1a and 1ab into functional proteins, playing an important role in viral replication. In this study, we demonstrated the expression of the SARS-CoV 3CLpro in Escherichia coli and Vero cells, and then characterized the in vitro trans-cleavage and the cell-based cis-cleavage by the 3CLpro. Mutational analysis of the 3CLpro demonstrated the importance of His41, Cys145, and Glu166 in the substrate-binding subsite S1 for keeping the proteolytic activity. In addition, alanine substitution of the cleavage substrates indicated that Gln-(P1) in the substrates mainly determined the cleavage efficiency. Therefore, this study not only established the quantifiable and reliable assay for the in vitro and cell-based measurement of the 3CLpro activity, but also characterized the molecular interaction of the SARS-CoV 3CLpro with the substrates. The results will be useful for the rational development of the anti-SARS drugs.

  15. MALT1 Protease Activation Triggers Acute Disruption of Endothelial Barrier Integrity via CYLD Cleavage.

    PubMed

    Klei, Linda R; Hu, Dong; Panek, Robert; Alfano, Danielle N; Bridwell, Rachel E; Bailey, Kelly M; Oravecz-Wilson, Katherine I; Concel, Vincent J; Hess, Emily M; Van Beek, Matthew; Delekta, Phillip C; Gu, Shufang; Watkins, Simon C; Ting, Adrian T; Gough, Peter J; Foley, Kevin P; Bertin, John; McAllister-Lucas, Linda M; Lucas, Peter C

    2016-09-27

    Microvascular endothelial cells maintain a tight barrier to prevent passage of plasma and circulating immune cells into the extravascular tissue compartment, yet endothelial cells respond rapidly to vasoactive substances, including thrombin, allowing transient paracellular permeability. This response is a cornerstone of acute inflammation, but the mechanisms responsible are still incompletely understood. Here, we demonstrate that thrombin triggers MALT1 to proteolytically cleave cylindromatosis (CYLD). Fragmentation of CYLD results in microtubule disruption and a cascade of events leading to endothelial cell retraction and an acute permeability response. This finding reveals an unexpected role for the MALT1 protease, which previously has been viewed mostly as a driver of pro-inflammatory NF-κB signaling in lymphocytes. Thus, MALT1 not only promotes immune cell activation but also acutely regulates endothelial cell biology, actions that together facilitate tissue inflammation. Pharmacologic inhibition of MALT1 may therefore have synergistic impact by targeting multiple disparate steps in the overall inflammatory response.

  16. Lesion Recognition and Cleavage by Endonuclease V

    PubMed Central

    Lin, Jun; Gao, Honghai; Schallhorn, Kathryn A.; Harris, Rebecca M.; Cao, Weiguo; Ke, Pu Chun

    2008-01-01

    Endonuclease V (endo V) recognizes and cleaves deoxyinosine in deaminated DNA. These enzymatic activities are precursors of DNA repair and are fueled by metal ions such as Ca2+ and Mg2+, with the former being associated with protein binding and the latter with DNA cleavage. Using the technique of fluorescence resonance energy transfer (FRET) we determined the single-molecule kinetics of endo V in a catalytic cycle using a substrate of deoxyinosine-containing single-stranded DNA (ssDNA). The ssDNA was labeled with TAMRA, a fluorescence donor, while the endo V was labeled with Cy5, a fluorescence acceptor. The time lapses of FRET, resulting from the sequential association, recognition, and dissociation of the deoxyinosine by the endo V, were determined at 5.9 s, 14.5 s, and 9.1 s, respectively, in the presence of Mg2+. In contrast, the process of deoxyinosine recognition appeared little affected by the metal type. The prolonged association and dissociation events in the presence of the Ca2+-Mg2+ combination, as compared to that of Mg2+ alone, support the hypothesis that endo V has two metal binding sites to regulate its enzymatic activities. PMID:17521169

  17. 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

    2013-12-05

    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.

  18. 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

  19. 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.

  20. 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.

  1. Rad18 E3 ubiquitin ligase activity mediates Fanconi anemia pathway activation and cell survival following DNA Topoisomerase 1 inhibition.

    PubMed

    Palle, Komaraiah; Vaziri, Cyrus

    2011-05-15

    Camptothecin (CPT) and related chemotherapeutic drugs induce formation of DNA Topoisomerase I (Top1) covalent or cleavage complexes (Top1ccs) that block leading-strand DNA synthesis and elicit DNA Double Stranded Breaks (DSB) during S phase. The Fanconi Anemia (FA) pathway is implicated in tolerance of CPT-induced DNA damage yet the mechanism of FA pathway activation by Top1 poisons has not been studied. We show here that the FA core complex protein FANCA and monoubiquitinated FANCD2 (an effector of the FA pathway) are rapidly mobilized to chromatin in response to CPT treatment in several human cancer cell lines and untransformed primary human dermal fibroblasts. FANCD2 depletion using siRNA leads to impaired recovery from CPT-induced inhibition or DNA synthesis, persistence of γH2AX (a DSB marker) and reduced cell survival following CPT treatment. The E3 ubiquitin ligase Rad18 is necessary for CPT-induced recruitment of FANCA and FANCD2 to chromatin. Moreover, Rad18-depletion recapitulates the DNA synthesis and survival defects of FANCD2-deficiency in CPT-treated cells. It is well-established that Rad18 promotes FA pathway activation and DNA damage tolerance in response to bulky DNA lesions via a mechanism involving PCNA monoubiquitination. In contrast, PCNA monoubiquitination is not involved in Rad18-mediated FA pathway activation or cell survival following acquisition of CPT-induced DSB. Moreover, while Rad18 is implicated in recombinational repair of DSB via an E3 ligase-independent mechanism, we demonstrate that Rad18 E3 ligase activity is essential for appropriate FA pathway activation and DNA damage tolerance after CPT treatment. Taken together, our results define a novel pathway of Rad18-dependent DSB repair that is dissociable from known Rad18-mediated DNA repair mechanisms based on its independence from PCNA ubiquitination and requirement for E3 ligase activity.

  2. 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.

  3. 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.

  4. OsSpo11-4, a rice homologue of the archaeal TopVIA protein, mediates double-strand DNA cleavage and interacts with OsTopVIB.

    PubMed

    An, Xiao Jing; Deng, Zhu Yun; Wang, Tai

    2011-01-01

    DNA topoisomerase VI from Archaea, a heterotetrameric complex composed of two TopVIA and two TopVIB subunits, is involved in altering DNA topology during replication, transcription and chromosome segregation by catalyzing DNA strand transfer through transient double-strand breaks. The sequenced yeast and animal genomes encode only one homologue of the archaeal TopVIA subunit, namely Spo11, and no homologue of the archaeal TopVIB subunit. In yeast, Spo11 is essential for initiating meiotic recombination and this function appears conserved among other eukaryotes. In contrast to yeast and animals, studies in Arabidopsis and rice have identified three Spo11/TopVIA homologues and one TopVIB homologue in plants. Here, we further identified two novel Spo11/TopVIA homologues (named OsSpo11-4 and OsSpo11-5, respectively) that exist just in the monocot model plant Oryza sativa, indicating that at least five Spo11/TopVIA homologues are present in the rice genome. To reveal the biochemical function of the two novel Spo11/TopVIA homologues, we first examined the interactions among OsSpo11-1, OsSpo11-4, OsSpo11-5, and OsTopVIB by yeast two-hybrid assay. The results showed that OsSpo11-4 and OsTopVIB can self-interact strongly and among the 3 examined OsSpo11 proteins, only OsSpo11-4 interacted with OsTopVIB. Pull-down assay confirmed the interaction between OsSpo11-4 and OsTopVIB, which indicates that OsSpo11-4 may interact with OsTopVIB in vivo. Further in vitro enzymatic analysis revealed that among the above 4 proteins, only OsSpo11-4 exhibited double-strand DNA cleavage activity and its enzymatic activity appears dependent on Mg(2+) and independent of OsTopVIB, despite its interaction with OsTopVIB. We further analyzed the biological function of OsSpo11-4 by RNA interference and found that down-regulated expression of OsSpo11-4 led to defects in male meiosis, indicating OsSpo11-4 is required for meiosis.

  5. DNA polymerase activity of tomato fruit chromoplasts.

    PubMed

    Serra, E C; Carrillo, N

    1990-11-26

    DNA polymerase activity was measured in chromoplasts of ripening tomato fruits. Plastids isolated from young leaves or mature red fruits showed similar DNA polymerase activities. The same enzyme species was present in either chloroplasts or chromoplasts as judged by pH and temperature profiles, sensitivities towards different inhibitors and relative molecular mass (Mr 88 kDa). The activities analyzed showed the typical behaviour of plastid-type polymerases. The results presented here suggest that chromoplast maintain their DNA synthesis potential in fruit tissue at chloroplast levels. Consequently, the sharp decrease of the plastid chromosome transcription observed at the onset of fruit ripening could not be due to limitations in the availability of template molecules. Other mechanisms must be involved in the inhibition of chromoplast RNA synthesis.

  6. Beta-secretase cleavage at amino acid residue 34 in the amyloid beta peptide is dependent upon gamma-secretase activity.

    PubMed

    Shi, Xiao-Ping; Tugusheva, Katherine; Bruce, James E; Lucka, Adam; Wu, Guo-Xin; Chen-Dodson, Elizabeth; Price, Eric; Li, Yueming; Xu, Min; Huang, Qian; Sardana, Mohinder K; Hazuda, Daria J

    2003-06-06

    The amyloid beta peptides (Abeta) are the major components of the senile plaques characteristic of Alzheimer's disease. Abeta peptides are generated from the cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. Beta-secretase (BACE), a type-I transmembrane aspartyl protease, cleaves APP first to generate a 99-amino acid membrane-associated fragment (CT99) containing the N terminus of Abeta peptides. Gamma-secretase, a multi-protein complex, then cleaves within the transmembrane region of CT99 to generate the C termini of Abeta peptides. The production of Abeta peptides is, therefore, dependent on the activities of both BACE and gamma-secretase. The cleavage of APP by BACE is believed to be a prerequisite for gamma-secretase-mediated processing. In the present study, we provide evidence both in vitro and in cells that BACE-mediated cleavage between amino acid residues 34 and 35 (Abeta-34 site) in the Abeta region is dependent on gamma-secretase activity. In vitro, the Abeta-34 site is processed specifically by BACE1 and BACE2, but not by cathepsin D, a closely related aspartyl protease. Moreover, the cleavage of the Abeta-34 site by BACE1 or BACE2 occurred only when Abeta 1- 40 peptide, a gamma-secretase cleavage product, was used as substrate, not the non-cleaved CT99. In cells, overexpression of BACE1 or BACE2 dramatically increased the production of the Abeta 1-34 species. More importantly, the cellular production of Abeta 1-34 species induced by overexpression of BACE1 or BACE2 was blocked by a number of known gamma-secretase inhibitors in a concentration-dependent manner. These gamma-secretase inhibitors had no effect on enzymatic activity of BACE1 or BACE2 in vitro. Our data thus suggest that gamma-secretase cleavage of CT99 is a prerequisite for BACE-mediated processing at Abeta-34 site. Therefore, BACE and gamma-secretase activity can be mutually dependent.

  7. 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.

  8. 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.

  9. Collision-activated cleavage of a peptide/antibiotic disulfide linkage: possible evidence for intramolecular disulfide bond rearrangement upon collisional activation.

    PubMed

    Fagerquist, Clifton K

    2004-01-01

    Ceftiofur is an important veterinary beta-lactam antibiotic whose bioactive metabolite, desfuroylceftiofur, has a free thiol group. Desfuroylceftiofur (DFC) was reacted with two peptides, [Arg8]-vasopressin and reduced glutathione, both of which have cysteine residues to form disulfide-linked peptide/antibiotic complexes. The products of the reaction, [vasopressin + (DFC-H) + (DFC-H) + H]+, [(vasopressin+H) + (DFC-H) + H]+ and [(glutathione-H) + (DFC-H) + H]+, were analyzed using collision-activated dissociation (CAD) with a quadrupole ion trap tandem mass spectrometer. MS/MS of [vasopressin + (DFC-H) + (DFC-H) + H]+ resulted in facile dissociative loss of one and two covalently bound DFC moieties. Loss of one DFC resulted from either homolytic or heterolytic dissociation of the peptide/antibiotic disulfide bond with equal or unequal partitioning of the two sulfur atoms between the fragment ion and neutral loss. Hydrogen migration preceded heterolytic dissociation. Loss of two DFC moieties from [vasopressin + (DFC-H) + (DFC-H) + H]+ appears to result from collision-activated intramolecular disulfide bond rearrangement (IDBR) to produce cyclic [vasopressin + H]+ (at m/z 1084) as well as other cyclic fragment ions at m/z 1084 +/- 32 and +64. The cyclic structure of these ions could only be inferred as MS/MS may result in rearrangement to non-cyclic structures prior to dissociative loss. IDBR was also detected from MS(3) experiments of [vasopressin + (DFC-H) + (DFC-H) + H]+ fragment ions. MS/MS of [(glutathione-H) + (DFC-H) + H]+ resulted in cleavage of the peptide backbone with retention of the DFC moiety as well as heterolytic cleavage of the peptide/antibiotic disulfide bond to produce the fragment ion: [(DFC-2H) + H]+. These results demonstrate the facile dissociative loss by CAD of DFC moieties covalently attached to peptides through disulfide bonds. Published in 2004 by John Wiley & Sons, Ltd.

  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. 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

  12. High-specificity single-tube multiplex genotyping using Ribo-PAP PCR, tag primers, alkali cleavage of RNA/DNA chimeras and MALDI-TOF MS.

    PubMed

    Mauger, Florence; Gelfand, David H; Gupta, Amar; Bodepudi, Veeraiah; Will, Stephen G; Bauer, Keith; Myers, Thomas W; Gut, Ivo G

    2013-01-01

    Here, we describe a high-throughput, single-tube, allele-specific ribonucleotide analog pyrophosphorolysis-activated polymerization (ribo-PAP) PCR multiplex genotyping and resequencing method. An RNA/DNA chimeric PCR product is generated using genomic DNA as starting template, a panel of allele-selective 5'-tagged primers, a reverse primer, one nucleotide in the ribo-form (90-100%), the other nucleotides in the deoxy-form, a DNA polymerase capable of incorporating ribonucleotides, a suitable buffer and thermal cycling. The RNA/DNA chimeric PCR products are fragmented by treatment with alkali and analyzed by mass spectrometry. All allele-selective primers have a 5' repetitive motif where each repeat unit has a unique, distinct mass upon reverse copying and alkali fragmentation. The mass of the complement repeat fragment or flag identifies the primer or primers that were recruited in the ribo-PAP PCR. The method readily identifies homozygous and heterozygous positions in simplex or duplex ribo-PAP PCR. Many different tags can be analyzed simultaneously. The assay can genotype several SNPs in a single tube. It thus constitutes the simplest genotyping protocol with multiplex analysis. This novel genotyping and resequencing protocol was applied to different genomic loci: NOS1 and H19 in 30 individuals in simplex ribo-PAP PCR and at two SLCO1B1 loci in 95 individuals in duplex ribo-PAP PCR.

  13. Physical association of the 2,6-diamino-4-hydroxy-5N-formamidopyrimidine-DNA glycosylase of Escherichia coli and an activity nicking DNA at apurinic/apyrimidinic sites.

    PubMed Central

    O'Connor, T R; Laval, J

    1989-01-01

    The 2,6-diamino-4-hydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase of Escherichia coli, which is coded for by the fpg gene, excises purine bases with ring-opened imidazoles. In addition to the DNA glycosylase activity, we report that the Fapy-DNA glycosylase of E. coli has an associated activity, resistant to EDTA, that nicks DNA at apurinic/apyrimidinic (AP) sites. The levels of Fapy-DNA glycosylase and AP-nicking activity were parallel in crude lysates of E. coli HB101 harboring different plasmids constructed from the fpg gene. The fpg gene is different from the xth, nth, and nfo genes of E. coli, whose gene products also cleave DNA at AP sites. The Fapy-DNA glycosylase was purified to electrophoretic homogeneity. During this purification, the Fapy-DNA glycosylase copurified with an AP-nicking activity using chromatographic separations based on ion-exchange, molecular weight exclusion, and hydrophobicity. The cleavage at AP sites by the Fapy-DNA glycosylase left a 5'-phosphomonoester nucleotide at one terminus. In addition, DNA containing reduced AP sites was not nicked by the Fapy-DNA glycosylase. These data suggest that the mechanism of cleavage involved beta elimination. Therefore, this activity of the Fapy-DNA glycosylase nicking DNA at AP sites should be referred to as an AP lyase. The 3' terminus did not prime nick-translation by E. coli DNA polymerase I. However, the 3' terminus becomes a substrate for nick-translation if first allowed to react with calf intestine phosphatase or the E. coli exonuclease III. These data suggest that the repair of the Fapy lesion at least to some extent results in the formation of both 5'- and 3'-phosphomonoester nucleotides and the release of the deoxyribose. Images PMID:2664776

  14. Kinetic analysis of the cleavage of natural and synthetic substrates by the Serratia nuclease.

    PubMed

    Friedhoff, P; Meiss, G; Kolmes, B; Pieper, U; Gimadutdinow, O; Urbanke, C; Pingoud, A

    1996-10-15

    The extracellular nuclease from Serratia marcescens is a non-specific endonuclease that hydrolyzes double-stranded and single-stranded DNA and RNA with high specific activity. Steady-state and presteady-state kinetic cleavage experiments were performed with natural and synthetic DNA and RNA substrates to understand the mechanism of action of the Serratia nuclease. Most of the natural substrates are cleaved with similar Kcat and K(m) values, the Kcat/K(m) ratios being comparable to that of staphylococcal nuclease. Substrates with extreme structural features, like poly(dA).poly(dT) or poly(dG).poly(dC), are cleaved by the Serratia nuclease with a 50 times higher or 10 times lower K(m), respectively, as salmon testis DNA. Neither with natural DNA or RNA nor synthetic oligodeoxynucleotide substrates did we observe substrate inhibition for the Serratia nuclease as reported recently. Experiments with short oligodeoxynucleotides confirmed previous results that for moderately good cleavage activity the substrate should contain at least five phosphate residues. Shorter substrates are still cleaved by the Serratia nuclease, albeit at a rate reduced by a factor of more than 100. Cleavage experiments with oligodeoxynucleotides substituted by a single phosphorothioate group showed that the negative charge of the pro-Rp-oxygen of the phosphate group 3' adjacent to the scissile phosphodiester bond is essential for cleavage, as only the Rp-phosphorothioate supports cleavage at the 5' adjacent phosphodiester bond. Furthermore, the modified bond itself is only cleaved in the Rp-diastereomer, albeit 1000 times more slowly than the corresponding unmodified phosphodiester bond, which offers the possibility to determine the stereochemical outcome of cleavage. Pre-steady-state cleavage experiments demonstrate that it is not dissociation of products but association of enzyme and substrate or the cleavage of the phosphodiester bond that is the rate-limiting step of the reaction. Finally

  15. Synthesis, spectral and quantum chemical studies and use of (E)-3-[(3,5-bis(trifluoromethyl)phenylimino)methyl]benzene-1,2-diol and its Ni(II) and Cu(II) complexes as an anion sensor, DNA binding, DNA cleavage, anti-microbial, anti-mutagenic and anti-cancer agent

    NASA Astrophysics Data System (ADS)

    Ünver, Hüseyin; Boyacıoğlu, Bahadır; Zeyrek, Celal Tuğrul; Yıldız, Mustafa; Demir, Neslihan; Yıldırım, Nuray; Karaosmanoğlu, Oğuzhan; Sivas, Hülya; Elmalı, Ayhan

    2016-12-01

    We report the synthesis of a novel Schiff base (E)-3-[(3,5-bis(trifluoromethyl) phenylimino)methyl] benzene-1,2-diol from the reaction of 2,3-dihydroxybenzaldehyde with 3,5-bis(trifluoromethyl)aniline, and its Ni(II) and Cu(II) complexes. The molecular structure of the Schiff base was experimentally determined using X-ray single-crystal data and was compared to the structure predicted by theoretical calculations using density functional theory (DFT), Hartree-Fock (HF) and Möller-Plesset second-order perturbation (MP2). In addition, nonlinear optical (NLO) effects of the compound was predicted using DFT. The antimicrobial activities of the compounds were investigated for their minimum inhibitory concentration. UV-Vis spectroscopy studies of the interactions between the compounds and calf thymus DNA (CT-DNA) showed that the compounds interacts with CT-DNA via intercalative binding. A DNA cleavage study showed that the Cu(II) complex cleaved DNA without any external agents. The compounds inhibited the base pair mutation in the absence of S9 with high inhibition rate. In addition, in vitro cytotoxicity of the Ni(II) complex towards HepG2 cell line was assayed by the MTT method. Also, the colorimetric response of the Schiff base in DMSO to the addition of equivalent amount of anions (F-, Br-, I-, CN-, SCN-, ClO4-, HSO4-, AcO-, H2PO4-, N3- and OH-) was investigated. In this regard, while the addition of F-, CN-, AcO- and OH- anions into the solution containing Schiff base resulted in a significant color change, the addition of Br-, I-, SCN-, ClO4-, HSO4-, H2PO4- and N3- anions resulted in no color change. The most discernable color change in the Schiff base was caused by CN-, which demonstrated that the ligand can be used to selectively detect CN-.

  16. Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis

    PubMed Central

    Dong, Hui; Dumenil, Jack; Lu, Fu-Hao; Na, Li; Vanhaeren, Hannes; Naumann, Christin; Klecker, Maria; Prior, Rachel; Smith, Caroline; McKenzie, Neil; Saalbach, Gerhard; Chen, Liangliang; Xia, Tian; Gonzalez, Nathalie; Seguela, Mathilde; Inzé, Dirk; Dissmeyer, Nico; Li, Yunhai; Bevan, Michael W.

    2017-01-01

    The characteristic shapes and sizes of organs are established by cell proliferation patterns and final cell sizes, but the underlying molecular mechanisms coordinating these are poorly understood. Here we characterize a ubiquitin-activated peptidase called DA1 that limits the duration of cell proliferation during organ growth in Arabidopsis thaliana. The peptidase is activated by two RING E3 ligases, Big Brother (BB) and DA2, which are subsequently cleaved by the activated peptidase and destabilized. In the case of BB, cleavage leads to destabilization by the RING E3 ligase PROTEOLYSIS 1 (PRT1) of the N-end rule pathway. DA1 peptidase activity also cleaves the deubiquitylase UBP15, which promotes cell proliferation, and the transcription factors TEOSINTE BRANCED 1/CYCLOIDEA/PCF 15 (TCP15) and TCP22, which promote cell proliferation and repress endoreduplication. We propose that DA1 peptidase activity regulates the duration of cell proliferation and the transition to endoreduplication and differentiation during organ formation in plants by coordinating the destabilization of regulatory proteins. PMID:28167503

  17. 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

  18. 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

  19. 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.

  20. HIV-1 Vpu Accessory Protein Induces Caspase-mediated Cleavage of IRF3 Transcription Factor*

    PubMed Central

    Park, Sang Yoon; Waheed, Abdul A.; Zhang, Zai-Rong; Freed, Eric O.; Bonifacino, Juan S.

    2014-01-01

    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

  1. DNA synthesis and DNA polymerase activity of herpes simplex virus type 1 temperature-sensitive mutants.

    PubMed Central

    Aron, G M; Purifoy, D J; Schaffer, P A

    1975-01-01

    Fifteen temperature-sensitive mutants of herpes simplex virus type 1 were studied with regard to the relationship between their ability to synthesize viral DNA and to induce viral DNA polymerase (DP) activity at permissive (34 C) and nonpermissive (39 C) temperatures. At 34 C, all mutants synthesized viral DNA, while at 39 C four mutants demonstrated a DNA+ phenotype, three were DNA+/-, and eight were DNA-. DNA+ mutants induced levels of DP activity similar to thhose of the wild-type virus at both temperatures, and DNA+/- mutants induced reduced levels of DP activity at 39 C but not at 34 C. Among the DNA- mutants three were DP+, two were DP+/-, and three showed reduced DP activity at 34 C with no DP activity at 39 C. DNA-, DP- mutants induced the synthesis of a temperature-sensitive DP as determined by in vivo studies. PMID:169388

  2. The DNA-remodelling activity of DnaD is the sum of oligomerization and DNA-binding activities on separate domains

    PubMed Central

    Carneiro, Maria J. V. M.; Zhang, Wenke; Ioannou, Charikleia; Scott, David J.; Allen, Stephanie; Roberts, Clive J.; Soultanas, Panos

    2011-01-01

    Summary The Bacillus subtilis DnaD protein is an essential protein that has been implicated in the primosomal step of DNA replication, and recently in global DNA remodelling. Here we show that DnaD consists of two domains with distinct activities; an N-terminal domain (Nd) with oligomerization activity, and a C-terminal domain (Cd) with DNA-binding activity and a second DNA-induced oligomerization activity. Although Cd can bind to DNA and form large nucleoprotein complexes, it does not exhibit global DNA-remodelling activity. The presence of separate Nd does not restore this activity. Our data suggest that the global DNA-remodelling activity of DnaD is the sum of three separate oligomerization and DNA-binding activities residing on two distinct but linked domains. PMID:16677303

  3. Fluorescence studies, DNA binding properties and antimicrobial activity of a dysprosium(III) complex containing 1,10-phenanthroline.

    PubMed

    Khorasani-Motlagh, Mozhgan; Noroozifar, Meissam; Moodi, Asieh; Niroomand, Sona

    2013-10-05

    Luminescence and binding properties of dysprosium(III) complex containing 1,10-phenanthroline (phen), [Dy(phen)2(OH2)3Cl]Cl2⋅H2O with DNA has been studied by electronic absorption, emission spectroscopy and viscosity measurement. The thermodynamic studies suggest that the interaction process to be endothermic and entropically driven, which indicates that the dysprosium(III) complex might interact with DNA by a non intercalation binding mode. Additionally, the competitive fluorescence study with ethidium bromide and also the effect of iodide ion and salt concentration on fluorescence of the complex-DNA system is investigated. Experimental results indicate that the Dy(III) complex strongly binds to DNA, presumably via groove binding mode. Furthermore, the complex shows a potent antibacterial activity and DNA cleavage ability.

  4. Failure of cell cleavage induces senescence in tetraploid primary cells.

    PubMed

    Panopoulos, Andreas; Pacios-Bras, Cristina; Choi, Justin; Yenjerla, Mythili; Sussman, Mark A; Fotedar, Rati; Margolis, Robert L

    2014-10-15

    Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control. We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts become senescent in tetraploid G1 after drug- or small interfering RNA (siRNA)-induced failure of cell cleavage. In contrast, T-antigen-transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle after cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker and of the fluorescent ubiquitination-based cell cycle indicator/late cell cycle marker geminin. Arrest is not due to DNA damage, as the γ-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-β-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid. We conclude that tetraploid primary cells can become senescent without DNA damage and that induction of senescence is critical to tetraploidy arrest.

  5. Influenza HA Subtypes Demonstrate Divergent Phenotypes for Cleavage Activation and pH of Fusion: Implications for Host Range and Adaptation

    PubMed Central

    Galloway, Summer E.; Reed, Mark L.; Russell, Charles J.; Steinhauer, David A.

    2013-01-01

    The influenza A virus (IAV) HA protein must be activated by host cells proteases in order to prime the molecule for fusion. Consequently, the availability of activating proteases and the susceptibility of HA to protease activity represents key factors in facilitating virus infection. As such, understanding the intricacies of HA cleavage by various proteases is necessary to derive insights into the emergence of pandemic viruses. To examine these properties, we generated a panel of HAs that are representative of the 16 HA subtypes that circulate in aquatic birds, as well as HAs representative of the subtypes that have infected the human population over the last century. We examined the susceptibility of the panel of HA proteins to trypsin, as well as human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2). Additionally, we examined the pH at which these HAs mediated membrane fusion, as this property is related to the stability of the HA molecule and influences the capacity of influenza viruses to remain infectious in natural environments. Our results show that cleavage efficiency can vary significantly for individual HAs, depending on the protease, and that some HA subtypes display stringent selectivity for specific proteases as activators of fusion function. Additionally, we found that the pH of fusion varies by 0.7 pH units among the subtypes, and notably, we observed that the pH of fusion for most HAs from human isolates was lower than that observed from avian isolates of the same subtype. Overall, these data provide the first broad-spectrum analysis of cleavage-activation and membrane fusion characteristics for all of the IAV HA subtypes, and also show that there are substantial differences between the subtypes that may influence transmission among hosts and establishment in new species. PMID:23459660

  6. 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.

  7. Activation of ATM by DNA Damaging Agents

    DTIC Science & Technology

    2005-09-01

    serine 139. Pretreatment of cells with NAC partially, peroxide dismutase and glutathione peroxidase - 1 (37). This but significantly, attenuated the... Gy , concentrations of wortmannin (lanes 3-5) for 30 min prior to the addi- 2 h) (Fig. 4A). tion of 1 gm doxorubicin (lanes 2-5) and further incubation...AD Award Number: DAMD17-02- 1 -0318 TITLE: Activation of ATM by DNA Damaging Agents PRINCIPAL INVESTIGATOR: Ebba U. Kurz, Ph.D. Susan P. Lees-Miller

  8. 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.

  9. Activation of 12/23-RSS-dependent RAG cleavage by hSWI/SNF complex in the absence of transcription.

    PubMed

    Du, Hansen; Ishii, Haruhiko; Pazin, Michael J; Sen, Ranjan

    2008-09-05

    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.

  10. 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.

  11. 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.

  12. Exploring the DNA binding mode of transition metal based biologically active compounds.

    PubMed

    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, (1)H 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 (K(b)) 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.

  13. Inhibition of pea chloroplast DNA helicase unwinding and ATPase activities by DNA-interacting ligands.

    PubMed

    Tuteja, N; Phan, T N

    1998-03-27

    DNA helicases unwind the duplex DNA in an ATP dependent manner and thus play an essential role in DNA replication, repair, recombination and transcription. Any DNA-interacting ligand which will modulate DNA helicase activity may interrupt practically all kinds of DNA transactions. There are no studies on the effect of various cytotoxic DNA-interacting ligands on organelle helicases. We have determined the effect of camptothecin, VP-16 (etoposide), ellipticine, genistein, novobiocin, m-AMSA, actinomycin C1, ethidium bromide, daunorubicin and nogalamycin on unwinding and ATPase activities of purified chloroplast DNA helicase from pea (Pisum sativum). Our study has shown that DNA-intercalating ligands actinomycin C1, ethidium bromide, daunorubicin and nogalamycin were inhibiting the DNA unwinding activity with an apparent Ki of 2.9 microM, 3.0 microM, 1.4 microM and 1.0 microM, respectively. These four inhibitors also inhibited the ATPase activity of pea chloroplast DNA helicase. These results indicate that the intercalation of the inhibitors into DNA generates a complex that impedes the translocation of chloroplast DNA helicase, resulting in both inhibition of unwinding activity and ATP hydrolysis. This study would be useful for understanding the mechanism of organelle DNA helicase unwinding and the mechanism by which these DNA-interacting ligands inhibit cellular function.

  14. Proteolytic cleavage of chemerin protein is necessary for activation to the active form, Chem157S, which functions as a signaling molecule in glioblastoma.

    PubMed

    Yamaguchi, Yasuto; Du, Xiao-Yan; Zhao, Lei; Morser, John; Leung, Lawrence L K

    2011-11-11

    Chemerin is a chemoattractant involved in innate and adaptive immunity as well as an adipokine implicated in adipocyte differentiation. Chemerin circulates as an inactive precursor in blood whose bioactivity is closely regulated through proteolytic processing at its C terminus. We developed methodology for production of different recombinant chemerin isoforms (chem163S, chem157S, and chem155A) which allowed us to obtain large quantities of these proteins with purity of >95%. Chem158K was generated from chem163S by plasmin cleavage. Characterization by mass spectrometry and Edman degradation demonstrated that both the N and C termini were correct for each isoform. Ca(2+) mobilization assays showed that the EC(50) values for chem163S and chem158K were 54.2 ± 19.9 nm and 65.2 ± 13.2 nm, respectively, whereas chem157S had a ∼50-fold higher potency with an EC(50) of 1.2 ± 0.7 nm. Chem155A had no agonist activity and weak antagonist activity, causing a 50% reduction of chem157S activity at a molar ratio of 100:1. Similar results were obtained in a chemotaxis assay. Because chem158K is the dominant form in cerebrospinal fluid from patients with glioblastoma (GBM), we examined the significance of chemerin in GBM biology. In silico analysis showed chemerin mRNA was significantly increased in tissue from grade III and IV gliomas. Furthermore, U-87 MG cells, a human GBM line, express the chemerin receptors, chemokine-like receptor 1 and chemokine receptor-like 2, and chem157S triggered Ca(2+) flux. This study emphasized the necessity of appropriate C-terminal proteolytic processing to generate the likely physiologic form of active chemerin, chem157S, and suggested a possible role in malignant GBM.

  15. Photooxidative damage of guanine in DG and DNA by the radicals derived from the alpha cleavage of the electronically excited carbonyl products generated in the thermolysis of alkoxymethyl-substituted dioxetanes and the photolysis of alkoxyacetones.

    PubMed

    Adam, W; Arnold, M A; Saha-Möller, C R

    2001-01-26

    On thermolysis of the methoxy (MeO-TMD), tert-butoxy (tBuO-TMD), and hydroxy (HO-TMD) derivatives of 3,3,4,4-tetramethyl-1,2-dioxetane (TMD) in the presence of dG and calf-thymus DNA, the guanine is oxidized considerably more efficiently than the parent TMD. The same trend in the oxidative reactivity is observed for the photolysis of the corresponding oxy-substituted ketones versus acetone. The oxidative reactivity order in the dioxetane thermolysis, as well as in the ketone photolysis, parallels the ability of the excited ketones to release radicals (determined by spin trapping with DMPO and EPR spectroscopy) upon alpha cleavage (Norrish-type-I reaction). In the presence of molecular oxygen, the carbon-centered radicals are scavenged to produce peroxyl radicals, which are proposed as the reactive species in the oxidation of the guanine in dG and calf-thymus DNA.

  16. Vaccinia topoisomerase and Cre recombinase catalyze direct ligation of activated DNA substrates containing a 3'-para-nitrophenyl phosphate ester.

    PubMed

    Woodfield, G; Cheng, C; Shuman, S; Burgin, A B

    2000-09-01

    DNA topoisomerases and DNA site-specific recombinases are involved in a diverse set of cellular processes but both function by making transient breaks in DNA. Type IB topoisomerases and tyrosine recombinases cleave DNA by transesterification of an active site tyrosine to generate a DNA-3'-phosphotyrosyl-enzyme adduct and a free 5'-hydroxyl (5'-OH). Strand ligation results when the 5'-OH attacks the covalent complex and displaces the enzyme. We describe the synthesis of 3'-phospho-(para-nitrophenyl) oligonucleotides (3'-pNP DNAs), which mimic the natural 3'-phosphotyrosyl intermediate, and demonstrate that such pre-activated strands are substrates for DNA ligation by vaccinia topoisomerase and Cre recombinase. Ligation occurs by direct attack of a 5'-OH strand on the 3'-pNP DNA (i.e., without a covalent protein-DNA intermediate) and generates free para-nitrophenol as a product. The chromogenic DNA substrate allows ligation to be studied in real-time and in the absence of competing cleavage reactions and can be exploited for high-throughput screening of topoisomerase/recombinase inhibitors.

  17. Neutrophil proteinase cathepsin G is proteolytically active on the human platelet glycoprotein Ib-IX receptor: characterization of the cleavage sites within the glycoprotein Ib alpha subunit.

    PubMed Central

    Pidard, D; Renesto, P; Berndt, M C; Rabhi, S; Clemetson, K J; Chignard, M

    1994-01-01

    The proteolytic activity of the neutrophil serine-proteinase cathepsin G (CG) on platelet adherence receptors, the glycoprotein (GP) Ib-IX complex and the integrin alpha IIb beta 3, has been investigated. In the range 50 to 200 nmol/l, CG is a potent platelet agonist which induces shape change, granule exocytosis and aggregation. Investigation of the proteolysis of the receptors' subunits during the course of platelet activation by CG was performed by immunoblot analysis of platelet proteins using a panel of specific antibodies. Exposure of platelets for 3 min at 37 degrees C to CG at a concentration that induces full cell activation resulted in an extensive cleavage of the N-terminal region of the extracellular domain of GPIb alpha, the largest (relative molecular mass, M(r), 143,000) of the three subunits constituting the GPIb-IX complex. In contrast, no detectable proteolytic modification of the two other subunits, GPIb beta and GPIX, was detected. Similarly, we observed that neither of the two subunits of the alpha IIb beta 3 receptor were proteolytically modified by CG. Cleavage of GPIb alpha by CG leaves a remnant of the polypeptide chain with M(r) approx. 106,000 in the plasma membrane, while releasing into the extracellular milieu the N-terminal domain with M(r) in the range 40,000 to 46,000. N-terminal sequencing of the CG-derived fragments of GPIb alpha indicated that the Leu275-Tyr276 peptide bond was the primary cleavage site for this proteinase. Proteolysis of GPIb alpha was already detectable at concentrations of CG as low as 25 nmol/l, while with 200 nmol/l the cleavage was detected as soon as 10 s after exposure of platelets to the proteinase. Comparison of the kinetics and concentration dependency for the proteolysis of GPIb alpha and for the activation of platelets by CG showed that cleavage of the GPIb-IX receptor is an early event that accompanies exocytosis and aggregation. Quantitative evaluation of the conversion of GPIb alpha into its

  18. Simian sarcoma virus-encoded gag-related protein: in vitro cleavage by Friend leukemia virus-associated proteolytic activity.

    PubMed

    Hafenrichter, R; Thiel, H J

    1985-05-01

    The simian sarcoma virus (SSV) encodes a gag-related 65,000-Da protein (SSV p65) which is not processed in SSV nonproducer cells (SSV-NP cells) (H.-J. Thiel, T. J. Matthews, E. M. Broughton, K. J. Weinhold, D. P. Bolognesi, T. Graf, and H. Beug (1981a), Virology 114, 124-131). In order to cleave SSV p65, retroviral particles containing this antigen were incubated with extracts from the heterologous helper virus Friend leukemia virus (FLV). Superinfection of SSV-NP cells by FLV has been previously shown to result in processing of SSV p65 in vivo (H.-J. Thiel, F. Weiland, R. Hafenrichter, T. J. Matthews, and K. J. Weinhold (1982), Virology 123, 229-234). In vitro cleavage was most efficient in the presence of a nonionic detergent (greater than 0.1% Nonidet-P40) and a reducing agent (greater than 5 mM dithiothreitol) at a pH of 7.0. The products, termed SSV p55 (p15, p12, p30), SSV p30, SSV p25 (p15, p12), and SSV p10, were characterized by (1) molecular weight, (2) kinetics experiments, (3) incorporation of different radiolabeled amino acids, and (4) comparison with SSAV structural proteins. Kinetics experiments with two amino acids ([3H]leucine, [35S]cysteine) revealed that initial processing of SSV p65 produced SSV p55 and SSV p10, with subsequent processing of SSV p55 occurring thereafter. In contrast to the Moloney system, the major intermediate p40 (p30, p10) could not be clearly demonstrated. A direct comparison of SSAV p10 and the cleavage product SSV p10 by SDS-PAGE suggests that SSAV pr65gag and SSV p65 differ slightly by molecular weight.

  19. 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.

  20. The protease activity of yeast separase (esp1) is required for anaphase spindle elongation independently of its role in cleavage of cohesin.

    PubMed

    Baskerville, Chris; Segal, Marisa; Reed, Steven I

    2008-04-01

    Separase is a caspase-family protease required for the metaphase-anaphase transition in eukaryotes. In budding yeast, the separase ortholog, Esp1, has been shown to cleave a subunit of cohesin, Mcd1 (Scc1), thereby releasing sister chromatids from cohesion and allowing anaphase. However, whether Esp1 has other substrates required for anaphase has been controversial. Whereas it has been reported that cleavage of Mcd1 is sufficient to trigger anaphase in the absence of Esp1 activation, another study using a temperature-sensitive esp1 mutant concluded that depletion of Mcd1 was not sufficient for anaphase in the absence of Esp1 function. Here we revisit the issue and demonstrate that neither depletion of Mcd1 nor ectopic cleavage of Mcd1 by Tev1 protease is sufficient to support anaphase in an esp1 temperature-sensitive mutant. Furthermore, we demonstrate that the catalytic activity of the Esp1 protease is required for this Mcd1-independent anaphase function. These data suggest that another protein, possibly a spindle-associated protein, is cleaved by Esp1 to allow anaphase. Such a function is consistent with the previous observation that Esp1 localizes to the mitotic spindle during anaphase.

  1. 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

  2. Ataxia telangiectasia mutated activation by transcription- and topoisomerase I-induced DNA double-strand breaks.

    PubMed

    Sordet, Olivier; Redon, Christophe E; Guirouilh-Barbat, Josée; Smith, Susan; Solier, Stéphanie; Douarre, Céline; Conti, Chiara; Nakamura, Asako J; Das, Benu B; Nicolas, Estelle; Kohn, Kurt W; Bonner, William M; Pommier, Yves

    2009-08-01

    Ataxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA damage response (DDR). As neurons have high rates of transcription that require topoisomerase I (TOP1), we investigated whether TOP1 cleavage complexes (TOP1cc)-which are potent transcription-blocking lesions-also produce transcription-dependent DNA double-strand breaks (DSBs) with ATM activation. We show the induction of DSBs and DDR activation in post-mitotic primary neurons and lymphocytes treated with camptothecin, with the induction of nuclear DDR foci containing activated ATM, gamma-H2AX (phosphorylated histone H2AX), activated CHK2 (checkpoint kinase 2), MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53 binding protein 1). The DSB-ATM-DDR pathway was suppressed by inhibiting transcription and gamma-H2AX signals were reduced by RNase H1 transfection, which removes transcription-mediated R-loops. Thus, we propose that Top1cc produce transcription arrests with R-loop formation and generate DSBs that activate ATM in post-mitotic cells.

  3. Synthesis of Aryl-1,2,4,5-tetrazinane-3-thiones, in vitro DNA binding studies, nuclease activity and its antimicrobial activity

    NASA Astrophysics Data System (ADS)

    Tabassum, Sartaj; Parveen, Mehtab; Ali, Akhtar; Alam, Mahboob; Ahmad, Anis; Khan, Asad U.; Khan, Rais Ahmad

    2012-08-01

    A series of small molecules with a tetrazine scaffold (5-8) was designed and synthesized as metal free DNA cleaving agent. These compounds (5-8) were characterized by using various spectroscopic (via; IR, 1H, 13C NMR and ESI-MS) and analytical methods. The interaction studies of (5-8) with CT DNA were carried out by using various biophysical techniques, which showed high binding affinity of 7 and 8 towards CT DNA. Gel electrophoresis pattern demonstrated that the compounds are efficient artificial nuclease and prefers minor groove binding site. The mechanistic pattern showed that compounds follow hydrolytic pathway for DNA cleavage. In vitro antibacterial screening of these tetrazine-thiones (5-8) gives remarkable results both against gram-positive and gram-negative strains. These compounds also possess moderate antifungal activity when compared with the standard drugs.

  4. Characterization of cleavage intermediate and star sites of RM.Tth111II

    PubMed Central

    Zhu, Zhenyu; Guan, Shengxi; Robinson, Derek; Fezzazi, Hanna El; Quimby, Aine; Xu, Shuang-yong

    2014-01-01

    Tth111II is a thermostable Type IIGS restriction enzyme that recognizes DNA sites CAARCA (R = A or G) and cleaves downstream at N11/N9. Here, the tth111IIRM gene was cloned and expressed in E. coli, and Tth111II was purified. The purified enzyme contains internally-bound S-adenosylmethionine (SAM). When the internal SAM was removed, the endonuclease activity was stimulated by adding SAM or its analog sinefungin. The cleavage intermediate is mostly top-strand nicked DNA on a single-site plasmid. Addition of duplex oligos with a cognate site stimulates cleavage activity of the one-site substrate. Tth111II cleaves a two-site plasmid DNA with equal efficiency regardless of site orientation. We propose the top-strand nicking is carried out by a Tth111II monomer and bottom-strand cleavage is carried out by a transient dimer. Tth111II methylates cleavage product-like duplex oligos CAAACAN9, but the modification rate is estimated to be much slower than the top-strand nicking rate. We cloned and sequenced a number of Tth111II star sites which are 1-bp different from the cognate sites. A biochemical pathway is proposed for the restriction and methylation activities of Tth111II. PMID:24452415

  5. Sequence dependent modulating effect of camptothecin on the DNA-cleaving activity of the calf thymus type I topoisomerase.

    PubMed Central

    Gromova, I I; Buchman, V L; Abagyan, R A; Ulyanov, A V; Bronstein, I B

    1990-01-01

    High-resolution mapping of topol cleavages in the regions of human DNA including the oncogene c-Ha-ras and p53, has revealed three kinds of topol cleavage sites: cleavage sites not affected by camptothecin; cleavage sites reinforced only in the presence of camptothecin, and cleavage sites which weaken in the presence of camptothecin. Statistical analysis of sequences revealed certain nucleotide or dinucleotide preferences for three groups studied. The preferences in camptothecin-reduced sites predominate upstream from the cleavage point, whereas in camptothecin-induced sites the situation is reversed. The influence of camptothecin on cleavage sites induced by two molecular forms of topol has been also studied. Images PMID:2155407

  6. Evaluation of DNA, BSA binding, and antimicrobial activity of new synthesized neodymium complex containing 29-dimethyl 110-phenanthroline.

    PubMed

    Moradi, Zohreh; Khorasani-Motlagh, Mozhgan; Rezvani, Ali Reza; Noroozifar, Meissam

    2017-02-21

    In order to evaluate biological potential of a novel synthesized complex [Nd(dmp)2Cl3.OH2] where dmp is 29-dimethyl 110-phenanthroline, the DNA-binding, cleavage, BSA binding, and antimicrobial activity properties of the complex are investigated by multispectroscopic techniques study in physiological buffer (pH 7.2).The intrinsic binding constant (Kb) for interaction of Nd(III) complex and FS-DNA is calculated by UV-Vis (Kb = 2.7 ± 0.07 × 10(5)) and fluorescence spectroscopy (Kb = 1.13 ± 0.03 × 10(5)). The Stern-Volmer constant (KSV), thermodynamic parameters including free energy change (ΔG°), enthalpy change (∆H°), and entropy change (∆S°), are calculated by fluorescent data and Vant' Hoff equation. The experimental results show that the complex can bind to FS-DNA and the major binding mode is groove binding. Meanwhile, the interaction of Nd(III) complex with protein, bovine serum albumin (BSA), has also been studied by using absorption and emission spectroscopic tools. The experimental results show that the complex exhibits good binding propensity to BSA. The positive ΔH° and ∆S° values indicate that the hydrophobic interaction is main force in the binding of the Nd(III) complex to BSA, and the complex can quench the intrinsic fluorescence of BSA remarkably through a static quenching process. Also, DNA cleavage was investigated by agarose gel electrophoresis that according to the results cleavage of DNA increased with increasing of concentration of the complex. Antimicrobial screening test gives good results in the presence of Nd(III) complex system.

  7. 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.

  8. Dipeptidyl peptidase IV-activated prodrugs of anti-varicella zoster virus bicyclic nucleoside analogues containing different self-cleavage spacer systems.

    PubMed

    Diez-Torrubia, Alberto; Cabrera, Silvia; De Meester, Ingrid; Camarasa, María-José; Balzarini, Jan; Velázquez, Sonsoles

    2012-09-01

    A new type of double prodrug of the antiviral family of bicyclic nucleoside analogues (BCNA) bearing cyclization self-cleavage spacers between the Val-Pro dipeptide sequence as well as the parent compound were synthesized and evaluated with regard to activation by the DPPIV/CD26 enzyme and for their stability in human and bovine serum. In buffer solution, carbamate and ester prodrugs were found to be chemically stable. Most prodrugs containing a dipeptidyl linker efficiently converted into the BCNA parent drug. In contrast, the Val-Pro alkyldiamino prodrugs converted predominantly into their alkyldiamino prodrug intermediates in the presence of CD26 and human serum. A marked increase in water solubility was observed for all prodrugs. In contrast to the parent compound, a tetrapeptide prodrug containing the Val-Val dipeptide as a self-cleavage spacer released substantial amounts of the BCNA parent drug at the basolateral side of Caco-2 cell cultures and exhibited 15- to 20-fold increased bioavailability in mice relative to the poorly bioavailable parent compound.

  9. Natural and engineered nicking endonucleases—from cleavage mechanism to engineering of strand-specificity

    PubMed Central

    Chan, Siu-Hong; Stoddard, Barry L.; Xu, Shuang-yong

    2011-01-01

    Restriction endonucleases (REases) are highly specific DNA scissors that have facilitated the development of modern molecular biology. Intensive studies of double strand (ds) cleavage activity of Type IIP REases, which recognize 4–8 bp palindromic sequences, have revealed a variety of mechanisms of molecular recognition and catalysis. Less well-studied are REases which cleave only one of the strands of dsDNA, creating a nick instead of a ds break. Naturally occurring nicking endonucleases (NEases) range from frequent cutters such as Nt.CviPII (^CCD; ^ denotes the cleavage site) to rare-cutting homing endonucleases (HEases) such as I-HmuI. In addition to these bona fida NEases, individual subunits of some heterodimeric Type IIS REases have recently been shown to be natural NEases. The discovery and characterization of more REases that recognize asymmetric sequences, particularly Types IIS and IIA REases, has revealed recognition and cleavage mechanisms drastically different from the canonical Type IIP mechanisms, and has allowed researchers to engineer highly strand-specific NEases. Monomeric LAGLIDADG HEases use two separate catalytic sites for cleavage. Exploitation of this characteristic has also resulted in useful nicking HEases. This review aims at providing an overview of the cleavage mechanisms of Types IIS and IIA REases and LAGLIDADG HEases, the engineering of their nicking variants, and the applications of NEases and nicking HEases. PMID:20805246

  10. 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

  11. DNA evidence uncompromised by active oxygen.

    PubMed

    Castelló, Ana; Francés, Francesc; Verdú, Fernando

    2010-03-05

    Currently, forensic sciences can make use of the potential of instrumental analysis techniques to obtain information from the smallest, even invisible, samples. However, as laboratory techniques improve, so too should the procedures applied in the search for and initial testing of clues in order to be equally effective. This requires continuous revision so that those procedures may resolve the problems that samples present. As far as bloodstains are concerned, there are methods available that are recognized as being both highly sensitive and effective. Nevertheless, the marketing of new cleaning products, those that contain active oxygen, has raised doubts about the ability of those procedures to detect blood. It has been shown that stains washed with these detergents (and still visible) invalidated both the presumptive test (reduced phenolphthalein, luminol, and Bluestar) and that applied for determining human hemoglobin. These findings have caused considerable concern both within the forensic and scientific community, and among the general public, so obliging us to seek solutions. In this work, the effect of these new cleaning products on DNA analyses is studied. The results, encouraging ones, show that these detergents, despite invalidating all other tests, do not hinder the extraction, or the subsequent analysis, of DNA.

  12. Single-molecule study of DNA polymerization activity of HIV-1 reverse transcriptase on DNA templates.

    PubMed

    Kim, Sangjin; Schroeder, Charles M; Xie, X Sunney

    2010-02-05

    HIV-1 RT (human immunodeficiency virus-1 reverse transcriptase) is a multifunctional polymerase responsible for reverse transcription of the HIV genome, including DNA replication on both RNA and DNA templates. During reverse transcription in vivo, HIV-1 RT replicates through various secondary structures on RNA and single-stranded DNA (ssDNA) templates without the need for a nucleic acid unwinding protein, such as a helicase. In order to understand the mechanism of polymerization through secondary structures, we investigated the DNA polymerization activity of HIV-1 RT on long ssDNA templates using a multiplexed single-molecule DNA flow-stretching assay. We observed that HIV-1 RT performs fast primer extension DNA synthesis on single-stranded regions of DNA (18.7 nt/s) and switches its activity to slow strand displacement synthesis at DNA hairpin locations (2.3 nt/s). Furthermore, we found that the rate of strand displacement synthesis is dependent on the GC content in hairpin stems and template stretching force. This indicates that the strand displacement synthesis occurs through a mechanism that is neither completely active nor passive: that is, the opening of the DNA hairpin is driven by a combination of free energy released during dNTP (deoxyribonucleotide triphosphate) hydrolysis and thermal fraying of base pairs. Our experimental observations provide new insight into the interchanging modes of DNA replication by HIV-1 RT on long ssDNA templates.

  13. Role of the Arabidopsis DNA glycosylase/lyase ROS1 in active DNA demethylation

    PubMed Central

    Agius, Fernanda; Kapoor, Avnish; Zhu, Jian-Kang

    2006-01-01

    DNA methylation is a stable epigenetic mark for transcriptional gene silencing in diverse organisms including plants and many animals. In contrast to the well characterized mechanism of DNA methylation by methyltransferases, the mechanisms and function of active DNA demethylation have been controversial. Genetic evidence suggested that the DNA glycosylase domain-containing protein ROS1 of Arabidopsis is a putative DNA demethylase, because loss-of-function ros1 mutations cause DNA hypermethylation and enhance transcriptional gene silencing. We report here the biochemical characterization of ROS1 and the effect of its overexpression on the DNA methylation of target genes. Our data suggest that the DNA glycosylase activity of ROS1 removes 5-methylcytosine from the DNA backbone and then its lyase activity cleaves the DNA backbone at the site of 5-methylcytosine removal by successive β- and δ-elimination reactions. Overexpression of ROS1 in transgenic plants led to a reduced level of cytosine methylation and increased expression of a target gene. These results demonstrate that ROS1 is a 5-methylcytosine DNA glycosylase/lyase important for active DNA demethylation in Arabidopsis. PMID:16864782

  14. High-Throughput Genotyping of Green Algal Mutants Reveals Random Distribution of Mutagenic Insertion Sites and Endonucleolytic Cleavage of Transforming DNA.

    PubMed

    Zhang, Ru; Patena, Weronika; Armbruster, Ute; Gang, Spencer S; Blum, Sean R; Jonikas, Martin C

    2014-04-01

    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.

  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. Multiple DNA binding activities of the novel site-specific recombinase, Piv, from Moraxella lacunata.

    PubMed

    Tobiason, D M; Lenich, A G; Glasgow, A C

    1999-04-02

    The recombinase, Piv, is essential for site-specific DNA inversion of the type IV pilin DNA segment in Moraxella lacunata and Moraxella bovis. Piv shows significant homology with the transposases of the IS110/IS492 family of insertion elements, but, surprisingly, Piv contains none of the conserved amino acid motifs of the lambda Int or Hin/Res families of site-specific recombinases. Therefore, Piv may mediate site-specific recombination by a novel mechanism. To begin to determine how Piv may assemble a synaptic nucleoprotein structure for DNA cleavage and strand exchange, we have characterized the interaction of Piv with the DNA inversion region of M. lacunata. Gel shift and nuclease/chemical protection assays, competition and dissociation rate analyses, and cooperativity studies indicate that Piv binds two distinct recognition sequences. One recognition sequence, found at multiple sites within and outside of the invertible segment, is bound by Piv protomers with high affinity. The second recognition sequence is located at the recombination cross-over sites at the ends of the invertible element; Piv interacts with this sequence as an oligomer with apparent low affinity. A model is proposed for the role of the different Piv binding sites of the M. lacunata inversion region in the formation of an active synaptosome.

  17. Hybrid Selection of Small RNAs by Using Simian Virus 40 DNA: Evidence that the Simian Virus 40-Associated Small RNA Is Synthesized by Specific Cleavage from Large Viral Transcripts

    PubMed Central

    Alwine, James C.

    1982-01-01

    The simian virus 40 (SV40)-associated small RNA (SAS-RNA), approximately 64 nucleotides, is virally encoded within a region of the viral late (+) DNA strand which encodes no known protein. The SAS-RNA arises in abundance late in SV40 lytic infection. Previous data indicate that the synthesis of the SAS-RNA may be under the control of the normal late viral promoter; i.e., inhibition of transcription from the late promoter results in cessation of SAS-RNA synthesis. The synthesis of SAS-RNA was examined to determine whether the SAS-RNA is the product of cleavage from noncoding regions of nuclear late transcripts or an independent transcription product like 5S RNA, or the adenovirus VA-RNAs. The data described below suggest that SAS-RNA is cleaved from large late transcripts. In vitro transcription of DNA fragments containing the SAS-RNA coding region yielded no SAS-RNA synthesis; this result was supported by DNA sequence analysis, which indicated no promoter-like regions either within or flanking the SAS-RNA coding region. In support of a cleavage mechanism, the SAS-RNA has a 3′-phosphate end, an occurrence which is indicative of nuclease cleavage. In addition, 5′-end labeling of the SAS-RNA was possible only after calf alkaline phosphatase treatment; this indicates that the SAS-RNA is not capped. Hybrid selection analysis was used to demonstrate that separation of the SAS-RNA coding region from the normal late promoter resulted in elimination of SAS-RNA synthesis. This was demonstrated in SV40-transformed cells in which integration of a single copy of SV40 breaks the continuity of the late coding region, so that the SAS-RNA coding region is physically separated from the normal late promoter. The lack of SAS-RNA synthesis indicates that the SAS-RNA coding region cannot function as a primary transcription unit. The same result and conclusion were obtained by using a permissive cell line transformed by SV40 (COS-1 cells); here it was found that the integrated SAS

  18. 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

  19. Design, assembly, and activity of antisense DNA nanostructures.

    PubMed

    Keum, Jung-Won; Ahn, Jin-Ho; Bermudez, Harry

    2011-12-16

    Discrete DNA nanostructures allow simultaneous features not possible with traditional DNA forms: encapsulation of cargo, display of multiple ligands, and resistance to enzymatic digestion. These properties suggested using DNA nanostructures as a delivery platform. Here, DNA pyramids displaying antisense motifs are shown to be able to specifically degrade mRNA and inhibit protein expression in vitro, and they show improved cell uptake and gene silencing when compared to linear DNA. Furthermore, the activity of these pyramids can be regulated by the introduction of an appropriate complementary strand. These results highlight the versatility of DNA nanostructures as functional devices.

  20. A new specific DNA endonuclease activity in yeast mitochondria.

    PubMed

    Sargueil, B; Delahodde, A; Hatat, D; Tian, G L; Lazowska, J; Jacq, C

    1991-02-01

    Two group I intron-encoded proteins from the yeast mitochondrial genome have already been shown to have a specific DNA endonuclease activity. This activity mediates intron insertion by cleaving the DNA sequence corresponding to the splice junction of an intronless strain. We have discovered in mitochondrial extracts from the yeast strain 777-3A a new DNA endonuclease activity which cleaves the fused exon A3-exon A4 junction sequence of the CO XI gene.

  1. SINGLE-MOLECULE STUDY OF DNA POLYMERIZATION ACTIVITY OF HIV-1 REVERSE TRANSCRIPTASE ON DNA TEMPLATES

    PubMed Central

    Kim, Sangjin; Schroeder, Charles M.; Xie, X. Sunney

    2009-01-01

    Human Immunodeficiency Virus-1 reverse transcriptase (HIV-1 RT) is a multifunctional polymerase responsible for reverse transcription of the HIV genome, including DNA replication on both RNA and DNA templates. During reverse transcription in vivo, HIV-1 RT replicates through various secondary structures on RNA and single-stranded DNA templates without the need for a nucleic acid unwinding protein, such as a helicase. In order to understand the mechanism of polymerization through secondary structures, we investigated the DNA polymerization activity of HIV-1 RT on long single-stranded DNA templates using a multiplexed single-molecule DNA flow-stretching assay. We observed that HIV-1 RT performs fast primer extension DNA synthesis on single-stranded regions of DNA (18.7 nt/s) and switches its activity to slow strand displacement synthesis at DNA hairpin locations (2.3 nt/s). Furthermore, we found that the rate of strand displacement synthesis is dependent on the GC content in hairpin stems and template stretching force. This indicates that the strand displacement synthesis occurs through a mechanism that is neither completely active nor passive, i.e. the opening of the DNA hairpin is driven by a combination of free energy released during dNTP hydrolysis and thermal fraying of base pairs. Our experimental observations provide new insight into the interchanging modes of DNA replication by HIV-1 RT on long single-stranded DNA templates. PMID:19968999

  2. Potent inhibition of DNA unwinding and ATPase activities of pea DNA helicase 45 by DNA-binding agents.

    PubMed

    Pham, Xuan Hoi; Tuteja, Narendra

    2002-06-07

    Pea DNA helicase 45 (PDH45) is an ATP-dependent DNA unwinding enzyme, with intrinsic DNA-dependent ATPase activity [Plant J. 24 (2000) 219]. We have determined the effect of various DNA-binding agents, such as daunorubicin, ethidium bromide, ellipticine, cisplatin, nogalamycin, actinomycin C1, and camptothecin on the DNA unwinding and ATPase activities of the plant nuclear DNA helicase PDH45. The results show that all the agents except actinomycin C1, and camptothecin inhibited the helicase (apparent K(i) values ranging from 1.5 to 7.0 microM) and ATPase (apparent K(i) values ranging from 2.5 to 11.9 microM) activities. This is the first study to show the effect of various DNA-binding agents on the plant nuclear helicase and also first to demonstrate inhibition of any helicase by cisplatin. Another striking finding that the actinomycin C1 and ellipticine act differentially on PDH45 as compared to pea chloroplast helicase suggests that the mechanism of DNA unwinding could be different in nucleus and chloroplast. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of PDH45, resulting in both the inhibitions of unwinding activity and ATP hydrolysis. This study would be useful to obtain a better understanding of the mechanism of plant nuclear DNA helicase unwinding and the mechanism by which these agents can disturb genome integrity.

  3. 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

  4. Unusual DNA packaging characteristics in endoreduplicated Caenorhabditis elegans oocytes defined by in vivo accessibility to an endogenous nuclease activity

    PubMed Central

    2013-01-01

    Background Germ cells in animals are highly specialized to preserve the genome. A distinct set of chromatin structures must be properly established in germ cells to maintain cell fate and genome integrity. We describe DNA-surface interactions in activated Caenorhabditis elegans oocytes that are revealed through the activity of an endogenous nuclease ('endocleavage’). Results Our analysis began with an unexpected observation that a majority (>50%) of DNA from ovulated but unfertilized C. elegans oocytes can be recovered in fragments of approximately 500 base pairs or shorter, cleaved at regular intervals (10 to 11 nt) along the DNA helix. In some areas of the genome, DNA cleavage patterns in these endoreduplicated oocytes appear consistent from cell-to-cell, indicating coherent rotational positioning of the DNA in chromatin. Particularly striking in this analysis are arrays of sensitive sites with a periodicity of approximately 10 bp that persist for several hundred base pairs of genomic DNA, longer than a single nucleosome core. Genomic regions with a strong bias toward a 10-nt periodic occurrence of A(n)/T(n) (so-called PATC regions) appear to exhibit a high degree of rotational constraint in endocleavage phasing, with a strong tendency for the periodic A(n)/T(n) sites to remain on the face of the helix protected from nuclease digestion. Conclusion The present analysis provides evidence for an unusual structure in C. elegans oocytes in which genomic DNA and associated protein structures are coherently linked. PMID:24279402

  5. Exploring the DNA binding/cleavage, cellular accumulation and topoisomerase inhibition of 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinone Mannich bases and their platinum(II) complexes.

    PubMed

    Neves, Amanda P; Pereira, Michelle X G; Peterson, Erica J; Kipping, Ralph; Vargas, Maria D; Silva-Jr, Floriano P; Carneiro, J Walkimar M; Farrell, Nicholas P

    2013-02-01

    Several chlorido and amino Pt(2+) complexes of 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinone Mannich bases HL exhibiting moderate to high cytotoxicity against cancer cell lines were studied in order to investigate their modes of DNA binding, in vitro DNA strand breaks, mechanism of topoisomerase (Topo I) inhibition and cellular accumulation. DNA model base studies have shown that complex 1a [Pt(HL1)Cl(2)] was capable of binding covalently to 9-ethylguanine (9-EtG) and 5'-GMP. (1)H NMR and mass spectrometry studies have shown that both chlorides were substituted by 9-EtG ligands, whereas 5'-GMP was able to replace only one chlorido ligand, due to steric hindrance. The chlorido Pt(2+) complexes [Pt(HL)Cl(2)] highly accumulate in prostate (PC-3) and melanoma (MDA-MB-435) cell lines, being able to induce DNA strand breaks in vitro and inhibit Topo I by a catalytic mode. On the other hand, the free 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinones HL and the amino Pt(2+) complexes [Pt(L(-))(NH(3))(2)]NO(3) neither cause DNA strand breakage nor exhibit strong DNA interaction, nevertheless the latter were also found to be catalytic inhibitors of Topo I at 100μM. Thus, coordination of the Mannich bases HL to the "PtCl(2)" fragment substantially affects the chemical and biophysical properties of the pro-ligands, leading to an improvement of their DNA binding properties and generating compounds that cleave DNA and catalytically inhibit Topo I. Finally, the high cytotoxicity exhibited by the free (uncomplexed) 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinones might be associated with their decomposition in solution, which is not observed for the Pt(2+) complexes.

  6. Electronic Activation of a DNA Nanodevice Using a Multilayer Nanofilm.

    PubMed

    Jeong, Hyejoong; Ranallo, Simona; Rossetti, Marianna; Heo, Jiwoong; Shin, Jooseok; Park, Kwangyong; Ricci, Francesco; Hong, Jinkee

    2016-10-01

    A method to control activation of a DNA nanodevice by supplying a complementary DNA (cDNA) strand from an electro-responsive nanoplatform is reported. To develop functional nanoplatform, hexalayer nanofilm is precisely designed by layer-by-layer assembly technique based on electrostatic interaction with four kinds of materials: Hydrolyzed poly(β-amino ester) can help cDNA release from the film. A cDNA is used as a key building block to activate DNA nanodevice. Reduced graphene oxides (rGOs) and the conductive polymer provide conductivity. In particular, rGOs efficiently incorporate a cDNA in the film via several interactions and act as a barrier. Depending on the types of applied electronic stimuli (reductive and oxidative potentials), a cDNA released from the electrode can quantitatively control the activation of DNA nanodevice. From this report, a new system is successfully demonstrated to precisely control DNA release on demand. By applying more advanced form of DNA-based nanodevices into multilayer system, the electro-responsive nanoplatform will expand the availability of DNA nanotechnology allowing its improved application in areas such as diagnosis, biosensing, bioimaging, and drug delivery.

  7. 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

  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. 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

  10. DNA replication origin activation in space and time.

    PubMed

    Fragkos, Michalis; Ganier, Olivier; Coulombe, Philippe; Méchali, Marcel

    2015-06-01

    DNA replication begins with the assembly of pre-replication complexes (pre-RCs) at thousands of DNA replication origins during the G1 phase of the cell cycle. At the G1-S-phase transition, pre-RCs are converted into pre-initiation complexes, in which the replicative helicase is activated, leading to DNA unwinding and initiation of DNA synthesis. However, only a subset of origins are activated during any S phase. Recent insights into the mechanisms underlying this choice reveal how flexibility in origin usage and temporal activation are linked to chromosome structure and organization, cell growth and differentiation, and replication stress.

  11. A strong strand displacement activity of thermostable DNA polymerase markedly improves the results of DNA amplification.

    PubMed

    Ignatov, Konstantin B; Barsova, Ekaterina V; Fradkov, Arkady F; Blagodatskikh, Konstantin A; Kramarova, Tatiana V; Kramarov, Vladimir M

    2014-08-01

    The sensitivity and robustness of various DNA detection and amplification techniques are to a large extent determined by the properties of the DNA polymerase used. We have compared the performance of conventional Taq and Bst DNA polymerases to a novel Taq DNA polymerase mutant (SD DNA polymerase), which has a strong strand displacement activity, in PCR (including amplification of GC-rich and complex secondary structure templates), long-range PCR (LR PCR), loop-mediated amplification (LAMP), and polymerase chain displacement reaction (PCDR). Our results demonstrate that the strand displacement activity of SD DNA polymerase, in combination with the robust polymerase activity, provides a notable improvement in the sensitivity and efficiency of all these methods.

  12. Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

    PubMed

    Thomas, Marshall P; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron J; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-06-01

    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. In this study, 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 targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation 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. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps 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.

  13. 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.

  14. 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.

  15. Stimulation of the DNA unwinding activity of human DNA helicase II/Ku by phosphorylation.

    PubMed

    Ochem, Alexander E; Rechreche, Hocine; Skopac, Doris; Falaschi, Arturo

    2008-02-01

    The Ku autoantigen is a heterodimeric protein of 70- and 83-kDa subunits, endowed with duplex DNA end-binding capacity and DNA helicase activity (Human DNA Helicase II, HDH II). HDH II/Ku is well established as the DNA binding component, the regulatory subunit as well as a substrate for the DNA-dependent protein kinase DNA-PK, a complex involved in the repair of DNA double-strand breaks and in V(D)J recombination in eukaryotes. The effects of phosphorylation by this kinase on the helicase activity of Escherichia coli-produced HDH II/Ku were studied. The rate of DNA unwinding by recombinant HDH II/Ku heterodimer is stimulated at least fivefold upon phosphorylation by DNA-PK(cs). This stimulation is due to the effective transfer of phosphate residues to the helicase rather than the mere presence of the complex. In vitro dephosphorylation of HeLa cellular HDH II/Ku caused a significant decrease in the DNA helicase activity of this enzyme.

  16. The quinobenzoxazines: relationship between DNA binding and biological activity.

    PubMed

    Kwok, Y; Sun, D; Clement, J J; Hurley, L H

    1999-10-01

    The quinobenzoxazine compounds, derived from antibacterial quinolones, is active in vitro and in vivo against murine and human tumors. In this contribution, we show that the relative DNA binding affinity of the quinobenzoxazine compounds correlates with their cytotoxicity, their ability to inhibit gyrase-DNA complex formation, and the decatenation of kinetoplast DNA by human topoisomerase II. DNA binding studies with the descarboxy-A-62176 analogue indicate that the beta-keto acid moiety of the quinobenzoxazine compounds plays an important role in their interaction with DNA.

  17. TstI, a Type II restriction–modification protein with DNA recognition, cleavage and methylation functions in a single polypeptide

    PubMed Central

    Smith, Rachel M.; Pernstich, Christian; Halford, Stephen E.

    2014-01-01

    Type II restriction–modification systems cleave and methylate DNA at specific sequences. However, the Type IIB systems look more like Type I than conventional Type II schemes as they employ the same protein for both restriction and modification and for DNA recognition. Several Type IIB proteins, including the archetype BcgI, are assemblies of two polypeptides: one with endonuclease and methyltransferase roles, another for DNA recognition. Conversely, some IIB proteins express all three functions from separate segments of a single polypeptide. This study analysed one such single-chain protein, TstI. Comparison with BcgI showed that the one- and the two-polypeptide systems differ markedly. Unlike the heterologous assembly of BcgI, TstI forms a homotetramer. The tetramer bridges two recognition sites before eventually cutting the DNA in both strands on both sides of the sites, but at each site the first double-strand break is made long before the second. In contrast, BcgI cuts all eight target bonds at two sites in a single step. TstI also differs from BcgI in either methylating or cleaving unmodified sites at similar rates. The site may thus be modified before TstI can make the second double-strand break. TstI MTase acts best at hemi-methylated sites. PMID:24634443

  18. 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).

  19. Nuclease activity and interaction studies of unsymmetrical binuclear Ni(II) complexes with CT-DNA and BSA.

    PubMed

    Poornima, S; Gunasekaran, K; Kandaswamy, M

    2015-10-07

    New unsymmetrical binuclear nickel(II) complexes [Ni2L(1-5)] (ClO4)2 (1-5) were synthesized by using [NiL] [(3-((9E)-(2-((E)-(3-formyl-2-olato-5-methylbenzylideneamino)methyl) phenylimino)methyl)-3-formyl-5-methyl-2-olato)nickel(II)] with various diamines like 1,2-diaminoethane (L(1)), 1,3-diaminopropane (L(2)), 1,4-diaminobutane (L(3)), 1,2-diaminobenzene (L(4)) and 1,8-diaminonaphthalene (L(5)) and characterized by elemental analysis and spectroscopic methods. The molecular structure of binuclear nickel(II) complex 1 is determined by a single crystal X-ray diffraction method. Cyclic voltammograms of binuclear Ni(II) complexes exhibit two quasi-reversible reduction waves in the cathodic region and two oxidation waves in the anodic region. DNA binding, protein binding and DNA cleavage studies were investigated. The interactions of complexes (1-5) with calf thymus DNA were studied by spectroscopic techniques, including absorption and fluorescence methods. The binding affinities of complexes (1-5) with CT-DNA and nuclease activities are in the following order: 5> 4>3 >2>1 . Binuclear Ni(II) complex 1 cleaved the plasmid DNA by a hydrolytic pathway. The hydrolytic cleavage of DNA by the complexes is supported by evidence from free radical quenching and T4 ligase ligation. Binuclear Ni(II) complexes (1-5) displayed significant protein (bovine serum albumin) interactions. The experimental results showed that the interaction between binuclear Ni(II) complexes and BSA was mainly a static quenching process.

  20. 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.

  1. Heat Shock Protein 90 Modulates Lipid Homeostasis by Regulating the Stability and Function of Sterol Regulatory Element-binding Protein (SREBP) and SREBP Cleavage-activating Protein.

    PubMed

    Kuan, Yen-Chou; Hashidume, Tsutomu; Shibata, Takahiro; Uchida, Koji; Shimizu, Makoto; Inoue, Jun; Sato, Ryuichiro

    2017-02-17

    Sterol regulatory element-binding proteins (SREBPs) are the key transcription factors that modulate lipid biosynthesis. SREBPs are synthesized as endoplasmic reticulum-bound precursors that require proteolytic activation in the Golgi apparatus. The stability and maturation of precursor SREBPs depend on their binding to SREBP cleavage-activating protein (SCAP), which escorts the SCAP-SREBP complex to the Golgi apparatus. In this study, we identified heat shock protein (HSP) 90 as a novel SREBP regulator that binds to and stabilizes SCAP-SREBP. In HepG2 cells, HSP90 inhibition led to proteasome-dependent degradation of SCAP-SREBP, which resulted in the down-regulation of SREBP target genes and the reduction in intracellular triglyceride and cholesterol levels. We also demonstrated in vivo that HSP90 inhibition decreased SCAP-SREBP protein, down-regulated SREBP target genes, and reduced lipids levels in mouse livers. We propose that HSP90 plays an indispensable role in SREBP regulation by stabilizing the SCAP-SREBP complex, facilitating the activation of SREBP to maintain lipids homeostasis.

  2. Comparative assessments of CRISPR-Cas nucleases' cleavage efficiency in planta.

    PubMed

    Johnson, Ross A; Gurevich, Vyacheslav; Filler, Shdema; Samach, Aviva; Levy, Avraham A

    2015-01-01

    Custom-designed nucleases can enable precise plant genome editing by catalyzing DNA-breakage at specific targets to stimulate targeted mutagenesis or gene replacement. The CRISPR-Cas system, with its target-specifying RNA molecule to direct the Cas9 nuclease, is a recent addition to existing nucleases that bind and cleave the target through linked protein domains (e.g. TALENs and zinc-finger nucleases). We have conducted a comparative study of these different types of custom-designed nucleases and we have assessed various components of the CRISPR-Cas system. For this purpose, we have adapted our previously reported assay for cleavage-dependent luciferase gene correction in Nicotiana benthamiana leaves (Johnson et al. in Plant Mol Biol 82(3):207-221, 2013). We found that cleavage by CRISPR-Cas was more efficient than cleavage of the same target by TALENs. We also compared the cleavage efficiency of the Streptococcus pyogenes Cas9 protein based on expression using three different Cas9 gene variants. We found significant differences in cleavage efficiency between these variants, with human and Arabidopsis thaliana codon-optimized genes having the highest cleavage efficiencies. We compared the activity of 12 de novo-designed single synthetic guide RNA (sgRNA) constructs, and found their cleavage efficiency varied drastically when using the same Cas9 nuclease. Finally, we show that, for one of the targets tested with our assay, we could induce a germinally-transmitted deletion in a repeat array in A. thaliana. This work emphasizes the efficiency of the CRISPR-Cas system in plants. It also shows that further work is needed to be able to predict the optimal design of sgRNAs or Cas9 variants.

  3. Heterogeneity of mammalian DNA ligase detected on activity and DNA sequencing gels.

    PubMed Central

    Mezzina, M; Sarasin, A; Politi, N; Bertazzoni, U

    1984-01-01

    A new method to detect DNA ligase activity in situ after NaDodSO4 polyacrylamide gel electrophoresis has been developed. After renaturation of active polypeptides the ligase reaction occurs in situ by incubating the intact gel in the presence of Mg++ and ATP. Further treatment with alkaline phosphatase removes the unligated 5'-32P-end of oligo (dT) used as a substrate and active polypeptides having ligase activity are identified by autoradiography. Analysis on DNA sequencing gels of the oligo (dT) reaction products present in the activity bands ensures that the radioactive material detected in activity gels or in standard in vitro ligase assays corresponds unambiguously to a ligase activity. Using these methods, we have analysed the purified phage T4 DNA ligase, and the activities present in crude extracts and in purified fractions from monkey kidney (CV1-P) cells. The purified T4 enzyme yields one or two active peptides with Mr values of 60,000 and 70,000. Crude extracts from CV1-P cells contain several polypeptides having DNA ligase activity. Partial purification of these extracts shows that DNA ligase I isolated from hydroxylapatite column is enriched in polypeptides with Mr 200,000, 150,000 and 120,000, while DNA ligase II is enriched in those with Mr 60,000 and 70,000. Images PMID:6377238

  4. DC-159a Shows Inhibitory Activity against DNA Gyrases of Mycobacterium leprae

    PubMed Central

    Yamaguchi, Tomoyuki; Yokoyama, Kazumasa; Nakajima, Chie

    2016-01-01

    Background Fluoroquinolones are a class of antibacterial agents used for leprosy treatment. Some new fluoroquinolones have been attracting interest due to their remarkable potency that is reportedly better than that of ofloxacin, the fluoroquinolone currently recommended for treatment of leprosy. For example, DC-159a, a recently developed 8-methoxy fluoroquinolone, has been found to be highly potent against various bacterial species. Nonetheless, the efficacy of DC-159a against Mycobacterium leprae is yet to be examined. Methodology/Principal Findings To gather data that can support highly effective fluoroquinolones as candidates for new remedies for leprosy treatment, we conducted in vitro assays to assess and compare the inhibitory activities of DC-159a and two fluoroquinolones that are already known to be more effective against M. leprae than ofloxacin. The fluoroquinolone-inhibited DNA supercoiling assay using recombinant DNA gyrases of wild type and ofloxacin-resistant M. leprae revealed that inhibitory activities of DC-159a and sitafloxacin were at most 9.8- and 11.9-fold higher than moxifloxacin. Also the fluoroquinolone–mediated cleavage assay showed that potencies of those drugs were at most 13.5- and 9.8-fold higher than moxifloxacin. In addition, these two drugs retained their inhibitory activities even against DNA gyrases of ofloxacin-resistant M. leprae. Conclusions/Significance The results indicated that DC-159a and sitafloxacin are more effective against wild type and mutant M. leprae DNA gyrases than moxifloxacin, suggesting that these antibacterial drugs can be good candidates that may supersede current fluoroquinolone remedies. DC-159a in particular is very promising because it is classified in a subgroup of fluoroquinolones that is known to be less likely to cause adverse effects. Our results implied that DC-159a is well worth further investigation to ascertain its in vivo effectiveness and clinical safety for humans. PMID:27681932

  5. Reduced DNA topoisomerase II activity in ataxia-telangiectasia cells.

    PubMed Central

    Singh, S P; Mohamed, R; Salmond, C; Lavin, M F

    1988-01-01

    Considerable evidence supports a defect at the level of chromatin structure or recognition of that structure in cells from patients with the human genetic disorder ataxia-telangiectasia. Accordingly, we have investigated the activities of enzymes that alter the topology of DNA in Epstein Barr Virus-transformed lymphoblastoid cells from patients with this syndrome. Reduced activity of DNA topoisomerase II, determined by unknotting of P4 phage DNA, was observed in partially purified extracts from 5 ataxia-telangiectasia cell lines. The levels of enzyme activity was reduced substantially in 4 of these cell lines and to a lesser extent in the other cell line compared to controls. DNA topoisomerase I, assayed by relaxation of supercoiled DNA, was found to be present at comparable levels in both cell types. Reduced activity of topoisomerase II in ataxia-telangiectasia is compatible with the molecular, cellular and clinical changes described in this syndrome. Images PMID:2836804

  6. Environmental DNA (eDNA) Detection Probability Is Influenced by Seasonal Activity of Organisms

    PubMed Central

    de Souza, Lesley S.; Godwin, James C.; Renshaw, Mark A.; Larson, Eric

    2016-01-01

    Environmental DNA (eDNA) holds great promise for conservation applications like the monitoring of invasive or imperiled species, yet this emerging technique requires ongoing testing in order to determine the contexts over which it is effective. For example, little research to date has evaluated how seasonality of organism behavior or activity may influence detection probability of eDNA. We applied eDNA to survey for two highly imperiled species endemic to the upper Black Warrior River basin in Alabama, US: the Black Warrior Waterdog (Necturus alabamensis) and the Flattened Musk Turtle (Sternotherus depressus). Importantly, these species have contrasting patterns of seasonal activity, with N. alabamensis more active in the cool season (October-April) and S. depressus more active in the warm season (May-September). We surveyed sites historically occupied by these species across cool and warm seasons over two years with replicated eDNA water samples, which were analyzed in the laboratory using species-specific quantitative PCR (qPCR) assays. We then used occupancy estimation with detection probability modeling to evaluate both the effects of landscape attributes on organism presence and season of sampling on detection probability of eDNA. Importantly, we found that season strongly affected eDNA detection probability for both species, with N. alabamensis having higher eDNA detection probabilities during the cool season and S. depressus have higher eDNA detection probabilities during the warm season. These results illustrate the influence of organismal behavior or activity on eDNA detection in the environment and identify an important role for basic natural history in designing eDNA monitoring programs. PMID:27776150

  7. Environmental DNA (eDNA) Detection Probability Is Influenced by Seasonal Activity of Organisms.

    PubMed

    de Souza, Lesley S; Godwin, James C; Renshaw, Mark A; Larson, Eric

    2016-01-01

    Environmental DNA (eDNA) holds great promise for conservation applications like the monitoring of invasive or imperiled species, yet this emerging technique requires ongoing testing in order to determine the contexts over which it is effective. For example, little research to date has evaluated how seasonality of organism behavior or activity may influence detection probability of eDNA. We applied eDNA to survey for two highly imperiled species endemic to the upper Black Warrior River basin in Alabama, US: the Black Warrior Waterdog (Necturus alabamensis) and the Flattened Musk Turtle (Sternotherus depressus). Importantly, these species have contrasting patterns of seasonal activity, with N. alabamensis more active in the cool season (October-April) and S. depressus more active in the warm season (May-September). We surveyed sites historically occupied by these species across cool and warm seasons over two years with