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Sample records for dna cleavage studies

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

    SciTech Connect

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

    1994-06-15

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

  2. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2009-09-01

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

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

    NASA Astrophysics Data System (ADS)

    Sampath, Krishnan; Sathiyaraj, Subbaiyan; Jayabalakrishnan, Chinnasamy

    2013-03-01

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

  5. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2010-12-01

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

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

    NASA Astrophysics Data System (ADS)

    Gupta, Lokesh Kumar; Chandra, Sulekh

    2008-11-01

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

  8. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

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

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

    PubMed

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

    2013-09-01

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

  11. 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.

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-12-01

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

  17. 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

  18. 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.

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

    PubMed

    Shimizu, M; Inoue, H; Ohtsuka, E

    1994-01-18

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

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

    PubMed Central

    Sander, M; Hsieh, T S

    1985-01-01

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

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

    PubMed

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

    2009-03-01

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

  2. 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

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

    SciTech Connect

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

    1990-01-01

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

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

    PubMed

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

    2015-10-01

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

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

    PubMed Central

    Meluzzi, Dario; Arya, Gaurav

    2016-01-01

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

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

    PubMed

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

    1997-05-01

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

  7. Synthesis, 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.

  8. Conventional and microwave-assisted synthesis, characterization, DFT calculations, in vitro DNA binding and cleavage studies of potential chemotherapeutic diorganotin(IV) mandelates.

    PubMed

    Mridula; Nath, Mala

    2016-09-01

    Diorganotin(IV) complexes of the general formulae {[R2Sn(L)]2O}(R=Me (1), n-Bu (2), and n-Oct (3); L=anion of mandelic acid) and {[R2Sn(L)]2Cl2}(R=Ph (4)) have been synthesized by conventional thermal method (1a-3a), except 4a and by microwave-assisted reactions (1b-4b). The elemental analysis, IR, NMR ((1)H, (13)C and (119)Sn) and ESI-MS/DART-mass spectral studies revealed that dimeric 1:1 complexes with SnOSn bridges (1-3) are formed possessing distorted trigonal bipyramidal geometry around the Sn atoms, except 4b which exhibits octahedral geometry with SnClSn bridges. The proposed geometries have been validated by density functional theory calculations. Thermal behavior of 1b-4b, studied by using thermogravimetry (TG), differential thermal analysis (DTA) and derivative thermogravimetric (DTG) techniques, indicated that all except 4b are stable up to 200°C. In vitro interaction studies of 1b-4b with CT-DNA were performed by UV-Vis, fluorescence titrations and results suggest that the complexes are binding to DNA via an intercalative mode. The binding affinity and quenching ability were quantified in terms of intrinsic binding constant (Kb) (3.74×10(4)M(-1), 2b; >3.67×10(4)M(-1), 4b; >3.03×10(4)M(-1), 3b; >0.72×10(4)M(-1), 1b) and Stern-Volmer quenching constant (Ksv) (2.16×10(5), 2b; >1.73×10(5), 4b; >1.66×10(5)3b; >1.51×10(5), 1b) which showed high binding affinity of 2b with CT-DNA. The cleavage studies of 1b-4b with pBR322 plasmid DNA was ascertained by agarose gel electrophoresis. They exhibited effective cleavage of supercoiled plasmid DNA into its nicked form (1b, 3b, 4b) and even into its linear form in presence of 2b. PMID:27423117

  9. 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.

  10. 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

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

    PubMed

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

    2016-03-01

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

  12. 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.

  13. 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.

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

    PubMed

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

    2013-04-15

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

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

    PubMed

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

    2015-02-01

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

  17. 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.

  18. The pattern of DNA cleavage intensity around indels.

    PubMed

    Chen, Wei; Zhang, Liqing

    2015-01-01

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

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

    PubMed

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

    2016-01-01

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

  20. 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

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

    PubMed

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

    2016-08-01

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

  2. 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.

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

    PubMed Central

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

    1995-01-01

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

  4. 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.

  5. 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.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2016-03-15

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

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

    NASA Astrophysics Data System (ADS)

    Arish, D.; Nair, M. Sivasankaran

    2011-11-01

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

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

    PubMed

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

    2016-05-01

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

  11. 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.

  12. 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

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

    PubMed

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

    2015-07-28

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

  14. 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.

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    2015-11-01

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

  17. 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

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

    PubMed Central

    2016-01-01

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

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

    PubMed Central

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

    1976-01-01

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

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

    PubMed Central

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

    1987-01-01

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

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

    PubMed

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

    2016-05-17

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

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

    SciTech Connect

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

    2005-04-29

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

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

    PubMed Central

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

    2009-01-01

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

  4. 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.

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

    PubMed

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

    2004-12-24

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

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

    PubMed Central

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

    1982-01-01

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

  7. 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

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

    PubMed Central

    Deiss, L P; Frenkel, N

    1986-01-01

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

  9. 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.

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

    PubMed

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

    2013-08-15

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

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

    PubMed Central

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

    2013-01-01

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

  12. 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.

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

    PubMed

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

    2013-01-01

    The effects of nucleotide analogue substitution on the cleavage efficiencies of type II restriction endonucleases have been investigated. Six restriction endonucleases (EcoRV, SpeI, XbaI, XhoI, PstI and SphI) were investigated respectively regarding their cleavage when substrates were substituted by 2'-O-methyl nucleotide (2'-OMeN) and phosphorothioate (PS). Substitutions were made in the recognition sequence and the two nucleotides flanking the recognition sequence for each endonuclease. The endonuclease cleavage efficiencies were determined using FRET-based assay. Results demonstrated a position-dependent inhibitory effect of substitution on the cleavage efficiency for all the six endonucleases. In general, the 2'-OMeN substitutions had greater impact than the PS substitutions on the enzymatic activities. Nucleotides of optimal substitutions for protection against RE cleavage were identified. Experimental results and conclusions in this study facilitate our insight into the DNA-protein interactions and the enzymatic cleavage mechanism, particularly for those whose detailed structure information is not available. In addition, the information could benefit the development of bioengineering and synthetic biology. PMID:24260216

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

    NASA Astrophysics Data System (ADS)

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

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

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

    PubMed Central

    Dreyer, G B; Dervan, P B

    1985-01-01

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

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

    PubMed

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

    2016-05-19

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

  17. 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

  18. 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.

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

    PubMed Central

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

    1981-01-01

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2005-04-01

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

  2. 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

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

    PubMed

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

    2010-02-01

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

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed

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

    2014-12-11

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    1986-01-01

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

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

    PubMed Central

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

    2005-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  12. 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

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

    PubMed

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

    2015-12-01

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

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

    PubMed Central

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

    2015-01-01

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

  15. 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

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

    PubMed

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

    2012-06-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2012-09-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2007-10-29

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

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

    PubMed

    Li, Cuiping; Wang, Hailin

    2015-08-01

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

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2002-07-18

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

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

    PubMed

    Krystosek, A

    1999-04-01

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

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

    PubMed

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

    2005-08-21

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

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

    PubMed

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

    2001-05-01

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

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

    PubMed Central

    Zhang, Ming; Swanson, Patrick C

    2009-01-01

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

  9. 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.

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

    PubMed

    Sampath, Krishnan; Sathiyaraj, Subbaiyan; Jayabalakrishnan, Chinnasamy

    2013-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-08-01

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

  12. 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.

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

    PubMed

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

    2004-09-01

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

  14. 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.

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

    PubMed

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

    2007-12-24

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

  16. 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

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

    PubMed

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

    2008-07-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2005-03-01

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

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

    PubMed Central

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

    1990-01-01

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

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

    PubMed

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

    2016-12-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2014-02-01

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

  4. 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

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

    PubMed

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

    2016-06-01

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

  6. 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.

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

    PubMed

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

    2009-09-01

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

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

    PubMed Central

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

    2005-01-01

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

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

    PubMed

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

    2014-12-16

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

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

    PubMed Central

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

    1981-01-01

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

  11. 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

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

    PubMed Central

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

    1980-01-01

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

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

    PubMed

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

    2016-08-15

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

  14. Spectroscopic evaluation for VO(II), Ni(II), Pd(II) and Cu(II) complexes derived from thiosemicarbazide: A special emphasis on EPR study and DNA cleavage

    NASA Astrophysics Data System (ADS)

    El-Metwally, Nashwa M.; Al-Hazmi, Gamil A. A.

    2013-04-01

    Some thiosemicarbazide complexes were prepared and deliberately investigated by all allowed tools. The ligand coordinates as a mono negative bidentate towards VO(II) and Ni(II) as well as a neutral bidentate towards Pd(II) and Cu(II) ions. Electronic spectral data beside the magnetic measurements facilitate the structural geometry proposal. EPR spectra of Cu(II) and VO(II) complexes were recorded in their solid state. Spin Hamiltonian parameters and molecular orbital coefficient for Cu(II) and VO(II) complexes were calculated and supporting the octahedral geometry of Cu(II) complex and a square pyramidal for VO(II) one. The biological activity investigation was studied by the use of all prepared compounds. The VO(II) and Cu(II) complexes display the susceptible biotoxicity against a gram-positive bacterium. Also, Cu(II) complex displays the same toxicity against gram-negative bacteria used. The effect of all compounds on DNA were photographed. A successive degradation for the DNA target was observed with Pd(II) and Ni(II) complexes beside their original ligand.

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2014-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2006-02-01

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

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

    PubMed

    Buchtík, Roman; Trávníček, Zdeněk; Vančo, Ján

    2012-11-01

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

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

    PubMed

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

    1999-06-15

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

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

    PubMed

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

    2009-02-16

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

  1. 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.

  2. Co(II), Cu(II), Cd(II), Fe(III) and U(VI) complexes containing a NSNO donor ligand: Synthesis, characterization, optical band gap, in vitro antimicrobial and DNA cleavage studies

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

    A new series of [Co(HPTP)Cl(H2O)2], [Cu(HPTP)Cl], [Cd(HPTP)Cl](H2O)4, [Fe(PTP)Cl(H2O)2](H2O), [UO2(HPTP)(OAc)(H2O)2] complexes of Schiff-bases derived from 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 analyses, spectral (IR, UV-vis, 1H NMR, 13C NMR and ESR), magnetic and thermal studies. IR spectra show that H2PTP is coordinated to the metal ions in a mono or binegative tridentate manner. The electronic spectra of the complexes and their magnetic moments provide information about geometries. The room temperature solid state ESR spectra of the Cu(II) complexes show dx2-y2 as a ground state, suggesting square-planar geometry around Cu(II) center. The molecular 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 3.25, 3.26, 3.34 and 3.27 eV for Co, Cu, Fe and U complexes, respectively. The synthesized ligand, in comparison to its metal complexes is screened for its antibacterial activity against bacterial species, Bacillus thuringiensis, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. The activity data show that the metal complexes to be more potent/antibacterial than the parent Schiff base ligand against one or more bacterial species. Finally, the biochemical studies showed that, Cu, Cd and Fe complexes have powerful and complete

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed

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

    1997-08-25

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

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

    PubMed

    Arjmand, Farukh; Muddassir, Mohd

    2011-03-01

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

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

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

    2002-01-01

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

  8. Study of mechanism of cleavage fracture at low temperature

    NASA Astrophysics Data System (ADS)

    Chen, J. H.; Wang, G. Z.

    1992-02-01

    In this investigation, a series of crack opening displacement (COD) tests were carried out at several low temperatures for C-Mn weld steel. Some of the specimens were loaded until fracture, and the mechanical properties and microscopic parameters on fracture surfaces were measured. Other specimens were unloaded before fracture at different applied loads. The distributions of the elongated cavities and the cleavage microcracks ahead of fatigue crack tips were observed in detail. Based on the experimental results, the combined criterion of a critical strain ɛ p ≥ ɛc) for initiating a crack nucleus, a critical stress triaxiality (σ m/σ ≥ tc) for preventing it from blunting, and a critical normal stress (σ yy/σf) for the cleavage extension was proposed again, and the critical values of ɛp and σm/-σ for the C-Mn weld steel were measured. The reason why the minimum COD value could not be zero is explained. The mechanism of generation of the lower limit COD value on the lower shelf of the toughness transition curve is proposed.

  9. 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.

  10. 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.

  11. 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.

  12. 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

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

    PubMed

    Hamann, Ingrit; Schwerdtle, Tanja; Hartwig, Andrea

    2009-10-01

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

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

    PubMed Central

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

    1997-01-01

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

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

    PubMed Central

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

    2014-01-01

    . Previously, we described a series of N-α-methylbenzyl-N′-aryl thiourea analogs that target the VZV portal protein (pORF54) and prevent viral replication in vitro. To better understand the mechanism of action of these compounds, it is important to define the structural and functional characteristics of the VZV portal protein. In contrast to HSV, no VZV mutants have been described for any of the seven essential DNA encapsidation genes. The VZV ORF54 deletion mutant described in this study represents the first VZV encapsidation mutant reported to date. We demonstrate that the deletion mutant can serve as a platform for the isolation of portal mutants via recombineering and provide a strategy for more in-depth studies of VZV portal structure and function. PMID:24807720

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

    PubMed

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

    2016-05-23

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

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

    PubMed

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

    2007-09-01

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

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

    PubMed Central

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

    2007-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-10-01

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

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

    PubMed

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

    2007-12-01

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

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

    PubMed

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

    2015-03-01

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

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

    PubMed

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

    2016-08-16

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

  3. 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

  4. A novel method for detecting apoptosis shows that hepatocytes undergo a time dependent increase in DNA cleavage and chromatin condensation which is augmented after TGF-beta 1 treatment.

    PubMed

    Cain, K; Inayat-Hussain, S H; Couet, C; Qin, H M; Oberhammer, F A

    1996-04-01

    This study describes a new method for quantitating apoptosis in hepatocyte monolayers in which nuclei were isolated from the cells and DNA strand breaks detected by in situ end-labeling and flow cytometry. Most (97%) nuclei from untreated hepatocytes had low end-labelling and were derived from non-apoptotic cells. Approximately 2-3% of the nuclei had high end-labelling and originated from apoptotic hepatocytes. The numbers of these nuclei increased linearly from 3 to 85% between 0 and 48 h after treatment with transforming growth factor-beta 1 (TGF-beta 1). However, a morphological assessment of apoptosis with Hoechst H33258 showed that the proportion of apoptotic nuclei plateaued at 18-19% between 24 and 48 h after TGF-beta 1 treatment. Thus, the in situ end-labeling technique also detected DNA cleavage in nuclei which did not have an obvious apoptotic morphology. Confocal microscopy of low and high end-labelled nuclei which had been separated by fluorescent cell sorting showed that nuclei with high levels of end-labeling exhibited a wide diversity of morphologies. These included nuclei with little or no chromatin condensation and nuclei with characteristic apoptotic morphology. In addition, nuclei from untreated hepatocytes contained low levels of DNA cleavage, which were localized in areas of condensed chromatin and increased according to the time in culture. Thus, hepatocytes undergo a progressive and cumulative process of DNA cleavage/chromatin condensation which is markedly enhanced by TGF-beta 1. PMID:8900474

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

    PubMed

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

    2016-02-15

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

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

    PubMed Central

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

    2009-01-01

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

  7. 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

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

    PubMed

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

    2014-01-01

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

  9. 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

  10. Mass spectrometric and theoretical studies on dissociation of the Ssbnd S bond in the allicin: Homolytic cleavage vs heterolytic cleavage

    NASA Astrophysics Data System (ADS)

    Zhang, Xiang

    2012-08-01

    On the basis of the tandem mass spectrometry (ESI-MS/MS) technique and DFT calculations, an experimental and theoretical investigation has been conducted into the gas-phase dissociation of the S1sbnd S1' bond in the allicin as well as that of the Ssbnd C (S1sbnd C2, S1'sbnd C2') bond. Meanwhile, the influence of protonation, alkali metal ion and electron transfer on the dissociation of the S1sbnd S1' bond has been taken into account. ESI-MS/MS experiments and DFT calculations show that in the neutral allicin, [allicin + Li]+ and [allicin + Na]+, the S1sbnd S1' bond favors homolytic cleavage, while in the allicin radical cation and protonated allicin, the S1sbnd S1' bond prefers heterolytic cleavage. In addition, alkali metal ions can strengthen the S1sbnd S1' bond in the allicin, while protonation or the loss of an electron will weaken the S1sbnd S1' bond.

  11. Model studies of DNA photoreactivation

    NASA Astrophysics Data System (ADS)

    Scannell, Michael P.

    1997-12-01

    This research was undertaken with the goal of understanding DNA damage and repair, specifically damage caused by the ultraviolet (UV) component of sunlight. The main type of DNA damage by UV irradiation is dimerization of adjacent thymines. This occurs through a (2+2) cycloaddition resulting in a cyclobutyl linkage between the thymines. These mutagenic lesions are repaired by an enzyme called photolyase, which repairs the dimers through a complex photochemical reaction. The work presented here is divided into three main topics. The first topic (Chapter 3) describes the measurement of the enthalpy of cleavage of dimethylthymine dimer. The enthalpy for the cleavage reaction of cis-syn 1,3-dimethylthymine dimer (DMTD) was measured by photothermal beam deflection calorimetry (PBD), and fluorescence quenching. These results show that the enthalpy of cleavage of the cyclobutyl ring is -19 kcal/mol. For the second topic (Chapters 4 and 5), the interactions of various pyrimidines and their corresponding cis-syn cyclobutane dimers with a series of excited-state electron donors were examined with the goal of understanding the energetics and mechanism of the repair step. For each substrate there is a good correlation between the excited state oxidation potential (E ox/sp/*) and the quenching rate constant (k q). The value for k q increases as E ox/sp/* becomes more negative, asymptotically approaching a value that is at or below the solvent diffusion limit. The data from this study were fit to the Rehm-Weller model of electron transfer. Reduction potentials for each of the substrates could be extracted from this analysis: -2.20 V (vs. SCE) for DMTD; -2.14 V for DMT; -2.17 V for DMCD; and -2.16 for DMC. The reduction potential of trans-syn dimethylthymine was also measured. This dimer shows a remarkably low reduction potential when compared to the cis-syn dimer. This is attributed to unfavorable charge-charge dipole interactions in the cis-syn dimer not presence in the trans

  12. Copper.Lys-Gly-His-Lys mediated cleavage of tRNA(Phe): studies of reaction mechanism and cleavage specificity.

    PubMed

    Bradford, Seth; Kawarasaki, Yuta; Cowan, J A

    2009-06-01

    The reactivity of [Cu2+.Lys-Gly-His-Lys-NH2]2+ and [Cu2+.Lys-Gly-His-Lys]+ toward tRNA(Phe) has been evaluated. The amidated and carboxylate forms of the copper peptides display complex binding behavior with strong and weak sites evident (K(D1)(app) approximately 71 microM, K(D2)(app) approximately 211 microM for the amide form; and K(D1)(app) approximately 34 microM, K(D2)(app) approximately 240 microM for the carboxylate form), while Cu2+(aq) yielded K(D1)(app) approximately 81 microM and K(D2)(app) approximately 136 microM. The time-dependence of the reaction of [Cu2+.Lys-Gly-His-Lys]+ and [Cu2+.Lys-Gly-His-Lys-NH2]2+ with tRNA(Phe) yielded k(obs) approximately 0.075 h(-1) for both complexes. HPLC analysis of the reaction products demonstrated guanine as the sole base product. Mass spectrometric data shows a limited number of cleavage fragments with product peak masses consistent with chemistry occurring at a discrete site defined by the structurally contiguous D and TPsiC loops, and in a domain where high affinity magnesium centers have previously been observed to promote hydrolysis of the tRNA(Phe) backbone. This cleavage pattern is more selective than that previously observed by Long and coworkers for nickel complexes of a series of C-terminally amidated peptides (Gly-Gly-His, Lys-Gly-His, and Arg-Gly-His), and may reflect variations in structural recognition and a distinct reaction path by the nickel derivatives. The data emphasizes the optimal positioning of the metal-associated reactive oxygen species, relative to scissile bonds, as a major criterion for development of efficient catalytic nucleases or therapeutics. PMID:19386364

  13. 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

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

    PubMed

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

    2012-08-28

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

  15. Studying DNA in the Classroom.

    ERIC Educational Resources Information Center

    Zarins, Silja

    1993-01-01

    Outlines a workshop for teachers that illustrates a method of extracting DNA and provides instructions on how to do some simple work with DNA without sophisticated and expensive equipment. Provides details on viscosity studies and breaking DNA molecules. (DDR)

  16. 2-Chlorotrityl chloride resin. Studies on anchoring of Fmoc-amino acids and peptide cleavage.

    PubMed

    Barlos, K; Chatzi, O; Gatos, D; Stavropoulos, G

    1991-06-01

    The esterification of 2-chlorotrityl chloride resin with Fmoc-amino acids in the presence of DIEA is studied under various conditions. High esterification yields are obtained using 0.6 equiv. Fmoc-amino acid/mmol resin in DCM or DCE, in 25 min, at room temperature. The reaction proceeds without by product formation even in the case of Fmoc-Asn and Fmoc-Gln. The quantitative and easy cleavage of amino acids and peptides from 2-chlorotrityl resin, by using AcOH/TFE/DCM mixtures, is accomplished within 15-60 min at room temperature, while t-butyl type protecting groups remain unaffected. Under these exceptionally mild conditions 2-chlorotrityl cations generated during the cleavage of amino acids and peptides from resin do not attack the nucleophilic side chains of Trp, Met, and Tyr. PMID:1917309

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

    PubMed

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

    2012-12-01

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

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

    PubMed

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

    2014-01-24

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

  19. 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.

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

    PubMed Central

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

    2014-01-01

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

  1. Site specific cleavage of thetaX-174 replicative form DNA after modification by N-acetoxy-N-2-acetylaminofluorene

    SciTech Connect

    Bases, R.; Mendez, F.; Mendez, L.

    1983-01-01

    Three kinds of structural disturbances were found in an 88 base pair (bp) fragment of thetaX-174 DNA after exposure to N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF). Frequent strand scissions at two specific guanine sites on the 5' /sup 32/P-end-labeled fragment were identified by base sequence analysis. Scissions at these two sites were induced at neutral pH and they were not increased by treatment with apurinic endonuclease. They are an immediate consequence of N-Aco-AAF action and are not primarily apurinic sites. Alkali treatment with 1 M piperidine at 90/sup 0/C induced strand scissions at every guanine, demonstrating adduct slices, depurination and strand scissions. Adducted DNA was sensitive to single-strand specific nuclease digestion, suggesting unwound DNA. These studies indicate the prediliction of N-Aco-AAF for certain DNA sites and they suggest three kinds of DNA modifications which can be expected after adduction by this carcinogen. Some of the sites may be premutational carcinogen-induced DNA structural modifications.

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

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

    2014-12-16

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

  4. 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.

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

    PubMed

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

    2006-01-01

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

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed Central

    Gomathi, Ramadoss; Ramu, Andy; Murugan, Athiappan

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-10-01

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

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

    PubMed

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

    2011-10-15

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

  10. Modeling study on the cleavage step of the self-splicing reaction in group I introns

    NASA Technical Reports Server (NTRS)

    Setlik, R. F.; Garduno-Juarez, R.; Manchester, J. I.; Shibata, M.; Ornstein, R. L.; Rein, R.

    1993-01-01

    A three-dimensional model of the Tetrahymena thermophila group I intron is used to further explore the catalytic mechanism of the transphosphorylation reaction of the cleavage step. Based on the coordinates of the catalytic core model proposed by Michel and Westhof (Michel, F., Westhof, E. J. Mol. Biol. 216, 585-610 (1990)), we first converted their ligation step model into a model of the cleavage step by the substitution of several bases and the removal of helix P9. Next, an attempt to place a trigonal bipyramidal transition state model in the active site revealed that this modified model for the cleavage step could not accommodate the transition state due to insufficient space. A lowering of P1 helix relative to surrounding helices provided the additional space required. Simultaneously, it provided a better starting geometry to model the molecular contacts proposed by Pyle et al. (Pyle, A. M., Murphy, F. L., Cech, T. R. Nature 358, 123-128. (1992)), based on mutational studies involving the J8/7 segment. Two hydrated Mg2+ complexes were placed in the active site of the ribozyme model, using the crystal structure of the functionally similar Klenow fragment (Beese, L.S., Steitz, T.A. EMBO J. 10, 25-33 (1991)) as a guide. The presence of two metal ions in the active site of the intron differs from previous models, which incorporate one metal ion in the catalytic site to fulfill the postulated roles of Mg2+ in catalysis. The reaction profile is simulated based on a trigonal bipyramidal transition state, and the role of the hydrated Mg2+ complexes in catalysis is further explored using molecular orbital calculations.

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

    PubMed

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

    2013-04-15

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

  12. Atomic Force Microscopy Studies on DNA Structural Changes Induced by Vincristine Sulfate and Aspirin

    NASA Astrophysics Data System (ADS)

    Zhu, Yi; Zeng, Hu; Xie, Jianming; Ba, Long; Gao, Xiang; Lu, Zuhong

    2004-04-01

    We report that atomic force microscopy (AFM) studies on structural variations of a linear plasmid DNA interact with various concentrations of vincristine sulfate and aspirin. The different binding images show that vincrinstine sulfate binding DNA chains caused some loops and cleavages of the DNA fragments, whereas aspirin interaction caused the width changes and conformational transition of the DNA fragments. Two different DNA structural alternations could be explained by the different mechanisms of the interactions with these two components. Our work indicates that the AFM is a powerful tool in studying the interaction between DNA and small molecules.

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

    PubMed

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

    2015-09-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    1988-01-01

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

  16. Theoretical study of the mechanism of the rearrangement-cleavage reactions of allylenammonium salts

    SciTech Connect

    Kletskii, M.E.; Minkin, V.I.; Babayan, A.T.

    1988-03-10

    In a theoretical study of the mechanism of the rearrangement-cleavage of tetra-substituted ammonium salts containing 1,2- and 3,4-unsaturated groups using the CNDO/2 and MINDO/3 semiempirical methods, calculations were carried out to determine the electron density in trimethylvinylammonium, trimethylethynylammonium, and phosphonium cations and the minimal energy pathways for the 3,3-sigmatropic shift reactions in the vinylallylammonium cation and of the product of the /alpha/-addition of a hydride ion to it (nucleophilic catalysis model). According to the calculation data, the nucleophilic attack of enammonium compounds in basic media is directed toward the /alpha/-position of the 1,2-unsaturated group. The driving force for the rearrangement-cleavage is the attack of the /alpha/-position by the nucleophile which results in intramolecular C-alkylation by a concerted 3,3-sigmatropic shift mechanism. The calculated activation barrier of the catalyzed reaction (15.8 kcal/mole) is 7.4 kcal/mole lower than in the absence of nucleophilic action.

  17. N-H bond cleavage of ammonia on graphene-like B36 borophene: DFT studies.

    PubMed

    Rostami, Zahra; Soleymanabadi, Hamed

    2016-04-01

    Ammonia N-H bond cleavage at metal-free substrates has attracted great attention because of its industrial importance. Here, we investigate the dissociative adsorption of ammonia onto the surface of a B36 borophene sheet by means of density functional theory calculations. We show that the N-H bond may be broken at the edges of B36 even at room temperature, regarding the small energy barrier of 14.1-19.3 kcal mol(-1) at different levels of theory, and more negative Gibbs free energy change. Unlike basis set size, the kind of exchange correlation functional significantly affects the electronic properties of the studied systems. Also, by increasing the percentage of Hartree Fock (HF) exchange of density functionals, the activation and adsorption energies are lowered. A linear relationship between the highest occupied molecular orbital or lowest unoccupied molecular orbital of B36 borophene and the %HF exchange of functionals is predicted. Our work reveals that pure whole boron nanosheets may be promising metal-free materials in N-H bond cleavage, which would raise the potential application of these sheets. PMID:26969676

  18. 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.

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

    PubMed

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

    2015-02-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2015-02-01

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

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

    PubMed Central

    Yu, Zhen; Han, Menglu

    2015-01-01

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

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

    PubMed

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

    2006-05-01

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

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

    PubMed

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

    2015-06-01

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

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

    PubMed

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

    2005-11-25

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2014-05-22

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

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

    SciTech Connect

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

    1991-10-15

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

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

    PubMed Central

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

    2009-01-01

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

  10. 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.

  11. 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.

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

    PubMed

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

    2016-07-01

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

  13. A mechanistic study of Trichoderma reesei Cel7B catalyzed glycosidic bond cleavage.

    PubMed

    Zhang, Yu; Yan, Shihai; Yao, Lishan

    2013-07-25

    An ONIOM study is performed to illustrate the mechanism of Trichoderma reesei Cel7B catalyzed p-nitrophenyl lactoside hydrolysis. In both the glycosylation and deglycosylation steps, the reaction proceeds in a concerted way, meaning the nucleophilic attack and the glycosidic bond cleavage occur simultaneously. The glycosylation step is rate limiting with a barrier of 18.9 kcal/mol, comparable to the experimental value derived from the kcat measured in this work. The function of four residues R108, Y146, Y170, and D172, which form a hydrogen-bond network involving the substrate, is studied by conservative mutations. The mutants, including R108K, Y146F, Y170F, and D172N, decrease the enzyme activity by about 150-8000-fold. Molecular dynamics simulations show that the mutations disrupt the hydrogen-bond network, cause the substrate to deviate from active binding and hinder either the proton transfer from E201 to O4(+1) or the nucleophilic attack from E196 to C1(-1). PMID:23822607

  14. Interaction of DNA with Simple and Mixed Ligand Copper(II) Complexes of 1,10-Phenanthrolines as Studied by DNA-Fiber EPR Spectroscopy

    PubMed Central

    Chikira, Makoto; Ng, Chew Hee; Palaniandavar, Mallayan

    2015-01-01

    The interaction of simple and ternary Cu(II) complexes of 1,10-phenanthrolines with DNA has been studied extensively because of their various interesting and important functions such as DNA cleavage activity, cytotoxicity towards cancer cells, and DNA based asymmetric catalysis. Such functions are closely related to the DNA binding modes of the complexes such as intercalation, groove binding, and electrostatic surface binding. A variety of spectroscopic methods have been used to study the DNA binding mode of the Cu(II) complexes. Of all these methods, DNA-fiber electron paramagnetic resonance (EPR) spectroscopy affords unique information on the DNA binding structures of the complexes. In this review we summarize the results of our DNA-fiber EPR studies on the DNA binding structure of the complexes and discuss them together with the data accumulated by using other measurements. PMID:26402668

  15. Theoretical studies on Si-C bond cleavage in organosilane precursors during polycondensation to organosilica hybrids.

    PubMed

    Shirai, Soichi; Goto, Yasutomo; Mizoshita, Norihiro; Ohashi, Masataka; Tani, Takao; Shimada, Toyoshi; Hyodo, Shi-aki; Inagaki, Shinji

    2010-05-20

    Molecular orbital theory calculations were carried out to predict the occurrence of Si-C bond cleavage in various organosilane precursors during polycondensation to organosilica hybrids under acidic and basic conditions. On the basis of proposed mechanisms for cleavage of the Si-C bonds, the proton affinity (PA) of the carbon atom at the ipso-position and the PA of the carbanion generated after Si-C cleavage were chosen as indices for Si-C bond stability under acidic and basic conditions, respectively. The indices were calculated using a density functional theory (DFT) method for model compounds of organosilane precursors (R-Si(OH)(3)) having organic groups (R) of benzene (Ph), biphenyl (Bp), terphenyl (Tph), naphthalene (Nph), N-methylcarbazole (MCz), and anthracene (Ant). The orders for the predicted stability of the Si-C bond were Ph > Nph > Bp > Ant > Tph > MCz for acidic conditions and Ph > MCz > Bp > Nph > Tph > Ant for basic conditions. These behaviors were primarily in agreement with experimental results where cleavage of the Si-C bonds occurred for Tph (both acidic and basic), MCz (acidic), and Ant (basic). The Si-C bond cleavage of organosilane precursors during polycondensation is qualitatively predicted from these indices based on our theoretical approach. PMID:20429568

  16. Magnetic resonance imaging-based anatomical study of the multifidus-longissimus cleavage planes in the lumbar spine

    PubMed Central

    Li, Haijun; Yang, Lei; Chen, Jinhua; Xie, Hao; Tian, Weizhong; Cao, Xiaojian

    2016-01-01

    Purpose: The Wiltse approach allows spinal surgeries to be performed with minimal soft tissue trauma. The purpose of this study was to investigate the anatomy of the natural cleavage plane between multifidus and longissimus at different levels based on MRI images. Methods: MRI cross-sectional scans from L1 to S1 were collected from 205 out patients (103 males, 102 females). Based on the images, some parameters were defined and measured to describe the locations, curvature and directions of Wiltse approach. Besides, differences of these parameters between genders and segments were compared. Results: Among the total of 2460 one-sided images, cleavage planes between multifidus and longissimus were not able to be identified in 105 images. The locations, directions and curvature of the cleavage plane differed significantly among different segments but followed some regular pattern from L1-S1. The simultaneous rotation of the plane around its deepest points to the midline from S1 to L1 and the plane seemed to be the most curved at L3 and relatively straight for L5 and S1. Conclusions: With a better understanding of the natural cleavage plane between multifidus and longissimus, performers can correctly plan the distance of skin incisions from the midline and the direction of muscle dissection at each vertebral level, thus reducing trauma in the operation. PMID:27069544

  17. Analytical methods to determine the comparative DNA binding studies of curcumin-Cu(II) complexes

    NASA Astrophysics Data System (ADS)

    Rajesh, Jegathalaprathaban; Rajasekaran, Marichamy; Rajagopal, Gurusamy; Athappan, Periakaruppan

    2012-11-01

    DNA interaction studies of two mononuclear [1:1(1); 1:2(2)] copper(II) complexes of curcumin have been studied. The interaction of these complexes with CT-DNA has been explored by physical methods to propose modes of DNA binding of the complexes. Absorption spectral titrations of complex 1 with CT-DNA shows a red-shift of 3 nm with the DNA binding affinity of Kb, 5.21 × 104 M-1 that are higher than that obtained for 2 (red-shift, 2 nm; Kb, 1.73 × 104 M-1) reveal that the binding occurs in grooves as a result of the interaction is via exterior phosphates. The CD spectra of these Cu(II) complexes show a red shift of 3-10 nm in the positive band with increase in intensities. This spectral change of induced CD due to the hydrophobic interaction of copper complexes with DNA is the characteristic of B to A conformational change. The EB displacement assay also reveals the same trend as observed in UV-Vis spectral titration. The addition of complexes 1 and 2 to the DNA bound ethidium bromide (EB) solutions causes an obvious reduction in emission intensities indicating that these complexes competitively bind to DNA with EB. The positive shift of both the Epc and E0' accompanied by reduction of peak currents in differential pulse voltammogram (DPV), upon adding different concentrations of DNA to the metal complexes, are obviously in favor of strong binding to DNA. The super coiled plasmid pUC18 DNA cleavage ability of Cu(II) complexes in the presence of reducing agent reveals the single strand DNA cleavage (ssDNA) is observed. The hydroxyl radical (HOrad ) and the singlet oxygen are believed to be the reactive species responsible for the cleavage.

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

    PubMed

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

    2011-08-01

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

  19. Single Molecule Study of DNA Organization and Recombination

    NASA Astrophysics Data System (ADS)

    Xiao, Botao

    We have studied five projects related to DNA organization and recombination using mainly single molecule force-spectroscopy and statistical tools. First, HU is one of the most abundant DNA-organizing proteins in bacterial chromosomes and participates in gene regulation. We report experiments that study the dependence of DNA condensation by HU on force, salt and HU concentration. A first important result is that at physiological salt levels, HU only bends DNA, resolving a previous paradox of why a chromosome-compacting protein should have a DNA-stiffening function. A second major result is quantitative demonstration of strong dependencies of HU-DNA dissociation on both salt concentration and force. Second, we have used a thermodynamic Maxwell relation to count proteins driven off large DNAs by tension, an effect important to understanding DNA organization. Our results compare well with estimates of numbers of proteins HU and Fis in previous studies. We have also shown that a semi-flexible polymer model describes our HU experimental data well. The force-dependent binding suggests mechano-chemical mechanisms for gene regulation. Third, the elusive role of protein H1 in chromatin has been clarified with purified H1 and Xenopus extracts. We find that H1 compacts DNA by both bending and looping. Addition of H1 enhances chromatin formation and maintains the plasticity of the chromatin. Fourth, the topology and mechanics of DNA twisting are critical to DNA organization and recombination. We have systematically measured DNA extension as a function of linking number density from 0.08 to -2 with holding forces from 0.2 to 2.4 pN. Unlike previous proposals, the DNA extension decreases with negative linking number. Finally, DNA recombination is a dynamic process starting from enzyme-DNA binding. We report that the Int-DBD domain of lambda integrase binds to DNA without compaction at low Int-DBD concentration. High concentration of Int-DBD loops DNA below a threshold force

  20. Synthesis, photochemical properties and DNA binding studies of dna cleaving agents based on chiral dipyridine dihydrodioxins salts

    NASA Astrophysics Data System (ADS)

    Shamaev, Alexei

    activated by UV-light. The mechanism of o-quinone release and intramolecular ET was studied in detail by methods of Ultrafast Transient Absortion Spectroscopy and supported by high-level quantum mechanical calculations. The binding properties of chiral intercalators based on PDHD to various DNA oligonucleotides were studied by various methods and DNA cleavage properties indicating strong binding and cleaving ability of the synthesized PDHDs. Also, a new method for synthesis of cyclohexa[e]pyrenes which possibly capable of intramolecular ET and electron transfer-oxidative stress (ET-OS) DNA cleavage was developed and partially accomplished.

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

    PubMed

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

    1997-08-01

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

  2. Cleavages and co-operation in the UK alcohol industry: A qualitative study

    PubMed Central

    2012-01-01

    Background It is widely believed that corporate actors exert substantial influence on the making of public health policy, including in the alcohol field. However, the industry is far from being monolithic, comprising a range of producers and retailers with varying and diverse interests. With a focus on contemporary debates concerning the minimum pricing of alcohol in the UK, this study examined the differing interests of actors within the alcohol industry, the cleavages which emerged between them on this issue and how this impacted on their ability to organise themselves collectively to influence the policy process. We conducted 35 semi-structured interviews between June and November 2010 with respondents from all sectors of the industry as well as a range of non-industry actors who had knowledge of the alcohol policy process, including former Ministers, Members of the UK Parliament and the Scottish Parliament, civil servants, members of civil society organisations and professionals. Methods The paper draws on an analysis of publicly available documents and 35 semi-structured interviews with respondents from the alcohol industry (on- and off-trade including retailers, producers of wines, spirits and beers and trade associations) and a range of non-industry actors with knowledge of the alcohol policy process (including former Ministers, Members of Parliament and of the Scottish Parliament, civil servants, members of civil society organisations and professional groups). Interviews were recorded, transcribed and analysed using Nvivo qualitative analysis software. Processes of triangulation between data sources and different types of respondent sought to ensure we gained as accurate a picture as possible of industry participation in the policy process. Results Divergences of interest were evident between producers and retailers and within the retail sector between the on and off trade. Divisions within the alcohol industry, however, existed not only between these

  3. Photophysical and photochemical studies of a novel amphiphilic zinc phthalocyanine and its interaction with calf thymus DNA.

    PubMed

    Yuan, Linxin; Gui, Li; Wang, Yue; Zhang, Quanquan; Zhou, Lin; Wei, Shaohua

    2016-04-01

    β-tetra (aminophenoxy) sulfonic substituted zinc phthalocyanines (SNZnPc), a novel amphiphilic zinc phthalocyanine (Pc), was synthesized. The photophysical, photochemical, and photobiology properties were studied. Results indicated that the synthesized SNZnPc has good amphiphilic property and high reactive oxygen species (ROSs) generation ability. Furthermore, SNZnPc has strong affinity to calf thymus DNA (CT-DNA) through intercalation ways and can effectively cleavage CT-DNA after irradiation by light with appropriate wavelength. PMID:26775097

  4. Photophysical and photochemical studies of a novel amphiphilic zinc phthalocyanine and its interaction with calf thymus DNA

    NASA Astrophysics Data System (ADS)

    Yuan, Linxin; Gui, Li; Wang, Yue; Zhang, Quanquan; Zhou, Lin; Wei, Shaohua

    2016-04-01

    β-tetra (aminophenoxy) sulfonic substituted zinc phthalocyanines (SNZnPc), a novel amphiphilic zinc phthalocyanine (Pc), was synthesized. The photophysical, photochemical, and photobiology properties were studied. Results indicated that the synthesized SNZnPc has good amphiphilic property and high reactive oxygen species (ROSs) generation ability. Furthermore, SNZnPc has strong affinity to calf thymus DNA (CT-DNA) through intercalation ways and can effectively cleavage CT-DNA after irradiation by light with appropriate wavelength.

  5. Fuzzy logic sensing of G-quadruplex DNA and its cleavage reagents based on reduced graphene oxide.

    PubMed

    Huang, Wei Tao; Zhang, Jian Rong; Xie, Wan Yi; Shi, Yan; Luo, Hong Qun; Li, Nian Bing

    2014-07-15

    Herein, by combining the merits of nanotechnology and fuzzy logic theory, we develop a simple, label-free, and general strategy based on an organic dye-graphene hybrid system for fluorescence intelligent sensing of G-quadruplexes (G4) formation, hydroxyl radical (HO∙), and Fe(2+) in vitro. By exploiting acridine orange (AO) dyes-graphene as a nanofilter and nanoswitch and the ability of graphene to interact with DNA with different structures, our approach can efficiently distinguish, quantitatively detect target analytes. In vitro assays with G4DNA demonstrated increases in fluorescence intensity of the AO-rGO system with a linear range of 16-338 nM and a detection limit as low as 2.0 nM. The requenched fluorescence of the G4TBA-AO-rGO system has a non-linear response to Fenton reagent. But this requenching reduces the fluorescence intensity in a manner proportional to the logarithm to the base 10 of the concentration of Fenton reagent in the range of 0.1-100 μM and 100-2000 μM, respectively. Furthermore, we develop a novel and intelligent sensing method based on fuzzy logic which mimics human reasoning, solves complex and non-linear problems, and transforms the numerical output into the language description output for potential application in biochemical systems, environmental monitoring systems, and molecular-level fuzzy logic computing system. PMID:24561526

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

    PubMed

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

    2001-11-01

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

  7. New Oxidovanadium Complexes Incorporating Thiosemicarbazones and 1, 10-Phenanthroline Derivatives as DNA Cleavage, Potential Anticancer Agents, and Hydroxyl Radical Scavenger.

    PubMed

    Ying, Peng; Zeng, Pengfei; Lu, Jiazheng; Chen, Hongyuan; Liao, Xiangwen; Yang, Ning

    2015-10-01

    Four novel oxidovanadium(IV) complexes, [VO(hntdtsc)(PHIP)] (1) (hntdtsc = 2-hydroxy-1-naphthaldehyde thiosemicarbazone, PHIP= 2-phenyl-imidazo[4,5-f]1,10-phenanthroline), [VO(hntdtsc)(DPPZ)](2)(DPPZ= dipyrido[3,2-a:2',3'-c]phenazine), [VO(satsc)(PHIP)](3) (satsc=salicylaldehyde thiosemicarbazone), and [VO(satsc)(DPPZ)](4), have been prepared and characterized. The chemical nuclease activities and photocleavage reactions of the complexes were tested. All four complexes can efficiently cleave pBR322 DNA, and complex 1 has the best cleaving ability. The antitumor properties of these complexes were examined with three different tumor cell lines using MTT assay. Their antitumor mechanism has been analyzed using cell cycle analysis, fluorescence microscopy of apoptosis, and Annexin V-FITC/PI assay. The results showed that the growth of human neuroblastoma (SH-SY5Y, SK-N-SH) and human breast adenocarcinoma (MCF-7) cells were inhibited significantly with very low IC50 values. Complex 1 was found to be the most potent antitumor agent among the four complexes. It can cause G0/G1 phase arrest of the cell cycle and exhibited significant induced apoptosis in SK-N-SH cells and displayed typical morphological apoptotic characteristics. In addition, they all displayed reasonable abilities to scavenge hydroxyl radical, and complex 1 was the best inhibitor. PMID:25659415

  8. Determining the Architecture of a Protein-DNA Complex by Combining FeBABE Cleavage Analyses, 3-D Printed Structures, and the ICM Molsoft Program.

    PubMed

    James, Tamara; Hsieh, Meng-Lun; Knipling, Leslie; Hinton, Deborah

    2015-01-01

    Determining the structure of a protein-DNA complex can be difficult, particularly if the protein does not bind tightly to the DNA, if there are no homologous proteins from which the DNA binding can be inferred, and/or if only portions of the protein can be crystallized. If the protein comprises just a part of a large multi-subunit complex, other complications can arise such as the complex being too large for NMR studies, or it is not possible to obtain the amounts of protein and nucleic acids needed for crystallographic analyses. Here, we describe a technique we used to map the position of an activator protein relative to the DNA within a large transcription complex. We determined the position of the activator on the DNA from data generated using activator proteins that had been conjugated at specific residues with the chemical cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). These analyses were combined with 3-D models of the available structures of portions of the activator protein and B-form DNA to obtain a 3-D picture of the protein relative to the DNA. Finally, the Molsoft program was used to refine the position, revealing the architecture of the protein-DNA within the transcription complex. PMID:26404142

  9. 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

  10. Water-soluble DNA minor groove binders as potential chemotherapeutic agents: synthesis, characterization, DNA binding and cleavage, antioxidation, cytotoxicity and HSA interactions.

    PubMed

    Fu, Xia-Bing; Liu, Dan-Dan; Lin, Yuan; Hu, Wei; Mao, Zong-Wan; Le, Xue-Yi

    2014-06-21

    Two new water-soluble copper(ii)-dipeptide complexes: [Cu(glygly)(PyTA)]ClO4·1.5H2O (1) and [Cu(glygly)(PzTA)]ClO4·1.5H2O (2) (glygly = glycylglycine anion, PyTA = 2,4-diamino-6-(2'-pyridyl)-1,3,5-triazine and PzTA = 2,4-diamino-6-(2'-pyrazino)-1,3,5-triazine), utilizing two interrelated DNA base-like ligands (PyTA and PzTA), have been synthesized and characterized. The structure elucidation for 1 performed by single crystal X-ray diffraction showed a one dimensional chain conformation in which the central copper ions arrange in a five-coordinate distorted square-pyramidal geometry. Spectroscopic titration, viscosity and electrophoresis measurements revealed that the complexes bound to DNA via an outside groove binding mode, and cleaved pBR322 DNA efficiently in the presence of ascorbate, probably via an oxidative mechanism with the involvement of ˙OH and ˙O2(-). Notably, the complexes exhibited considerable in vitro cytotoxicity against four human carcinoma cell lines (HepG2, HeLa, A549 and U87) with IC50 values ranging from 41.68 to 159.17 μM, in addition to their excellent SOD mimics (IC50 ~ 0.091 and 0.114 μM). Besides, multispectroscopic evidence suggested their HSA-binding at the cavity containing Trp-214 in subdomain IIA with moderate affinity, mainly via hydrophobic interaction. Further, the molecular docking technique utilized for ascertaining the mechanism and mode of action towards DNA and HSA theoretically verified the experimental results. PMID:24770345

  11. 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.

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

    PubMed

    Naik, Abani Kanta; Raghavan, Sathees C

    2012-01-20

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

  13. Mixed-ligand copper(ii) Schiff base complexes: the role of the co-ligand in DNA binding, DNA cleavage, protein binding and cytotoxicity.

    PubMed

    Lian, Wen-Jing; Wang, Xin-Tian; Xie, Cheng-Zhi; Tian, He; Song, Xue-Qing; Pan, He-Ting; Qiao, Xin; Xu, Jing-Yuan

    2016-05-31

    Four novel mononuclear Schiff base copper(ii) complexes, namely, [Cu(L)(OAc)]·H2O (), [Cu(HL)(C2O4)(EtOH)]·EtOH (), [Cu(L)(Bza)] () and [Cu(L)(Sal)] () (HL = 1-(((2-((2-hydroxypropyl)amino)ethyl)imino)methyl)naphthalene-2-ol), Bza = benzoic acid, Sal = salicylic acid), were synthesized and characterized by X-ray crystallography, elemental analysis and infrared spectroscopy. Single-crystal diffraction analysis revealed that all the complexes were mononuclear molecules, in which the Schiff base ligand exhibited different coordination modes and conformations. The N-HO and O-HO inter- and intramolecular hydrogen bonding interactions linked these molecules into multidimensional networks. Their interactions with calf thymus DNA (CT-DNA) were investigated by UV-visible and fluorescence spectrometry, as well as by viscosity measurements. The magnitude of the Kapp values of the four complexes was 10(5), indicating a moderate intercalative binding mode between the complexes and DNA. Electrophoresis results showed that all these complexes induced double strand breaks of pUC19 plasmid DNA in the presence of H2O2 through an oxidative pathway. In addition, the fluorescence spectrum of human serum albumin (HSA) with the complexes suggested that the quenching mechanism of HSA by the complexes was a static process. Moreover, the antiproliferative activity of the four complexes against HeLa (human cervical carcinoma) and HepG-2 (human liver hepatocellular carcinoma) cells evaluated by colorimetric cell proliferation assay and clonogenic assay revealed that all four complexes had improved cytotoxicity against cancer cells. Inspiringly, complex , with salicylic acid as the auxiliary ligand, displayed a stronger anticancer activity, suggesting that a synergistic effect of the Schiff base complex and the nonsteroidal anti-inflammatory drug may be involved in the cell killing process. The biological features of mixed-ligand copper(ii) Schiff base complexes and how acetic auxiliary

  14. Anhydride formation is not a valid mechanism for peptide cleavage by carboxypeptidase-A: a semiempirical reaction pathway study

    NASA Astrophysics Data System (ADS)

    Vardi-Kilshtain, Alexandra; Shoham, Gil; Goldblum, Amiram

    The mechanism of action of zinc metalloproteinases has been studied by following the direct nucleophilic pathway, which has been frequently suggested but not yet examined by computational methods, and comparing it to other pathways. We computed the reaction enthalpies for the direct nucleophilic attack by Glu270 in the active site model of carboxypeptidase-A on a model substrate's peptide carbonyl and followed this pathway through mixed anhydride formation and subsequent anhydride cleavage by water. The starting molecular coordinates originate in our own high-resolution crystal structure and the computations have been conducted with the minimal neglect of differential overlap (MNDO) Hamiltonian, modified to include the d-orbitals of zinc and the effects of multiple hydrogen bonding, thus labelled MNDO/d/H. Compared to our recent results for two other candidate pathways for this mechanism, both of the General-Acid-General-Base type, we conclude that the direct nucleophilic or 'anhydride' pathway has a much higher energy barrier at the rate determining step, which is a proton transfer, than previously calculated paths. We argue that the 'anhydride' pathway is thus not a valid one for the cleavage of peptides by carboxypeptidase-A.

  15. RNA Study Using DNA Nanotechnology.

    PubMed

    Tadakuma, Hisashi; Masubuchi, Takeya; Ueda, Takuya

    2016-01-01

    Transcription is one of the fundamental steps of gene expression, where RNA polymerases (RNAPs) bind to their template genes and make RNAs. In addition to RNAP and the template gene, many molecules such as transcription factors are involved. The interaction and the effect of these factors depend on the geometry. Molecular layout of these factors, RNAP and gene is thus important. DNA nanotechnology is a promising technology that allows controlling of the molecular layout in the range of nanometer to micrometer scale with nanometer resolution; thus, it is expected to expand the RNA study beyond the current limit. PMID:26970193

  16. Mutation, DNA strand cleavage and nitric oxide formation caused by N-nitrosoproline with sunlight: a possible mechanism of UVA carcinogenicity.

    PubMed

    Arimoto-Kobayashi, Sakae; Ando, Yoshiko; Horai, Yumi; Okamoto, Keinosuke; Hayatsu, Hikoya; Lowe, Jillian E; Green, Michael H L

    2002-09-01

    N-Nitrosoproline (NPRO) is endogenously formed from proline and nitrite. NPRO has been reported to be nonmutagenic and noncarcinogenic. In this study, we have detected the direct mutagenicity of NPRO plus natural sunlight towards Salmonella typhimurium. Furthermore, formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a mutagenic lesion, was observed in calf thymus DNA treated with NPRO plus simulated sunlight. The treatment with NPRO and sunlight induced single strand breaks in the superhelical replicative form of phage M13mp2 DNA. Single-strand DNA breaks also occurred in the human fibroblast cells on treatment with NPRO plus UVA, as detected by the comet assay. An analysis using scavengers suggested that both reactive oxygen species and NO radical mediate the strand breaks. The formation of nitric oxide was observed in NPRO solution irradiated with UVA. We analyzed the photodynamic spectrum of mutation induction and DNA breakage using monochromatic radiation at a series of wavelengths between 300 and 400 nm. Both mutation frequencies and DNA breakage were highest at the absorption maximum of NPRO, 340 nm. The co-mutagenic and co-toxic actions of NPRO and sunlight merit attention as possible mechanisms increasing the carcinogenic risk from UVA irradiation. PMID:12189198

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

    PubMed

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

    2014-07-01

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

  18. Comparative study of HOCl-inflicted damage to bacterial DNA ex vivo and within cells.

    PubMed

    Suquet, Christine; Warren, Jeffrey J; Seth, Nimulrith; Hurst, James K

    2010-01-15

    The prospects for using bacterial DNA as an intrinsic probe for HOCl and secondary oxidants/chlorinating agents associated with it has been evaluated using both in vitro and in vivo studies. Single-strand and double-strand breaks occurred in bare plasmid DNA that had been exposed to high levels of HOCl, although these reactions were very inefficient compared to polynucleotide chain cleavage caused by the OH.-generating reagent, peroxynitrite. Plasmid nicking was not increased when intact Escherichia coli were exposed to HOCl; rather, the amount of recoverable plasmid diminished in a dose-dependent manner. At concentration levels of HOCl exceeding lethal doses, genomic bacterial DNA underwent extensive fragmentation and the amount of precipitable DNA-protein complexes increased several-fold. The 5-chlorocytosine content of plasmid and genomic DNA isolated from HOCl-exposed E. coli was also slightly elevated above controls, as measured by mass spectrometry of the deaminated product, 5-chlorouracil. However, the yields were not dose-dependent over the bactericidal concentration range. Genomic DNA recovered from E. coli that had been subjected to phagocytosis by human neutrophils occasionally showed small increases in 5-chlorocytosine content when compared to analogous cellular reactions where myeloperoxidase activity was inhibited by azide ion. Overall, the amount of isolable 5-chlorouracil from the HOCl-exposed bacterial cells was far less than the damage manifested in polynucleotide bond cleavage and cross-linking. PMID:19850004

  19. Photoleucine Survives Backbone Cleavage by Electron Transfer Dissociation. A Near-UV Photodissociation and Infrared Multiphoton Dissociation Action Spectroscopy Study

    NASA Astrophysics Data System (ADS)

    Shaffer, Christopher J.; Martens, Jonathan; Marek, Aleš; Oomens, Jos; Tureček, František

    2016-04-01

    We report a combined experimental and computational study aimed at elucidating the structure of N-terminal fragment ions of the c type produced by electron transfer dissociation of photo-leucine (L*) peptide ions GL*GGKX. The c 4 ion from GL*GGK is found to retain an intact diazirine ring that undergoes selective photodissociation at 355 nm, followed by backbone cleavage. Infrared multiphoton dissociation action spectra point to the absence in the c 4 ion of a diazoalkane group that could be produced by thermal isomerization of vibrationally hot ions. The c 4 ion from ETD of GL*GGK is assigned an amide structure by a close match of the IRMPD action spectrum and calculated IR absorption. The energetics and kinetics of c 4 ion dissociations are discussed.

  20. Photoleucine Survives Backbone Cleavage by Electron Transfer Dissociation. A Near-UV Photodissociation and Infrared Multiphoton Dissociation Action Spectroscopy Study.

    PubMed

    Shaffer, Christopher J; Martens, Jonathan; Marek, Aleš; Oomens, Jos; Tureček, František

    2016-07-01

    We report a combined experimental and computational study aimed at elucidating the structure of N-terminal fragment ions of the c type produced by electron transfer dissociation of photo-leucine (L*) peptide ions GL*GGKX. The c 4 ion from GL*GGK is found to retain an intact diazirine ring that undergoes selective photodissociation at 355 nm, followed by backbone cleavage. Infrared multiphoton dissociation action spectra point to the absence in the c 4 ion of a diazoalkane group that could be produced by thermal isomerization of vibrationally hot ions. The c 4 ion from ETD of GL*GGK is assigned an amide structure by a close match of the IRMPD action spectrum and calculated IR absorption. The energetics and kinetics of c 4 ion dissociations are discussed. Graphical Abstract ᅟ. PMID:27059977

  1. Photoleucine Survives Backbone Cleavage by Electron Transfer Dissociation. A Near-UV Photodissociation and Infrared Multiphoton Dissociation Action Spectroscopy Study

    NASA Astrophysics Data System (ADS)

    Shaffer, Christopher J.; Martens, Jonathan; Marek, Aleš; Oomens, Jos; Tureček, František

    2016-07-01

    We report a combined experimental and computational study aimed at elucidating the structure of N-terminal fragment ions of the c type produced by electron transfer dissociation of photo-leucine (L*) peptide ions GL*GGKX. The c 4 ion from GL*GGK is found to retain an intact diazirine ring that undergoes selective photodissociation at 355 nm, followed by backbone cleavage. Infrared multiphoton dissociation action spectra point to the absence in the c 4 ion of a diazoalkane group that could be produced by thermal isomerization of vibrationally hot ions. The c 4 ion from ETD of GL*GGK is assigned an amide structure by a close match of the IRMPD action spectrum and calculated IR absorption. The energetics and kinetics of c 4 ion dissociations are discussed.

  2. Synthesis, characterization and multi-spectroscopic DNA interaction studies of a new platinum complex containing the drug metformin

    NASA Astrophysics Data System (ADS)

    Shahabadi, Nahid; Heidari, Leila

    2014-07-01

    A new platinum(II) complex; [Pt(Met)(DMSO)Cl]Cl in which Met = metformin and DMSO: dimethylsulfoxide, was synthesized and characterized by 1H NMR, IR, UV-Vis spectra, molar conductivity and computational methods. Binding interaction of this complex with calf thymus (CT) DNA has been investigated by using absorption, emission, circular dichroism, viscosity measurements, differential pulse voltammetry and cleavage studies by agarose gel electrophoresis. UV-Vis absorption studies showed hyperchromism. CD studies showed less perturbation on the base stacking and helicity bands in the CD spectrum of CT-DNA (B → C structural transition). In fluorimeteric studies, the Pt(II) complex can bind with DNA-NR complex and forms a new non-fluorescence adduct. The anodic peak current in the differential pulse voltammogram of the Pt(II) complex decreased gradually with the addition of DNA. Cleavage experiments showed that the Pt(II) complex does not induce any cleavage under the experimental setup. Finally all results indicated that Pt(II) complex interact with DNA via groove binding mode.

  3. Mass Spectrometry and Theoretical Studies on N-C Bond Cleavages in the N-Sulfonylamidino Thymine Derivatives

    NASA Astrophysics Data System (ADS)

    Kobetić, Renata; Kazazić, Snježana; Kovačević, Borislav; Glasovac, Zoran; Krstulović, Luka; Bajić, Miroslav; Žinić, Biserka

    2015-05-01

    The reactivity of new biologically active thymine derivatives substituted with 2-(arylsulfonamidino)ethyl group at N1 and N3 position was investigated in the gas phase using CID experiments (ESI-MS/MS) and by density functional theory (DFT) calculations. Both derivatives show similar chemistry in the negative mode with a retro-Michael addition (Path A-) being the most abundant reaction channel, which correlate well with the fluoride induced retro-Michael addition observed in solution. The difference in the fragmentation of N-3 substituted thymine 5 and N-1 substituted thymine 1 in the positive mode relates to the preferred cleavage of the sulfonyl group ( m/z 155, Path B) in N-3 isomer and the formation of the acryl sulfonamidine 3 ( m/z 309) via Path A in N-1 isomer. Mechanistic studies of the cleavage reaction conducted by DFT calculations give the trend of the calculated activation energies that agree well with the experimental observations. A mechanism of the retro-Michael reaction was interpreted as a McLafferty type of fragmentation, which includes Hβ proton shift to one of the neighboring oxygen atoms in a 1,5-fashion inducing N1(N3)-Cα bond scission. This mechanism was found to be kinetically favorable over other tested mechanisms. Significant difference in the observed fragmentation pattern of N-1 and N-3 isomers proves the ESI-MS/MS technique as an excellent method for tracking the fate of similar sulfonamidine drugs. Also, the observed N-1 and/or N-3 thymine alkylation with in situ formed reactive acryl sulfonamidine 3 as a Michael acceptor may open interesting possibilities for the preparation of other N-3 substituted pyrimidines.

  4. Double-stranded DNA-templated cleavage of oligonucleotides containing a P3'->N5' linkage triggered by triplex formation: the effects of chemical modifications and remarkable enhancement in reactivity.

    PubMed

    Ito, Kosuke Ramon; Kodama, Tetsuya; Tomizu, Masaharu; Negoro, Yoshinori; Orita, Ayako; Osaki, Tomohisa; Hosoki, Noritsugu; Tanaka, Takaya; Imanishi, Takeshi; Obika, Satoshi

    2010-11-01

    We recently reported double-stranded DNA-templated cleavage of oligonucleotides as a sequence-specific DNA-detecting method. In this method, triplex-forming oligonucleotides (TFOs) modified with 5'-amino-2',4'-BNA were used as a DNA-detecting probe. This modification introduced a P3'→N5' linkage (P-N linkage) in the backbone of the TFO, which was quickly cleaved under acidic conditions when it formed a triplex. The prompt fission of the P-N linkage was assumed to be driven by a conformational strain placed on the linkage upon triplex formation. Therefore, chemical modifications around the P-N linkage should change the reactivity by altering the microenvironment. We synthesized 5'-aminomethyl type nucleic acids, and incorporated them into TFOs instead of 5'-amino-2',4'-BNA to investigate the effect of 5'-elongation. In addition, 2',4'-BNA/LNA or 2',5'-linked DNA were introduced at the 3'- and/or 5'-neighboring residues of 5'-amino-2',4'-BNA to reveal neighboring residual effects. We evaluated the triplex stability and reaction properties of these TFOs, and found out that chemical modifications around the P-N linkage greatly affected their reaction properties. Notably, 2',5'-linked DNA at the 3' position flanking 5'-amino-2',4'-BNA brought significantly higher reactivity, and we succeeded in indicating that a TFO with this modification is promising as a DNA analysis tool. PMID:20615902

  5. Micromanipulation Study of DNA, DNA-protein Interactions, and Chromosomes

    NASA Astrophysics Data System (ADS)

    Marko, John F.

    2002-03-01

    Physical - and to a large degree mechanical - properties of nucleic acids and proteins are essential to their functions in living cells. Therefore understanding the micromechanics of biomolecules is important to understanding cell machinery. I will review the rapid progress that has been made over the last decade in studying physical properties of single DNA molecules using micromanipulation techniques, and the future research directions for this field. I will then discuss how our lab has started to study whole chromosomes from cells with micromanipulation techniques. I focus on the use of in-situ enzyme reactions to show that DNA itself is the contiguous load-bearing element of the folded chromosome.

  6. 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.

  7. A new ternary copper(II) complex derived from 2-(2'-pyridyl)benzimidazole and glycylglycine: synthesis, characterization, DNA binding and cleavage, antioxidation and HSA interaction.

    PubMed

    Fu, Xia-Bing; Lin, Zi-Hua; Liu, Hai-Feng; Le, Xue-Yi

    2014-03-25

    A new ternary copper(II)-dipeptide complex [Cu(glygly)(HPB)(Cl)]⋅2H2O (glygly=glycylglycine anion, HPB=2-(2'-pyridyl)benzimidazole) has been synthesized and characterized. The DNA interaction of the complex was studied by spectroscopic methods, viscosity, and electrophoresis measurements. The antioxidant activity was also investigated using the pyrogallol autoxidation assay. Besides, the interaction of the complex with human serum albumin (HSA) in vitro was examined by multispectroscopic techniques. The complex partially intercalated to CT-DNA with a high binding constant (Kb=7.28×10(5) M(-1)), and cleaved pBR322 DNA efficiently via an oxidative mechanism in the presence of Vc, with the HO· and O2(-) as the active species, and the SOD as a promoter. Furthermore, the complex shows a considerable SOD-like activity with the IC50 value of 3.8386 μM. The complex exhibits desired binding affinity to HSA, in which hydrogen bond or vander Waals force played a major role. The alterations of HSA secondary structure induced by the complex were confirmed by UV-visible, CD, synchronous fluorescence and 3D fluorescence spectroscopy. PMID:24291450

  8. Studies of DNA-carbon nanotube interactions

    NASA Astrophysics Data System (ADS)

    Hughes, Mary Elizabeth

    2008-10-01

    Recently a new biomaterial consisting of a DNA-wrapped single-walled carbon nanotube, and known as a DNA/SWNT, has been discovered. The possible applications of this hybrid are varied and range from genomic sequencing to nanoscale electronics to molecular delivery. The realization of these potential applications requires more knowledge about the microscopic properties of this material. In this thesis, I present studies of: the orientation of nucleobases on the nanotube sidewall; the sequence and length dependence of the DNA-nanotube interaction; and solution conditions to manipulate the DNA/SWNT hybrid. The measurement of the UV optical absorbance of DNA/SWNT and the nucleotide absorbance from DNA/SWNT provide the first experimental confirmation that DNA binds to nanotubes through pi-stacking. Because the hypochromic absorbance typical of pi-stacked structures are expected to occur primarily for DNA dipole transitions that lie along the axis of the optically anisotropic SWNTs, the absorbance changes following binding of DNA to the nanotubes reveals the preferred orientation assumed by each of the four bound nucleotides with respect to the nanotube's long axis. The first observations of pronounced sequence- and length-dependent variations in the binding between ssDNA and SWNTs in aqueous solution are presented. These observations rely on the discovery that there exists a range of DNA lengths able to hybridize with SWNTs that can nevertheless be dissociated at temperatures below the boiling point of water. Quantitative results comparing the isochronal dissociation temperatures and binding energies of DNA/SWNT composed of differing DNA sequences and lengths are given. These results indicate variability and complexity in the binding mechanism responsible for the stability of the hybrid system that transcends simple models based on the sum of independent base-nanotube interactions. Binding energies between a DNA base and nanotube (0.05 to 0.09 eV per base) are similar

  9. 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.

  10. Label-Free and Sensitive Fluorescent Detection of Sequence-Specific Single-Strand DNA Based on S1 Nuclease Cleavage Effects

    PubMed Central

    Guan, Zheng; Liu, Jinchuan; Bai, Wenhui; Lv, Zhenzhen; Jiang, Xiaoling; Yang, Shuming; Chen, Ailiang; Lv, Guiyuan

    2014-01-01

    The ability to detect sequence-specific single-strand DNA (ssDNA) in complex, contaminant-ridden samples, using a fluorescent method directly without a DNA extraction and PCR step could simplify the detection of pathogens in the field and in the clinic. Here, we have demonstrated a simple label-free sensing strategy to detect ssDNA by employing its complementary ssDNA, S1 nuclease and nucleic acid fluorescent dyes. Upon clearing away redundant complementary ssDNA and possibly mismatched double strand DNA by using S1 nuclease, the fluorescent signal-to-noise ratio could be increased dramatically. It enabled the method to be adaptable to three different types of DNA fluorescent dyes and the ability to detect target ssDNA in complex, multicomponent samples, like tissue homogenate. The method can distinguish a two-base mismatch from avian influenza A (H1N1) virus. Also, it can detect the appearance of 50 pM target ssDNA in 0.5 µg·mL−1 Lambda DNA, and 50 nM target ssDNA in 5 µg·mL−1 Lambda DNA or in tissue homogenate. It is facile and cost-effective, and could be easily extended to detect other ssDNA with many common nucleic acid fluorescent dyes. PMID:25285445