Novel Halophyte EREBP/AP2-type DNA Binding Protein Improves Salt Tolerance in Transgenic Tobacco

EREBP/AP2-type proteins are members of a large DNA binding protein (DBP) family found in plants. Some members like APETALA2 and AtDREB/CBF can regulate flower development and response to environmental stresses, respectively. To characterize transcription factors involved in plant responses to salt stress, we constructed cDNA library from salt-treated halophyte (Atriplex hortensis) and isolated a novel gene encoding EREBP/AP2-type protein from this library. This cDNA contained an ORF of 723 bp and a long 3'-Untranslated-Region (UTR) of 655 bp. The deduced amino acid sequence showed one conserved DNA binding domain of EREBP/AP2, thus the corresponding gene was named AhDREB1 with a calculated molecular mass of 26.1 kD. AhDREB1 under the control of CaMV 35S promoter was then transformed into tobacco and nine independent transgenic lines were obtained and subjected to long term salt stress. The results suggested that overexpression of AhDREB1 improved the salt tolerance in transgenic tobacco through functioning as a regulatory molecule in response to salt stress. Analysis of Arabidopsis genome in database resulted in dozens of EREBP/AP2-type homologous proteins, of which seven members showed high similarity to AhDREB1. Secondary structure analysis predicted similar arrangement of a-helix in their DNA binding domains.

Inhibitor of DNA binding 2 accelerates nerve regeneration after sciatic nerve injury in mice

Inhibitor of DNA binding 2(Id2)can promote axonal regeneration after injury of the central nervous system.However,whether Id2 can promote axonal regeneration and functional recovery after peripheral nerve injury is currently unknown.In this study,we established a mouse model of bilateral sciatic nerve crush injury.Two weeks before injury,AAV9-Id2-3×Flag-GFP was injected stereotaxically into the bilateral ventral horn of lumbar spinal cord.Our results showed that Id2 was successfully delivered into spinal cord motor neurons projecting to the sciatic nerve,and the number of regenerated motor axons in the sciatic nerve distal to the crush site was increased at 2 weeks after injury,arriving at the tibial nerve and reinnervating a few endplates in the gastrocnemius muscle.By 1 month after injury,extensive neuromuscular reinnervation occurred.In addition,the amplitude of compound muscle action potentials of the gastrocnemius muscle was markedly recovered,and their latency was shortened.These findings suggest that Id2 can accelerate axonal regeneration,promote neuromuscular reinnervation,and enhance functional improvement following sciatic nerve injury.Therefore,elevating the level of Id2 in adult neurons may present a promising strategy for peripheral nerve repair following injury.The study was approved by the Experimental Animal Ethics Committee of Jinan University(approval No.20160302003)on March 2,2016.

A novel detection of single-stranded DNA binding protein based on ss-DNA modified chip using surface plasmon resonance microscopy

An ss-DNA gold chip was prepared based on self-assembly of the thiol-derivatized oligonucleotide, and used for the determination of single-stranded binding protein （SSB） by surface plasmon resonance microscopy （SPR）. The experiment results showed that SSB binds ss-DNA with high specificity, and relative signal of SPR response is proportional to the concentration of SSB in the range of 0.1-100 ng/mL with a detection limit （S/N = 3） of 0.07 ng/mL.

Endonuclease-based Method for Detecting the Sequence Specific DNA Binding Protein on Double-stranded DNA Microarray

The double-stranded DNA (dsDNA) probe contains two different protein binding sites. One is for DNA- binding proteins to be detected and the other is for a DNA restriction enzyme. The two sites were arranged together with no base interval. The working principle of the capturing dsDNA probe is described as follows: the capturing probe can be cut with the DNA restriction enzyme (such as EcoR I) to cause a sticky terminal, if the probe is not bound with a target protein, and the sticky terminal can be extended and labeled with Cy3-dUTP by DNA polymerase. When the probe is bound with a target protein, the probe is not capable to be cut by the restriction enzyme because of space obstruction. The amount of the target DNA binding proteins can be measured according to the variations of fluorescent signals of the corresponding probes.

Identification and characterization of GIP1,an Arabidopsis thaliana protein that enhances the DNA binding affinity and reduces the oligomeric state of G-box binding factors

Environmental control of the alcohol dehydrogenase(Adh)and other stress response genes in plants is in part brought about by transcriptional regulation involving the G-box cis-acting DNA element and bZIP G-box Binding Factors(GBFs).The mechanisms of GBF regulation and requirements for additional factors in this control process are not well understood.In an effort to identify potential GBF binding and control partners,maize GBF1 was used as bait in a yeast two-hybrid screen of an A.thaliana cDNA library.GBF Interacting Protein 1(GIP1)arose from the screen as a 496 amino acid protein with a predicted molecular weight of 53,748 kDa that strongly interacts with GBFs.Northern analysis of A.thaliana tissue suggests a 1.8-1.9 kb GIP1 transcript,predominantly in roots.Immunolocalization studies indicate that GIP1 protein is mainly localized to the nucleus.In vitro electrophoretic mobility shift assays using an Adh G-box DNA probe and recombinant A.thaliana GBF3 or maize GBF1,showed that the presence of GIP1 resulted in a tenfold increase in GBF DNA binding activity without altering the migration,suggesting a transient association between GIP1 and GBF.Addition of GIP1 to intentionally aggregated GBF converted GBF to lower molecular weight macromolecular complexes and GIP1 also refolded denatured rhodanese in the absence of ATP.These data suggest GIP1 functions to enhance GBF DNA binding activity by acting as a potent nuclear chaperone or crowbar,and potentially regulates the multimeric state of GBFs,thereby contributing to bZIP-mediated gene regulation.

Synthesis, Crystal Structure and DNA Binding Studies of α,α'-Bis(3,5-bismethyl-1-pyrozolyl)-carbene-acetyl-isopropenyl Hydrazine

The title compound α,α′-bis（3,5-bismethyl-pyrozole-N-yl）-carbene-acetyl-isopropenyl hydrazine （C16H22N6O, Mr = 314.40） has been prepared. It was characterized by elemental analysis as well as IR, MS, ^1H-NMR and ^13C-NMR spectra. Its crystal structure was determined by single-crystal X-ray diffraction, getting the following data： triclinic, space group P1^- with a = 6.9734（16）, b = 10.773（3）, c = 12.001（3）A, α = 75.311（4）, β = 82.695（4）, ）,γ = 77.143（4）°, Z = 2, V = 847.9（3）A^3, Dc = 1.231 g/cm^3, F（000） = 336 and/μ（MoKα） = 0.082 mm^-1 （λ= 0.71073A）. The results of crystal structure determination show that there exist intermolecular and intramolecular hydrogen bonds, resulting in a two-dimensional supramolecular framework of the title compound. The binding of the title compound to DNA was investigated by absorption, emission, and viscosity measurements. The title compound shows absorption hyperchromicity accompanied by a blue shift at about 254 nm. The binding constant Kb for the title compound has been determined to be 1.89 × 10^4 M^-1 from absorption measurements. The addition of the title compound to DNA pretreated with EB causes appreciable reduction in the emission intensity, indicating that the DNA-bound EB fluorophore is partially replaced by the title compound. The value of K is 3.093 × 10^4 M^-1. The relative viscosity of DNA decreased with the addition of the title compound. Results suggest that the title compound binds to DNA with a non-classical intercalative or groove interaction mode. The observed efficient nuclease activity of the title compound is interesting and may have further influences on the chemistry of DNA minor groove binders.

Synthesis, Characterization, Antimicrobial and DNA Binding Studies of a Tetradentate N2O2 Amino Acid Schiff Base and Its Coordination Compounds

Aminoethanoic acid undergoes condensation with 1,4-benzenedicarboxaldehyde to form an O, N, N, O donor Schiff base, N,N'-di(carboxymethylene) terephthalaldehyde, Ligand L. Coordination compounds of this Schiff base using Ni (II), Cu (II), VO (IV) and Co (II) were then obtained in-situ. The Schiff base and the complexes were evaluated for their antimicrobial and DNA binding abilities. Molecular docking studies of the ligand and synthesized compounds were also carried out. Evidence for the formation of the Schiff base coordination compounds and the coordinating atoms was obtained from 1H NMR, infrared and ultraviolet spectral data, EDX, EDTA complexometric titration and magnetic susceptibility measurement. The results obtained are consistent with octahedral geometry for Ni (II) complex, the metal ion coordinating to one molecule of Ligand L and with additional coordination with two oxygen atoms of two molecules of the solvent. A square-planar geometry was suggested for both Co (II), and Cu (II) complexes and a five-coordinate, square pyramidal geometry for the VO (IV) complex. The results further indicated that the carboxylic acid of Ligand L was not deprotonated both in the free base and also the complexes. In addition, the results showed that Compound 2 elicited the best antimicrobial activity potential. Generally, the compounds exhibited considerable good affinity to CT-DNA.

DNA binding mechanism of WhiB4 from Mycobacterium tuberculosis

Mycobacterium tuberculosis(Mtb),the pathogen of tuberculosis,has latently infected about one-third of the world's population and may lead to severe clinical symptoms and death.The WhiB4 protein,a transcription factor,plays a crucial role in the survival and pathology of Mtb.WhiB4 leads to the condensation of mycobacterial nucleoids and regulates the expression of genes involved in central metabolism,respiration,and maintaining redox homeostasis.Here,we report the solution structure of reduced apo-WhiB4 monomer,which consists of an unstructured N-terminal domain with four cysteine residues and a helix-turnhelix C-terminal domain that plays a major role in DNA binding.The C-terminal domain of WhiB4 binds DNA at the minor groove,with five positively charged lysine/arginine residues contacting DNA sugar-phosphate backbones through electrostatic interactions.AT-rich DNA sequences with narrower minor grooves are more preferred by WhiB4.The binding affinity of a single C-terminal domain of WhiB4 is weak.When oxidized,WhiB4 can form dimers and oligomers in different forms through disulfide bonds,which should significantly enhance its DNA binding ability through multivalent effect and change the local structure of target genes and influence their transcription.These structural features form the basis for WhiB4 to function as a redox-sensitive transcription factor in Mtb.

Synthesis, characterization and DNA binding of the complexes of rare earth with phenanthroline and demethylcantharate

Three novel rare earth complexes, [Ln2（DCA）2（phen）2]（NO3）2·6H2O （Ln（III）=Sm（III）（1）, Er（III）（2）, Yb（III）（3）; DCA2–=de-methylcantharate, 7-oxabicyclo[2.2.1] heptane-2,3-dicarboxylate, C8H8O52–; phen=1,10-phenanthroline, C12H8N2） were synthesized. The structures were characterized by elemental analysis, molar conductance, IR and TGA. The results suggested that the structural features of the complexes were： in each DCA2–, one carboxylate group, as bidentate bridging group, connected two rare earth ions; the other carboxylate group, as bidentate chelate group took part in the coordination with rare earth ion. And cyclic ether oxygen of DCA2– and nitrogen atoms of phen took part in the coordination. The probable coordination number was seven. The interaction of the complexes with DNA was studied by UV-spectra, fluorescence spectra and viscosity measurements. Following increasing the concentration of DNA, the UV absorption bands nearby 265 nm of the three complexes appeared hypochromism and red-shift phenomena. And the values of binding constants Kb were 1.89×105 L/mol （1）, 3.54×104 L/mol （2） and 3.83×104 L/mol （3）. The complexes could quench the fluorescence of EB-DNA system, and the values of equilibrium constants Ksq were 1.72（1）, 0.56（2） and 1.09（3）. The relative viscosity of DNA steadily decreased with increasing the concentration of complexes. So, we could infer that the complexes may partially insert into DNA. The study of agarose gel electrophoresis showed that the complexes could cleave plasmid DNA, and the process of the reaction was through unclassical redox mechanism.

DNA Binding and Oxidative Cleavage by a Water-soluble Carboxyl Manganese（Ⅲ） Corrole

The interaction of calf thymus DNA （CT DNA） and water-soluble manganese corrole, 5,10,15-tris（4-carboxyphenyl）corrolatomanganese（Ⅲ） （MnuTCPC） has been studied by absorption spectra, fluorescence spectra and CD spectra, as well as by viscosity measurements. Results revealed that this manganese corrole binds to CT DNA via an outside groove binding mode with intrinsic binding constant Ku of 4.67 × 104 Lomol L DNA cleavage activities of MnmTCPC in the presence of various oxidants were also investigated, MnmTCPC can cleave the supercoiled plas- mid pBR322 DNA to both nicked and linear form in the presence of hydrogen peroxide or tert-BuOOH, while no nuclease activity was observed by using KHSO5 as oxidant. Inhibitor tests revealed that hydroxyl radicals or singlet oxygen was not involved in MnTCPC mediated DNA oxidative cleavage. It is suggested that （oxo）manganese（V） corrole is the possibly active intermediate in this oxidative cleavage reactions.

Crystal structure and DNA binding studies of a cobalt(Ⅱ) complex containing mixed-ligands of 1,10-phenathroline and glycollic acid

A cobalt（11） complex containing mixed-ligands of 1,10-phenathroline（phen） and glycollic acid（GA）.｜Co（GA）_2（phen）｜ was synthesized and its structure was characterized by X-ray diffraction.The interaction of the complex with DNA was investigated by electronic absorption spectra and electrochemical methods.Electronic absorption spectrum experiments showed that after interaction with DNA.the characteristic absorption peaks of ｜Co（GA）_2（phen）｜ underwent hypochromic effect as well as redshift. Also,the binding strength of 3.8×10~4L/mol was estimated by titration method.Electrochemical assays revealed that the redox peak currents of the complex decreased obviously accompanied by a positive shift of the formal potential after association with DNA.All these results revealed that the synthesized cobalt complex bound with DNA via an intercalation mode.

MSH1 Is a Plant Organellar DNA Binding and Thylakoid Protein under Precise Spatial Regulation to Alter Development

As metabolic centers, plant organelles participate in maintenance, defense, and signaling. MSH1 is a plant- specific protein involved in organeUar genome stability in mitochondria and plastids. Plastid depletion of MSH1 causes heritable, non-genetic changes in development and DNA methylation. We investigated the rash I phenotype using hemi-complementation mutants and transgene-null segregants from RNAi suppres- sion lines to sub-compartmentalize MSH1 effects. We show that MSH1 expression is spatially regulated, specifically localizing to plastids within the epidermis and vascular parenchyma. The protein binds DNA and localizes to plastid and mitochondrial nucleoids, but fractionation and protein-protein interactions data indicate that MSH1 also associates with the thylakoid membrane. Plastid MSH1 depletion results in variegation, abiotic stress tolerance, variable growth rate, and delayed maturity. Depletion from mitochon- dria results in 7%-10% of plants altered in leaf morphology, heat tolerance, and mitochondrlal genome sta- bility. MSH1 does not localize within the nucleus directly, but plastid depletion produces non-genetic changes in flowering time, maturation, and growth rate that are heritable independent of MSH 1. MSH1 deple- tion alters non-photoactive redox behavior in plastids and a sub-set of mitochondrially altered lines. Ectopic expression produces deleterious effects, underlining its strict expression control. Unraveling the complexity of the MSH1 effect offers insight into triggers of plant-specific, transgenerational adaptation behaviors.

Antimicrobial, anticancer and DNA binding studies of transition metal(Ⅱ)complexes containing 2-phenyl-4-selenazole carboxylic acid

Three novel transition metal complexes [ML2（phen）]＇H20 （M= Mn, Co, Zn; HL= C10H702NSe, 2-phenyl-4-selenazole carboxylic acid, phen= 1,10-phenanthroline） 1-3 were synthesized and characterized by elemental analysis, IR spectra. Their crystal structures were deterimined by single-crystal X-ray diffraction method. The antibacterial activities of the complexes against five species of bacteria, Escherichia coli （E. coli）, Staphylococcus epidermidis （S. epidermidis）, Staphylococcus aureus （S. au- reus）, Acinetobacter baumanii （A. baumanii） and Streptococcus viridans （S. viridans）, were tested respectively. The anticancer activities of the complexes against human pancreatic cancer line PANC-28 and human hepatocarcinoma line HuH7 were also studied. The interactions between the complexes and DNA were studied by ethidium bromide （EB） fluorescent probe.

Anticancer activity and DNA binding property of the trimers of triphenylethylene-coumarin hybrids

Novel trimers of triphenylethylene-coumarin hybrid containing two amino side chains （5a-d and 6a-d） were designed and synthesized by the condensation of 1,3,5-benzenetricarboxylic acid with the varied amino monomeric hybrids catalyzed by HATU and DIPEA at room temperature. The extended trimeric compound 6a （R = piperidinyl） exhibited significant anti-proliferative activity against three cancer cells at IC5o of near 10 μmol/L. UV-vis, fluorescence （lifetime） and thermal denaturation exhibited that 6a had significant interaction with Ct-DNA by the intercalative mode of binding. The order of their anti- proliferative activities was 6（a, d） 〉 5（a, d） and （5-6）a 〉 （5-6）d, respectively, in accordance with that of their DNA binding properties, which suggested that the prolonged linker （six carbons） and piperidinyl ~roun on the side chains are beneficial to DNA binding and the anti-tumor activity.

Novel pyrazole fused heterocyclic ligands:Synthesis,characterization,DNA binding/cleavage activity and anti-BVDV activity

A series of novel pyrazole fused heterocyclic derivatives were synthesized via a two-step procedure or a one-pot two step method, and their catalytic DNA cleavage abilities and anti-BVDV activities were also evaluated. The results obtained indicated that compounds 3b-3c could catalyze the cleavage of supercoiled DNA （pUC 19 plasmid DNA） to nicked DNA under physiological conditions with high yields via a hydrolytic mechanism. The studies on anti-viral activities against bovine viral diarrhea virus （BVDV） demonstrated that some of the pyrazole derivatives showed pronounced anti-BVDV activity with interesting ECso values and no significant cytotoxicity. Among them, compound 31 showed the highest antiviral activity （ECso = 0.12 μmol/L） and was 10 fold more than that of the positive control ribavirin （ECso = 1.3 μmol/L）, which provided a potential candidate for the development of anti-BVDV agents.

Synthesis,DNA binding and cleavage studies of the copper(Ⅱ) complexes of PNA-cyclen conjugates

Two dinucleotide PNA-cyclen copper(II) complexes with α-PNA (P1) and classical PNA (P2) backbones were synthesized and characterized.The interactions between title complexes and DNA were investigated under physiological conditions.Fluorescence studies indicate that the binding ability of complex P1 to CT-DNA is as twice as that of P2.DNA melting experiments were also carried out and the results show that ΔTm caused by P1 is higher than that caused by P2.Agarose gel electrophoresis experiments demonstrate that P1 is an excellent chemical nuclease,which can cleavage plasmid DNA completely in 12 h.

Cytotoxic activity and DNA binding of naphthalimide derivatives with amino acid and dichloroacetamide functionalizations

A series of novel naphthalimide derivatives modified by amino acids and their dichloroacetamide derivatives at the 3-position have been synthesized. Their cytotoxic activities were preliminarily evaluated against He/a, A549 and K562 cells, which showed that the length of the side chains of the amino acids influenced the cytotoxic activities. Moreover, compound 7d showed a very good cytotoxic activity against A549 cells with an IC50 value of 4.78 μmol L-1, Furthermore, the UV-vis, fluorescence, and circular dichroism （CD） spectroscopies and thermal denaturation experiment indicated that compounds 6a, 6d and 7a, 7d, as DNA intercalators, exhibited binding affinities with calf-thymus DNA （Ct-DNA）.

Studies on the DNA binding and cleavage activity of a synthesized polyamide containing dipeptide Ser-His

The DNA binding and cleavage activity of a synthetic polyamide containing dipeptide Ser-His has been investigated by spectroscopic techniques,such as electronic absorbance and circular dichroism(CD)spectropolarimetry,as well as gel electrophoresis.The results show that the molecule has a strong interaction with DNA and can improve DNA-cleavage ability about 100 fold as compared with single dipeptide Ser-His.

Characterization, Crystal Structure and Initial DNA Binding Interaction of Two Cu（Ⅱ） Complexes with 4,5-Diazafluoren-9-one

Two new complexes [Cu（dafo）2（en）]（ClO4）2·2H2O （en=NH2CH2CH2NH2） 1 and [Cu（dafo）2（dap）]（ClO4）2·2H2O [dap=NH2CH2CH（CH3）NH2] 2 （dafo=4,5-diazafluoren-9-one） have been synthesized and characterized by elemental analysis, IR and UV spectra. Meanwhile, the complex 1 has been characterized by single crystal X-ray diffraction analysis. The initial DNA binding interactions of the complexes 1 and 2 have been investigated by UV spectra, emission spectra and cyclic voltammogram. Concluding the results of three methods used to measure the interaction of complexes 1 and 2 with DNA, the action mode of the two complexes with DNA is intercalation, and character of ligands and steric effect may affect the interaction of the complexes with DNA.

DNA Binding Properties and Cytotoxic Activities of Two Trinuclear Ruthenium（Ⅱ） Polypyridyl Complexes

The interactions between two trinuclear Ru（II） complexes and calf thymus DNA（CT DNA） were studied via absorption spectroscopy, reverse salt titrations, binding stoichiometry, DNA melting experiments, as well as viscosity measurement. The results indicate that complexes 1 and 2 bind to DNA via the interaction of the planar π-delocalized system of the complexes with intrinsic binding constants of 4.18 × 10^5 and 3.85 × 10^6 L/tool, respectively, and non-electrostatic binding free energy makes a predominant contribution to the binding free energy. The in vitro cytotoxic activity of complexes 1 and 2 was evaluated by the MTT[3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl- 2H-tetrazolium bromide] method. Complex I shows higher anticancer potency than complex 2 against four tumor cell lines. Further mechanism study indicates that complexes 1 and 2 can cause cell cycle arrest in the G2/M phase.