An Extracellullar Nuclease from Bacillus Firmus VKPACU-1: Specificity and Mode of Action

An extracellular nuclease from Bacillus firmus VKPACU-1 was multifunctional enzyme, this nuclease hydrolyzed poly U rapidly and more preferentially than the other homopolyribonucleotides. Hydrolysis of RNA this enzyme released mononucleotides in the order 5′UMP > 5′AMP > 5′GMP where as in hydrolysis of DNA the mononucleotides in the order of 5′dAMP > 5′dGMP > 5′dTMP and oligonucleotides. Uridylic linkages in RNA and adenylic linkages in DNA were preferentially cleaved by the nuclease. Nuclease produced oligonucleotides having only 3’ hydroxyl and 5’ phosphate termini. Present nuclease hydrolyzed RNA and DNA released oligonucleotides as major end products and mononucleotides, suggesting an endo mode of action.     

Evolution of 1, 3, 5-trisubstituted bipyrazole scaffold based platinum(II) complexes as a biological active agent

Abstract

Square planar mononuclear platinum(II) complexes having general formula [Pt(Lⁿ)Cl₂], (where, Lⁿ?=?L^1–4) were synthesized with neutral bidentate heterocyclic 1,3,5-trisubstituted bipyrazole based ligands. The synthesized compounds were characterized by physicochemical method such as TGA, molar conductance, micro-elemental analysis and magnetic moment, and spectroscopic method such as, FT-IR, UV–vis, ¹H NMR, ¹³C NMR and mass spectrometry. Biological applications of the compounds were carried out using in vitro brine shrimp lethality bioassay, in vitro antimicrobial study against five different pathogens, and cellular level cytotoxicity against Schizosaccharomyces pombe (S. Pombe) cells. Pt(II) complexes were tested for DNA interaction activities using electronic absorption titration, viscosity measurements study, fluorescence quenching technique and molecular docking assay. Binding constants (K_b) of ligands and complexes were observed in the range of 0.23–1.07?×?10⁵?M^?1 and 0.51–3.13?×?10⁵?M^?1, respectively. Pt(II) complexes (I–IV) display an excellent binding tendency to biomolecule (DNA) and possess comparatively high binding constant (K_b) values than the ligands. The DNA binding study indicate partial intercalative mode of binding in complex-DNA. The gel electrophoresis activity was carried out to examine DNA nuclease property of pUC19 plasmid DNA.     

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo,Orthologue of Human Progeroid WRN Exonuclease,and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.     

ATP‐dependent DNA binding,unwinding, and resection by the Mre11/Rad50 complex

ATP‐dependent DNA end recognition and nucleolytic processing are central functions of the Mre11/Rad50 (MR) complex in DNA double‐strand break repair. However, it is still unclear how ATP binding and hydrolysis primes the MR function and regulates repair pathway choice in cells. Here, Methanococcus jannaschii MR‐ATPγS‐DNA structure reveals that the partly deformed DNA runs symmetrically across central groove between two ATPγS‐bound Rad50 nucleotide‐binding domains. Duplex DNA cannot access the Mre11 active site in the ATP‐free full‐length MR complex. ATP hydrolysis drives rotation of the nucleotide‐binding domain and induces the DNA melting so that the substrate DNA can access Mre11. Our findings suggest that the ATP hydrolysis‐driven conformational changes in both DNA and the MR complex coordinate the melting and endonuclease activity.     

基因前定点敲入FLAG标签表达的FLAG-SND1蛋白参与胞内应激颗粒的形成

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-Cas9 nuclease) 基因编辑技术是近年来新兴的一种可以实现基因特异性敲除和敲入的技术。本文利用CRISPR/Cas9基因编辑系统,将3×FLAG标签定点敲入HeLa细胞SND1基因前方,使细胞内源性表达的SND1蛋白带有3×FLAG标签,并观察SND1与应激颗粒及加工体的定位情况。设计针对SND1基因起始密码子ATG附近的sgRNA,以px459为表达载体,构建出重组真核表达质粒。设计含有3×FLAG及待插入位置上下游150 bp同源臂的序列,经公司合成获得重组质粒。将2个质粒共同转染HeLa细胞,使用嘌呤霉素筛选阳性细胞,挑取单克隆后培养。Western 印迹表明,细胞表达3×FLAG-SND1融合蛋白质。提取细胞基因组DNA进行测序。测序无误获得稳定株后,用流式细胞术检测细胞周期和凋亡,发现与WT细胞相比无显著性差异。同时,使用0.5 mmol/L亚砷酸钠处理,细胞发生氧化应激,eIF2α蛋白磷酸化增加,胞浆中出现应激颗粒,SND1与应激颗粒标志蛋白TIAR存在共定位现象,但不存在与加工体蛋白DCP1α的共定位。     

Effect of siRNA nuclease stability on the in vitro and in vivo kinetics of siRNA-mediated gene silencing

Small interfering RNA (siRNA) molecules achieve sequence-specific gene silencing through the RNA interference (RNAi) mechanism. Here, live-cell and live-animal bioluminescent imaging (BLI) is used to directly compare luciferase knockdown by unmodified and nuclease-stabilized siRNAs in rapidly (HeLa) and slowly (CCD-1074Sk) dividing cells to reveal the impact of cell division and siRNA nuclease stability on the kinetics of siRNA-mediated gene silencing. Luciferase knockdown using unmodified siRNAs lasts approximately 1 week in HeLa cells and up to 1 month in CCD-1074Sk cells. There is a slight increase in the duration of luciferase knockdown by nuclease-stabilized siRNAs relative to unmodified siRNAs after cationic lipid transfection, but this difference is not observed after electroporation. In BALB/cJ mice, a fourfold increase in maximum luciferase knockdown is observed after hydrodynamic injection (HDI) of nuclease-stabilized siRNAs relative to unmodified siRNAs, yet the overall kinetics of the recovery after knockdown are nearly identical. By using a mathematical model of siRNA-mediated gene silencing, the trends observed in the experimental data can be duplicated by changing model parameters that affect the stability of the siRNAs before they reach the cytosolic compartment. Based on these findings, we hypothesize that the stabilization advantages of nuclease-stabilized siRNAs originate primarily from effects prior to and during internalization before the siRNAs can interact with the intracellular RNAi machinery.     

Carbohydrate-based heteronuclear complexes as topoisomerase Iα inhibitor: approach toward anticancer chemotherapeutics

Due to the critical role of cellular enzymes necessary for cell proliferation by deciphering topological hurdles in the process of DNA replication, topoisomerases have been one of the major targets in the anticancer drug development area. A need, therefore, arises for new metallodrugs that specifically recognizes DNA and inhibits the activity of topoisomerase enzymes, herein, we report the synthesis and characterization of new metal-based glycoconjugate entities containing heterobimetallic core Cu^II–Sn^IV (1) and Ni^II–Sn^IV (2) derived from N-glycoside ligand (L). The optimized structure of complex 1 and other significant vibrational modes have been explained using dispersion corrected B3LYP/DFT calculations. In vitro DNA binding profile of the L and both the complexes 1 and 2 were done by various biophysical studies. Complex 1 breaks pBR322 DNA via a hydrolytic means which was validated by T4 DNA enzymatic assay. To get a mechanistic insight of mode of action topoisomerase I (Topo I) inhibition assay was carried out. Also, we have taken the help of molecular modeling studies in accordance with experimental findings. In vitro cytotoxicity of the complex 1 was evaluated against a panel of cancer cells which exhibited remarkably good anticancer activity (GI₅₀ values <10 μg/ml). Moreover, intracellular localization of the complex 1 was visualized by confocal microscopy against HeLa cells.     

The effects of amino acid replacements of glycine 20 on conformational stability and catalysis of staphylococcal nuclease

Staphylococcal nuclease (SNase) is a well-established model for protein folding studies. Its three-dimensional structure has been determined. The enzyme, Ca2+, and DNA or RNA substrate form a ternary complex. Glycine 20 is the second position of the first beta-turn of SNase, which may serve as the folding initiation site for the SNase polypeptide. To study the role of Gly20 in the conformational stability and catalysis of SNase, three mutants, in which Gly20 was replaced by alanine, valine, or isoleucine, were constructed and studied by using circular dichroism spectra, intrinsic and ANS-binding fluorescence spectra, stability and activity assays. The mutations have little effect on the conformational integrity of the mutants. However, the catalytic activity is reduced drastically by the mutations, and the stability of the protein is progressively decreased in the order G20A相似文献   

Development of cell lines stably expressing staphylococcal nuclease fused to dengue 2 virus capsid protein for CTVI

Dengue virus (DV) is one of the most significanthuman viral pathogens transmitted by arthropod vectorsand now present in over 100 countries. Half of the world’spopulation live in areas at risk of dengue virus infection[1]. The infection of DV causes a spectrum of diseasesranging from a debilitating, self-limited illness (denguefever), and life-threaten syndromes (dengue haemorrhagicfever/dengue shock syndrome). Annually, the fourserotypes of DV collectively cause 50–100 million casesof inf…