DrugBank:BIOD00001 - FACTA Search

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Query: DrugBank:BIOD00001 (DNase I)
8,324 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Initial experiments in our laboratory have successfully reassembled infectious polyoma virions from dissociated virion products. Virions treated with ethyleneglycol-bis-N,N'-tetraacetic acid and the reducing agent beta-mercaptoethanol at pH 7.5 were dissociated to a 48S DNA-protein complex and capsomere subunits. The virion dissociation products were not infectious by plaque assay and lacked hemagglutination activity. These virion dissociation products were reassembled to intact virions by overnight dialysis against a reassembly buffer containing CaCl2, dimethyl sulfoxide, and Triton X-100 in phosphate-buffered saline at pH 7.4. The biophysical characteristics of the reassembled virions were identical to those of untreated virions in that the reassembled virions had a sedimentation value of 240S in sucrose gradients and a buoyant density of 1.315 g/cm3 in CsCl isopycnic gradients. The reassembled virions were intact as determined by electron microscopy and were found to be 60% resistant to DNase I treatment. Biologically, the reassembled purified virions were found to partially regain both hemagglutinating activity and plaque-forming ability.
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PMID:In vitro reassembly of infectious polyoma virions. 22 81

Juxtanuclear birefringent caps (FC) containing 10-nm filaments form during the early stages of baby hamster kidney (BHK-21) cell spreading. FC are isolated from spreading cells after replating by treatment with 0.6 M KCl, 1% Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.) and DNase I in phosphate-buffered saline. Purified FC are birefringent and retain the pattern of distribution of 10-nm filaments that is seen in situ. Up to 90% of the FC protein is resolved as two polypeptides of approximately 54,000 and 55,000 molecular weight on sodium dodecyl sulfate (SDS) polyacrylamide gels. The protein is immunologically and biochemically distinct from tubulin as determined by indirect immunofluorescence, double immunodiffusion, one-dimensional peptide mapping by limited proteolysis in SDS gels, and amino acid analysis. The BHK-21 FC amino acid composition, however, is very similar to that obtained for 10-nm filament protein derived from other sources including brain and smooth muscle. Partial disassembly of 10-nm filaments has been achieved by treatment of FC with 6 mM sodium-potassium phosphate buffer, pH 7.4. The solubilized components assemble into distinct 10-nm filaments upon the addition of 0.171 M sodium chloride.
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PMID:Biochemical and immunological analysis of rapidly purified 10-nm filaments from baby hamster kidney (BHK-21) cells. 56 63

A procedure for the de novo construction of nucleosome core particles from defined DNA sequences of prokaryotic origin is described. Efficient de novo reconstitution without added carrier DNA is demonstrated. DNase I and exonuclease III analysis of a nucleosome core prepared from a 154 base pair fragment extending from base 853 to base 1006 of pBR322 indicates a non-random positioning of the histone core along the DNA. As bacteria have no histones, their DNA cannot be expected to have a histone core positioning signal encoded in it, the efficient formation of a uniquely positioned core particle is not self evident. The possibility that a phosphate end group positions DNA fragments on the histone is considered. The de novo reconstitution of carrier-less defined nucleosome core particles should facilitate the physicochemical study of nucleosomes on the fine structural level.
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PMID:Construction of nucleosome cores from defined DNA sequences of prokaryotic origin. 141 62

A 45 kDa protein in Dictyostelium discoideum cells that was recognized by a phosphotyrosine-specific antibody was identified by its binding activity to DNase I and its 2D-electrophoretic behavior as actin. The reactivity of actin with the antibody was transiently enhanced for about 30 minutes shortly after starving cells were reintroduced into nutrient medium. This effect indicates a modification of actin that is regulated under physiological conditions. A similar effect was obtained when growing cells were treated with phenylarsine oxide (PAO), an inhibitor of phosphotyrosine phosphatases. This effect was reversed and the cells fully recovered upon addition of the PAO antagonist 2,3-dimercaptopropanol. Starved cells did not show this enhancement of antibody labelling, which indicates that the response to PAO depends on the developmental stage. Phosphorylated amino acid residues were identified after in vivo labelling with [32P]phosphate in the presence of PAO. Part of the radioactivity in the actin band was recovered as phosphotyrosine, another part as phosphoserine. PAO caused the cells to form elongated blebs, to round up and finally to become immobilized. Fluorescence labelling with phalloidin of cells that were fixed at different times of PAO treatment revealed a progressive decrease in the staining for actin filaments and showed that these alterations in cytoskeleton organization were readily reversible, in accordance with the reversal of tyrosine phosphorylation at actin.
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PMID:Stage-specific tyrosine phosphorylation of actin in Dictyostelium discoideum cells. 150 36

The crystal structure of a complex between DNase I and the self-complementary octamer duplex d(GGTATACC)2 has been solved using the molecular replacement method and refined to a crystallographic R-factor of 18.8% for all data between 6.0 and 2.3 A resolution. In contrast to the structure of the DNase I-d(GCGATCGC)2 complex solved previously, the DNA remains uncleaved in the crystal. The general architecture of the two complexes is highly similar. DNase I binds in the minor groove of a right-handed DNA duplex, and to the phosphate backbones on either side over five base-pairs, resulting in a widening of the minor groove and a concurrent bend of the DNA away from the bound enzyme. There is very little change in the structure of the DNase I on binding the substrate. Many other features of the interaction are conserved in the two complexes, in particular the stacking of a deoxyribose group of the DNA onto the side-chain of a tyrosine residue (Y76), which affects the DNA conformation and the binding of an arginine side-chain in the minor groove. Although the structures of the DNA molecules appear at first sight rather similar, detailed analysis reveals some differences that may explain the relative resistance of the d(GGTATACC)2 duplex to cleavage by DNase I: whilst some backbone parameters are characteristic of a B-conformation, the spatial orientation of the base-pairs in the d(GGTATACC)2 duplex is close to that generally observed in A-DNA. These results further support the hypothesis that the minor-groove width and depth and the intrinsic flexibility of DNA are the most important parameters affecting the interaction. The disposition of residues around the scissile phosphate group suggests that two histidine residues, H134 and H252, are involved in catalysis.
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PMID:X-ray structure of the DNase I-d(GGTATACC)2 complex at 2.3 A resolution. 151 54

The glp regulon of Escherichia coli encodes the proteins required for utilization of sn-glycerol 3-phosphate and its precursors. Transcription of the divergently transcribed glpTQ and glpACB operons is initiated at sites separated by 132 base pairs (bp) of DNA. These operons are controlled negatively by glp repressor and positively by the cAMP-cAMP receptor protein (CRP) complex. The locations of the binding sites for the glp repressor and for cAMP.CRP in the control regions of these operons were determined by DNase I footprinting. Binding of the glp repressor protected the region -32 to -51 (OT) in the glpTQ promoter, which was also the binding site for cAMP.CRP. Four repressor binding sites (-41 to -60 (OA1), -9 to -28 (OA2), +12 to -8 (OA3), and +52 to +33 (OA4)) and two cAMP.CRP binding sites (+11 to -11 and -30 to -51) were found in the glpACB promoter region. Comparison of the sequences of the repressor binding sites found in the glpTQ-glpACB control region with those operators previously described in the glpD operon allowed formulation of a consensus operator sequence which was the palindrome 5'-WATGTTCGWTAWC-GAACATW-3' (W is A or T). The role of each operator was assessed by measuring repression in constructs where individual operators were altered by site-directed mutagenesis. Alteration of OT did not significantly decrease repression of either operon. Each of the glpACB operators contributed to repression of both operons. These results suggest involvement of glpACB operator(s) in control of glpTQ expression perhaps via formation of a repression loop. Evidence supporting this hypothesis was obtained by measuring the degree of repression of the glpTQ promoter in constructs containing 6- or 10-bp insertions between the glpTQ and glpACB operators. A 6-bp insertion located within OA2 or between OT and OA1 eliminated repression of the glpTQ promoter, whereas significant repression was maintained in the case of a 10-bp insertion within OA2.
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PMID:Interaction at a distance between multiple operators controls the adjacent, divergently transcribed glpTQ-glpACB operons of Escherichia coli K-12. 155 20

The effects of the neutral salt concentration, pH, and coexistence of myosin on the denaturation of F-actin without ATP at low temperature were studied using the DNase I inhibition assay. The percent denaturation of F-actin gradually increased with a decrease in pH from 8.0 to 5.2, on incubation for 2 weeks in the presence of 50 mM KCl at 0 degrees C. This change was much faster in 0.5 M KCl and more than 75% of the F-actin became denatured on incubation for 1 week at pH 5.2. The buffer composition was found to exert a strong influence on the denaturation of F-actin. That is, there was a tendency for the denaturation of F-actin at pH 6.0 to be faster in MES[2-(N-morpholino)ethanesulfonic acid]-NaOH buffer than in sodium phosphate buffer, the critical concentrations of actin in 0.5 M KCl being 0.31 mg/ml for MES-NaOH buffer and 0.15 mg/ml for sodium phosphate buffer. A sigmoidal relationship was found between the percent denaturation of F-actin and the KCl concentration added, the greatest change occurring at KCl concentrations between 0.25 and 0.75 M. The time courses of the denaturation of F-actin showed that the percent denaturation rose at first and that in time the rate of the increase decreased. In the case of pH 8.0 and 0.5 M KCl, it took about 1 week for the denaturation rate to begin to drop. The pH of 6.0 further promoted the instability of F-actin exposed to high KCl concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Instability of F-actin in the absence of ATP: a small amount of myosin destabilizes F-actin. 163 56

In vitro, Pseudomonas aeruginosa TrpI protein activates transcription initiation at the trpBA promoter (trpPB) and represses initiation at its own promoter (trpPI), which diverges from, and overlaps, trpPB. Indoleglycerol phosphate (InGP) reduces the TrpI concentration required for binding to its strong binding site (site I), as measured by repression of trpPI; it also facilitates activation of trpPB, presumably because it enables TrpI to bind to a weaker binding site (site II) and thereby interact with RNA polymerase. The role of site II and InGP in regulation of the two promoters was investigated by constructing site II mutants. A 2 bp substitution affected the ability of TrpI to activate trpPB, but did not significantly affect TrpI binding to site II. A more extensive (8 bp) substitution inhibited TrpI-mediated activation of trpPB and TrpI-mediated protection of site II in a DNase I footprinting assay. However, the mutation did not alter the pattern of TrpI binding observed in gel retardation experiments. In particular, a more slowly-migrating complex (Complex 2) whose appearance was correlated with TrpI binding to site II was formed equally well on a wild-type or substituted DNA fragment. Based on the mutant phenotypes, we propose that a particular sequence of protein--protein and protein--DNA interactions is required for activation of trpPB by TrpI and InGP.
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PMID:Mutations in TrpI binding site II that differentially affect activation of the trpBA promoter of Pseudomonas aeruginosa. 175 20

We cloned the cryptic phn operon of a K-12 strain, phn(EcoK), and analyzed the nucleotide sequence of the phn region (11,672 bp). An mRNA start site upstream of the phnC gene was identified by S1 nuclease mapping. The pho regulon activator PhoB protects a pho box region near the mRNA start in DNase I footprinting and methylation protection experiments. The sequence of the cryptic phn(EcoK) operon was very similar to that of the functional phn operon of an Escherichia coli B strain, phn(EcoB) (C.-M. Chen, Q.-Z. Ye, Z. Zhu, B. L. Wanner, and C. T. Walsh, J. Biol. Chem. 265:4461-4471, 1990). The phnE(EcoK) gene has an 8-bp insertion, absent from the phnE(EcoB) gene, which causes a frameshift mutation. The spontaneous activation of the cryptic phn(EcoK) operon is accompanied by loss of this additional 8-bp insertion. Studies of the structure, regulation, and function of the phn region suggest that the phosphate starvation-inducible phn operon consists of 14 cistrons from phnC to phnP.
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PMID:Molecular analysis of the cryptic and functional phn operons for phosphonate use in Escherichia coli K-12. 184 May 80

The inactivation efficiency and repair of single-strand breaks was investigated using model strand breaks created by endonucleolytic incision of damaged DNA. Phi X-174 duplex transfecting DNA containing either thymine glycols, urea residues, or abasic (AP) sites was incubated with AP endonucleases that produce breaks on the 3' side, the 5' side, or both sides of the lesion. For each lesion, incubation with Escherichia coli endonuclease III results in a single-strand break containing a 3' alpha, beta-unsaturated aldehyde (4-hydroxy-2-pentenal), while treatment of AP- or urea-containing DNA with E. coli endonuclease IV results in a single-strand break containing a 5' deoxyribose or a 5' deoxyribosylurea moiety, respectively. Incubation of lesion-containing DNA with both enzymes results in a base gap. Ligatable nicks containing 3' hydroxyl and 5' phosphate moieties were produced by subjecting undamaged DNA to DNase I. When the biological activity of these DNAs was assessed in wild-type cells, ligatable nicks were not lethal, but each of the other strand breaks tested was lethal, having inactivation efficiencies between 0.12 and 0.14. These inactivation efficiencies are similar to those of the base lesions from which the strand breaks were derived. In keeping with the current model of base excision repair, when phi X duplex DNA containing strand breaks with a blocked 3' terminus was transfected into an E. coli double mutant lacking the major 5' cellular AP endonucleases, a greater than twofold decrease in survival was observed. Moreover, when this DNA was treated with a 5' AP endonuclease prior to transfection, the survival returned to that of wild type. As expected, when DNA containing strand breaks with a 5' blocked terminus or DNA containing base gaps was transfected into the double mutant lacking 5' AP endonucleases, the survival was the same as in wild-type cells. The decreased survival of transfecting DNA containing thymine glycols, urea, or AP sites observed in appropriate base excision repair-defective mutants was also obviated if the DNA was incubated with the homologous enzyme prior to transfection. Thus, in every case, with both base lesions and single-strand breaks, the lesion was repaired in the cell by the enzyme that recognizes it in vitro. Furthermore, the repair step in the cell could be eliminated if the appropriate enzyme was added in vitro prior to transfection.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Processing of model single-strand breaks in phi X-174 RF transfecting DNA by Escherichia coli. 185 23

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