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The Biochemical Journal Jun 1984Native DNAase (deoxyribonuclease) activities derived from mouse peritoneal cavity and peripheral blood components were separated, detected, and characterized by...
Native DNAase (deoxyribonuclease) activities derived from mouse peritoneal cavity and peripheral blood components were separated, detected, and characterized by electrophoresis into polyacrylamide gels containing DNA, followed by incubation of the gels, and staining of the substrate to reveal only the DNAase activities. Resident peritoneal macrophages contained 12 DNAase-II-like activities that were characteristic of that cell type, whereas lymphocytes and granulocytes each contained five DNAases. Induction of inflammation by peritoneal injection of thioglycollate resulted in changes in macrophage DNAase expression, including: increased total DNAase activity, a decrease in the number of activities from 12 to 11, increased activity of a specific subset of the enzymes, and a change in the apparent size of a specific subset of the enzymes. Electrophoretic and enzymic properties and sensitivity to endo-beta-N-acetylglucosaminidase H indicated that the macrophage activities probably represented charge variants of one or two parent peptide chains.
Topics: Animals; Ascitic Fluid; Deoxyribonucleases; Electrophoresis, Polyacrylamide Gel; Glycoside Hydrolases; In Vitro Techniques; Inflammation; Isoenzymes; Leukocytes; Macrophages; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Mice; Mice, Inbred AKR; Thioglycolates
PubMed: 6430283
DOI: 10.1042/bj2200561 -
FEBS Letters Aug 2013In Anabaena sp. PCC 7120, FurA is a global transcriptional regulator whose expression is strongly induced by NtcA in proheterocysts and remains stably expressed in...
In Anabaena sp. PCC 7120, FurA is a global transcriptional regulator whose expression is strongly induced by NtcA in proheterocysts and remains stably expressed in mature heterocysts. In the present study, overexpression of furA partially suppressed heterocyst differentiation by impairing morphogenesis at an early stage. Recombinant purified FurA specifically bound in vitro to the promoter regions of ntcA, while quantitative RT-PCR analyses indicated that furA overexpression strongly affected the transient increase of ntcA expression that occurs shortly after nitrogen step-down. Overall, the results suggest a connection between iron homeostasis and heterocyst differentiation via FurA, by modulating the expression of ntcA.
Topics: Anabaena; Bacterial Proteins; Base Sequence; Cell Differentiation; Deoxyribonuclease I; Deoxyribonucleases; Gene Expression Regulation, Bacterial; Homeostasis; Iron; Molecular Sequence Data; Promoter Regions, Genetic
PubMed: 23851073
DOI: 10.1016/j.febslet.2013.07.007 -
Proceedings of the National Academy of... Aug 1998Caspase-activated DNase (CAD) cleaves chromosomal DNA during apoptosis. Here, we report isolation of two classes of human CAD cDNAs from a human KT-3 leukemic cell cDNA...
Caspase-activated DNase (CAD) cleaves chromosomal DNA during apoptosis. Here, we report isolation of two classes of human CAD cDNAs from a human KT-3 leukemic cell cDNA library. One class of cDNA encoded a protein comprising 338 amino acids, which showed a marked similarity to its murine counterpart. In vitro transcription and translation of this cDNA resulted in a functional CAD protein when the protein was synthesized in the presence of its inhibitor (inhibitor of CAD). The other cDNA class contained many deletions, insertions, and point mutations in the sequence corresponding to the coding region, suggesting that it is derived from a pseudogene. The functional CAD gene was localized to human chromosome 1p36.3 by fluorescent in situ hybridization. The CAD mRNA was expressed in a limited number of human tissues, including pancreas, spleen, prostate, and ovary. The expression of the CAD mRNA in human cell lines correlated with their ability to show DNA fragmentation during apoptosis. Overexpression of CAD potentiated DNA fragmentation by apoptotic stimuli in these cell lines, indicating that CAD is responsible for the apoptotic DNA degradation.
Topics: Amino Acid Sequence; Animals; Apoptosis; Base Sequence; Cell Line; Chromosome Mapping; Chromosomes, Human, Pair 1; Cloning, Molecular; DNA, Complementary; Deoxyribonucleases; Humans; Mice; Molecular Sequence Data; Sequence Homology, Amino Acid
PubMed: 9689044
DOI: 10.1073/pnas.95.16.9123 -
The Journal of Biological Chemistry Jan 1986The chromatin structure of the promoter region of the human dihydrofolate reductase gene was determined using a variety of nucleases including DNase I, micrococcal...
The chromatin structure of the promoter region of the human dihydrofolate reductase gene was determined using a variety of nucleases including DNase I, micrococcal nuclease, several restriction endonucleases, exonuclease III, and Bal31. Two separate regions from -670 to -340 (the distal hypersensitive region) and from -170 to +150 (the proximal hypersensitive region) were shown to be essentially free of proteins as indicated by their accessibility to both endo- and exonucleases. Within the proximal hypersensitive region, one protein appears to be bound at the start site for transcription. A 170-base pair fragment between the two hypersensitive regions was highly resistant to all nucleases tested. Multiple barriers against exonuclease digestion and resistance to dissociation by high salt concentrations suggest that more than one protein is tightly bound to this region. The upstream sequence from -670 and the downstream sequence from +150 were shown to be packaged into nucleosomes. The selective accessibility of certain sites to micrococcal nuclease cutting indicates that the initial nucleosomes are phased upstream from the distal hypersensitive region. There appears to be a protein bound between the phased nucleosomes and the upstream boundary of the distal hypersensitive region. These results suggest that the normal nucleosome array is interrupted by about 900 base pairs of nucleosome-free DNA to which several nuclear proteins bind in a DNA sequence-specific manner.
Topics: Chromatin; DNA Restriction Enzymes; Deoxyribonuclease EcoRI; Deoxyribonuclease I; Deoxyribonucleases, Type II Site-Specific; Drug Resistance; Exodeoxyribonucleases; HeLa Cells; Humans; Methotrexate; Micrococcal Nuclease; Operon; Tetrahydrofolate Dehydrogenase
PubMed: 3003070
DOI: No ID Found -
European Journal of Biochemistry May 1977A deoxyribonuclease has been purified more than 2000-fold from the green algae, Chlamydomonas reinhardii. The enzyme is most active on denatured DNA. Optimum activity is...
A deoxyribonuclease has been purified more than 2000-fold from the green algae, Chlamydomonas reinhardii. The enzyme is most active on denatured DNA. Optimum activity is at pH 8.5, in 80 mM Tris-HCl buffer and 2 mM CaCl2. Other divalent cations can replace Ca2+ with varying lower efficiency. EDTA and inorganic phosphate are strongly inhibitory, while ATP and high concentrations of 2-mercaptoethanol are slightly inhibitory. The molecular weight is approximately 35 000, the Stokes radius is 2.7 nm, and the sedimentation coefficient 2.8 S. It is a single polypeptide chain, and the frictional ratio of 1.27 suggests it is only slightly asymetrical. The isoelectric point is 9.5. This enzyme has been termed exonuclease 1.
Topics: Adenosine Triphosphate; Calcium; Cations, Divalent; Cations, Monovalent; Cell Division; Chlamydomonas; Deoxyribonucleases; Edetic Acid; Hydrogen-Ion Concentration; Kinetics; Mercaptoethanol; Molecular Weight; Nucleic Acid Denaturation; Phosphates; Protein Conformation; Spermidine
PubMed: 18343
DOI: 10.1111/j.1432-1033.1977.tb11536.x -
Microbiology and Immunology 1985Molecular heterogeneity of the extracellular deoxyribonuclease (DNase) in group A streptococci was demonstrated in 42 clinical isolates. Although polyacrylamide gel...
Molecular heterogeneity of the extracellular deoxyribonuclease (DNase) in group A streptococci was demonstrated in 42 clinical isolates. Although polyacrylamide gel electrophoretic patterns of the extracellular DNase of all the isolates were heterogeneous, they could be divided into five main patterns with respect to the presence or absence of three DNase components including DNase B. By comparing the electrophoretic patterns of DNase in all the isolates with their T-types, we found that the patterns were quite characteristic for their T-types, especially in the prevalent T-types 12 and 1, and that the isolates of T-types 12 and 1 produced DNase B as their major extracellular DNase. Relative DNase B activity in the total extracellular DNase activity of group A, B, and G isolates was determined by the rapid method of neutralization with anti-DNase B antibody. The results showed neutralization of DNase activity in all the isolates of group A streptococci, largely corresponding to their T-types, but not of the isolates of groups B and G. These results indicate that the electrophoretic patterns of the extracellular DNase of group A streptococci are closely correlated with their T-types, suggesting the physicochemical taxonomic value of these properties.
Topics: Antibodies, Bacterial; Antigen-Antibody Reactions; Deoxyribonucleases; Electrophoresis, Polyacrylamide Gel; Extracellular Space; Streptococcus pyogenes
PubMed: 3892240
DOI: 10.1111/j.1348-0421.1985.tb00819.x -
The Journal of Investigative Dermatology Jan 2007The removal of keratinocyte (KC) nuclear DNA by deoxyribonucleases (DNases) is an important step in the formation of normal stratum corneum (SC). However, the molecular...
The removal of keratinocyte (KC) nuclear DNA by deoxyribonucleases (DNases) is an important step in the formation of normal stratum corneum (SC). However, the molecular identity of the DNA-degrading enzymes has so far remained elusive. Here we show that the endonuclease DNase1-like 2 (DNase1L2) is preferentially expressed in the epidermis and that its expression correlates with terminal differentiation of KC in vitro and in vivo. In biopsies of normal skin, DNase1L2 mRNA was regularly found in suprabasal KC and DNase1L2 protein was highly abundant in the stratum granulosum. In contrast to normal skin, DNase1L2 expression was downregulated in parakeratotic epidermis such as in psoriatic lesions. When DNase1L2 gene expression was knocked down by small interfering RNA in a human skin equivalent model, nuclei were maintained through all layers of the SC. Taken together, our data demonstrate that DNase1L2 plays an essential role in DNA degradation during terminal differentiation of epidermal KC.
Topics: Bowen's Disease; Cell Differentiation; Cells, Cultured; DNA; Deoxyribonuclease I; Deoxyribonucleases; Endodeoxyribonucleases; Epidermal Cells; Gene Expression Regulation; Humans; Keratinocytes; Psoriasis; RNA, Messenger
PubMed: 16902420
DOI: 10.1038/sj.jid.5700503 -
European Journal of Biochemistry Aug 1979A purification procedure described previously resulting in electrophoretically pure Bacillus subtilis ATP-dependent DNAse has now been modified by adding a fractionation...
A purification procedure described previously resulting in electrophoretically pure Bacillus subtilis ATP-dependent DNAse has now been modified by adding a fractionation stage with Polymin P to permit large-scale isolation of the enzyme. It has been found that the enzyme molecule (Mr = 300000) consists of two large subunits with Mr 155000 and 140000. The purified enzyme has three activities: (1) DNAse on linear single-stranded and double-stranded DNAs (2) DNA-unwinding and (3) ATPase. Circular DNAs were not affected by the enzyme. Study of the dependence of these activities on temperature, pH, and ATP and Mg2+ concentrations has revealed two different states of the enzyme. At low ATP concentrations and alkaline pH, it showed chiefly nuclease action, degrading considerable amounts of DNA to small fragments five residues long on average. At higher ATP concentrations and neutral pH (more physiological conditions) it predominantly unwound DNA. Simultaneously it cut preferentially one of the duplex strands to fragments more than 1000 residues in length. The results obtained suggest that the energy of the enzyme-cleaved ATP is mainly expended on unwinding rather than on degrading DNA molecules.
Topics: Adenosine Triphosphate; Bacillus subtilis; Deoxyribonucleases; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Substrate Specificity
PubMed: 39753
DOI: 10.1111/j.1432-1033.1979.tb13201.x -
Applied Microbiology Jun 1974The production of extracellular deoxyribonuclease was examined with anaerobic organisms isolated from clinical specimens. Nuclease activity was extraordinarily common....
The production of extracellular deoxyribonuclease was examined with anaerobic organisms isolated from clinical specimens. Nuclease activity was extraordinarily common. All strains of Fusobacterium, including eight species, as well as Bacteroides fragilis and B. melaninogenicus, displayed enzyme activity. Whereas the gram-positive bacteria were generally less productive, all strains of Clostridium perfringens, Peptostreptococcus intermedius, and P. anaerobius specifically produced deoxyribonuclease. The test is taxonomically valuable, particularly in the characterization of gram-positive cocci, since a deoxyribonuclease-producing coccus indicates P. intermedius or P. anaerobius. Additionally, possession of the enzyme may prove to be a useful correlate of the potential pathogenicity of anaerobes.
Topics: Anaerobiosis; Bacteria; Bacteriological Techniques; Bacteroides; Clostridium perfringens; Culture Media; DNA; Deoxyribonucleases; Fusobacterium; Veillonella
PubMed: 4364608
DOI: 10.1128/am.27.6.1031-1033.1974 -
Nucleic Acids Research 2005Type II restriction endonucleases protect bacteria against phage infections by cleaving recognition sites on foreign double-stranded DNA (dsDNA) with extraordinary...
Type II restriction endonucleases protect bacteria against phage infections by cleaving recognition sites on foreign double-stranded DNA (dsDNA) with extraordinary specificity. This capability arises primarily from large conformational changes in enzyme and/or DNA upon target sequence recognition. In order to elucidate the connection between the mechanics and the chemistry of DNA recognition and cleavage, we used a single-molecule approach to measure rate changes in the reaction pathway of EcoRV and BamHI as a function of DNA tension. We show that the induced-fit rate of EcoRV is strongly reduced by such tension. In contrast, BamHI is found to be insensitive, providing evidence that both substrate binding and hydrolysis are not influenced by this force. Based on these results, we propose a mechanochemical model of induced-fit reactions on DNA, allowing determination of induced-fit rates and DNA bend angles. Finally, for both enzymes a strongly decreased association rate is obtained on stretched DNA, presumably due to the absence of intradomain dissociation/re-association between non-specific sites (jumping). The obtained results should apply to many other DNA-associated proteins.
Topics: DNA; Deoxyribonuclease BamHI; Deoxyribonucleases, Type II Site-Specific; Hydrolysis; Kinetics; Models, Chemical; Models, Molecular; Nucleic Acid Conformation; Stress, Mechanical
PubMed: 15886396
DOI: 10.1093/nar/gki565