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European Journal of Biochemistry May 1982Transition of bovine ribonuclease A from its monomeric to a dimeric form changes the pattern of enzymic activity response to ionic strength [Sorrentino, S., Carsana, A.,...
Transition of bovine ribonuclease A from its monomeric to a dimeric form changes the pattern of enzymic activity response to ionic strength [Sorrentino, S., Carsana, A., Furia, A., Doskocil, J., and Libonati, M. (1980) Biochim. Biophys. Acta. 609, 40-52]. To see whether this phenomenon could be common to other enzyme-substrate systems, the action of various dimeric and monomeric enzymes (ox pancreas deoxyribonuclease, hog spleen acid deoxyribonuclease, bovine seminal ribonuclease, egg-white lysozyme, and papain) on polyelectrolytic substrates has been studied under different conditions of ionic strength. Dimerization of ox pancreas deoxyribonuclease, lysozyme and papain was obtained by cross-linkage with dimethyl suberimidate. The main results of the investigation, similar to those obtained with ribonuclease A, are the following. 1. Enzyme monomers and dimers show markedly different patterns of activity response to ionic strength at given pH values: the reactions catalyzed by monomeric enzymes are highly modulated by salt, whereas those catalyzed by dimeric enzymes are not. In particular, at the reaction optimum the monomeric form of an enzyme is significantly more active than the dimeric one. 2. The optimum of the reaction catalyzed by a dimeric enzyme is shifted to higher ionic strengths in comparison with that of the reaction catalyzed by a monomeric enzyme. A model is proposed that could explain these results on the basis of the influence of ionic strength on the intramolecular dynamics of the enzyme molecule and its non-specific interactions with polyelectrolytic substrates.
Topics: Animals; Cattle; Deoxyribonuclease I; Deoxyribonucleases; Egg White; Endonucleases; Hydrogen-Ion Concentration; Male; Muramidase; Osmolar Concentration; Pancreas; Papain; Ribonucleases; Semen; Spleen; Substrate Specificity; Swine
PubMed: 6282587
DOI: 10.1111/j.1432-1033.1982.tb05923.x -
Nucleic Acids Research Feb 1983We have detected a fraction that is rich in unmethylated HpaII and HhaI sites by end-labelling HpaII fragments of chicken DNA. The fraction is not obvious when DNA...
We have detected a fraction that is rich in unmethylated HpaII and HhaI sites by end-labelling HpaII fragments of chicken DNA. The fraction is not obvious when DNA fragments are stained with ethidium bromide as it amounts to less than 2% of the genome. The average frequency of sites for HpaII is over thirteen times greater in the unmethylated fraction than in total DNA. Partial digests indicate that the unmethylated sites are clustered in the genome. Similar unmethylated fractions were detected in six other vertebrates in both somatic and germ line DNA.
Topics: Animals; Brain Chemistry; Chickens; DNA; DNA Restriction Enzymes; Deoxyribonuclease HpaII; Deoxyribonucleases, Type II Site-Specific; Kidney; Liver; Methylation
PubMed: 6188105
DOI: 10.1093/nar/11.3.647 -
Nucleic Acids Research Aug 1989A method is described that allows cloning of human telomeres in S. cerevisiae by joining human telomeric restriction fragments to yeast artificial chromosome halves. The...
A method is described that allows cloning of human telomeres in S. cerevisiae by joining human telomeric restriction fragments to yeast artificial chromosome halves. The resulting chimeric yeast-human chromosomes propagate as true linear chromosomes, demonstrating that the human telomere structure is capable of functioning in yeast and suggesting that telomere functions are evolutionarily conserved between yeast and human. One cloned human telomere, yHT1, contains 4 kb of human genomic DNA sequence next to the tandemly repeating TTAGGG hexanucleotide. Genomic hybridizations using both cloned DNA and TTAGGG repeats have revealed a common structural organization of human telomeres. This 4 kb of genomic DNA sequence is present in most, but not all, human telomeres, suggesting that the region is not involved in crucial chromosome-specific functions. However, the extent of common features among the human telomeres and possible similarities in organization with yeast telomeres suggest that this region may play a role in general chromosome behavior such as telomere-telomere interactions. Unlike the simple telomeric TTAGGG repeats, our cloned human genomic DNA sequence does not cross-hybridize with rodent DNA. Thus, this clone allows the identifications of the terminal restriction fragments of specific human chromosomes in human-rodent hybrid cells.
Topics: Animals; Bacterial Proteins; Cell Line; Chromosome Mapping; Chromosomes, Fungal; Chromosomes, Human; Chromosomes, Human, Pair 21; Cloning, Molecular; Cricetinae; DNA; DNA Probes; DNA Restriction Enzymes; Deoxyribonuclease BamHI; Deoxyribonuclease EcoRI; Deoxyribonucleases, Type II Site-Specific; Genome, Human; Humans; Hybrid Cells; Nucleic Acid Hybridization; Repetitive Sequences, Nucleic Acid; Saccharomyces cerevisiae; Transformation, Genetic
PubMed: 2549507
DOI: 10.1093/nar/17.15.6109 -
Genome Research Jul 2012Zinc finger nucleases (ZFNs) are powerful tools of genome engineering but are limited by their inevitable reliance on error-prone nonhomologous end-joining (NHEJ) repair...
Zinc finger nucleases (ZFNs) are powerful tools of genome engineering but are limited by their inevitable reliance on error-prone nonhomologous end-joining (NHEJ) repair of DNA double-strand breaks (DSBs), which gives rise to randomly generated, unwanted small insertions or deletions (indels) at both on-target and off-target sites. Here, we present programmable DNA-nicking enzymes (nickases) that produce single-strand breaks (SSBs) or nicks, instead of DSBs, which are repaired by error-free homologous recombination (HR) rather than mutagenic NHEJ. Unlike their corresponding nucleases, zinc finger nickases allow site-specific genome modifications only at the on-target site, without the induction of unwanted indels. We propose that programmable nickases will be of broad utility in research, medicine, and biotechnology, enabling precision genome engineering in any cell or organism.
Topics: Cloning, Molecular; DNA Breaks, Double-Stranded; DNA Breaks, Single-Stranded; DNA End-Joining Repair; Deoxyribonuclease I; Deoxyribonucleases, Type II Site-Specific; Gene Targeting; Genetic Engineering; Genetic Vectors; Genome, Human; HEK293 Cells; Humans; INDEL Mutation; Sensitivity and Specificity; Zinc Fingers
PubMed: 22522391
DOI: 10.1101/gr.138792.112 -
The Journal of Biological Chemistry Nov 1984M13 RF DNA was synthesized in vitro in the presence of various single deoxynucleoside 5'-O-(1-thiotriphosphate) phosphorothioate analogues, and the three other...
M13 RF DNA was synthesized in vitro in the presence of various single deoxynucleoside 5'-O-(1-thiotriphosphate) phosphorothioate analogues, and the three other appropriate deoxynucleoside triphosphates using a M13 (+)-single-stranded template, Escherichia coli DNA polymerase I and T4 DNA ligase. The resulting DNAs contained various restriction endonuclease recognition sequences which had been modified at their cleavage points in the (-)-strand by phosphorothioate substitution. The behavior of the restriction enzymes AvaI, BamHI, EcoRI, HindIII, and SalI towards these substituted DNAs was investigated. EcoRI, BamHI, and HindIII were found to cleave appropriate phosphorothioate-substituted DNA at a reduced rate compared to normal M13 RF DNA, and by a two-step process in which all of the DNA is converted to an isolable intermediate nicked molecule containing a specific discontinuity at the respective recognition site presumably in the (+)-strand. By contrast, SalI cleaved substituted DNA effectively without the intermediacy of a nicked form. AvaI, however, is only capable of cleaving the unsubstituted (+)-strand in appropriately modified DNA.
Topics: Bacteriophage phi X 174; Base Sequence; Binding Sites; DNA Restriction Enzymes; DNA, Single-Stranded; DNA, Viral; Deoxyribonuclease BamHI; Deoxyribonuclease EcoRI; Deoxyribonuclease HindIII; Deoxyribonucleases, Type II Site-Specific; Organothiophosphates; Organothiophosphorus Compounds
PubMed: 6094546
DOI: No ID Found -
The Journal of Biological Chemistry Dec 1979Deoxyribonuclease (DNase) activities have been partially purified from human serum and pancreas. Several of their physical and enzymatic characteristics were determined... (Comparative Study)
Comparative Study
Deoxyribonuclease (DNase) activities have been partially purified from human serum and pancreas. Several of their physical and enzymatic characteristics were determined and compared in order to evaluate their relatedness. Human serum deoxyribonuclease has an isoelectric point in the range of 3.9 to 4.3 and a molecular weight of 33,000 to 38,000. Optimal enzymatic activity at pH 7.0 was dependent on both Mg2+ and Ca2+, whereas a pH optimum of from 5.5 to 5.8 was observed in the presence of Mg2+ and ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). The proportion of single strand or double strand breakage products at early stages of DNA digestion were variable functions of the composition of the buffers employed for the reactions. Single strand break age was predominant under all reaction conditions. Double strand breakage occurred with greatest frequency under neutral conditions in the presence of Mg2+ and Ca2+, was inhibited by the inclusion of 0.15 M NaCl, and did not occur at pH 5.8 in the presence of Mg2+, EGTA, and 0.15 M NaCl. Human pancreas deoxyribonuclease exhibited essentially the same physical properties and enzymatic characteristics as those of the human serum enzyme. Thus, human serum deoxyribonuclease may originate in this pancreas.
Topics: Calcium; Deoxyribonucleases; Egtazic Acid; Endonucleases; Humans; Kinetics; Magnesium; Pancreas
PubMed: 115887
DOI: No ID Found -
PloS One 2013Apoptosis and necrosis, two major forms of cell death, can be distinguished morphologically and biochemically. Internucleosomal DNA fragmentation (INDF) is a biochemical...
Apoptosis and necrosis, two major forms of cell death, can be distinguished morphologically and biochemically. Internucleosomal DNA fragmentation (INDF) is a biochemical hallmark of apoptosis, and caspase-activated DNase (CAD), also known as DNA fragmentation factor 40 kDa (DFF40), is one of the major effector endonucleases. DNase γ, a Mg(2+)/Ca(2+)-dependent endonuclease, is also known to generate INDF but its role among other apoptosis-associated endonucleases in cell death is unclear. Here we show that (i) INDF occurs even during necrosis in cell lines, primary cells, and in tissues of mice in vivo, and (ii) DNase γ, but not CAD, is the effector endonuclease for INDF in cells undergoing necrosis. These results document a previously unappreciated role for INDF in necrosis and define its molecular basis.
Topics: Animals; DNA Fragmentation; Deoxyribonucleases; Endodeoxyribonucleases; Humans; Mice; Necrosis; Poly-ADP-Ribose Binding Proteins; U937 Cells
PubMed: 24312463
DOI: 10.1371/journal.pone.0080223 -
The Journal of Biological Chemistry Dec 1986Porcine pancreatic DNase has been purified to homogeneity. The polypeptide exhibits a single band of Mr = 34,000 on sodium dodecyl sulfate-polyacrylamide gel...
Porcine pancreatic DNase has been purified to homogeneity. The polypeptide exhibits a single band of Mr = 34,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is a glycoprotein containing glucosamine. The results of end group analyses show leucine at the NH2 terminus and alanine at the COOH terminus. The enzymatic properties of the purified porcine DNase are very similar to those of bovine and ovine DNases. The sequence data on the tryptic and chymotryptic peptides derived from CNBr fragments of porcine DNase, along with the results of automated Edman degradation of the intact polypeptide and of the two largest CNBr fragments, indicate the complete amino acid sequence of porcine DNase to be as follows:L-R- I-A-F-N-I-R-T-F-G-E-T-K-M-S-N-A-T-S-N-Y-I-V-R-I-L-S-R-Y-D-I-A-L-I-Q- E-V-R-D-S-H-L-T-A-V-G-K-L-L-N-E-L-N-Q-D-D-P-N-N-Y-H-H-V-V-S-E-P-L-G-R- S-T-Y-K-E-R-Y-L-F-V-F-R-P-N-Q-V-S-V-L-D-S-Y-L-Y-D-D-G-C-E-P-C-G-N-D-T- F-N-R-E-P-S-V-V-K-F-S-S-P-F-T-Q-V-K-E-F-A-I-V-P-L-H-A-A-P-S-D-A-A-A-E- I-N-S-L-Y-D-V-Y-L-N-V-R-Q-K-W-D-L-Q-D-I-M-L-M-G-D-F-N-A-G-C-S-Y-V-T- T-S-H-W-S-S-I-R-L-R-E-S-P-P-F-Q-W-L-I-P-D-T-A-D-T-T-V-S-S-H-T-C-A-Y- D-R-I-V-V-A-G-P-L-L-Q-R-A-V-V-P-D-S-A-A-P-F-D-F-Q-A-A-F-G-L-S-Q-E-T- A-L-A-I-S-D-H-Y-P-V-E-V-T-L-K-R-A. The polypeptide consists of 262 amino acid residues. One of the two disulfide loops links Cys-101 and Cys-104 and the other Cys-173 and Cys-209. Two carbohydrate side chains are attached at Asn-18 and Asn-106.
Topics: Amino Acid Sequence; Amino Acids; Animals; Carbohydrates; Deoxyribonucleases; Hydrogen-Ion Concentration; Molecular Weight; Pancreas; Swine
PubMed: 3782104
DOI: No ID Found -
Proceedings of the National Academy of... Nov 1972DNA isolated from Mycoplasmatales viruses MVL51 and MVGs51 was infectious when mixed with Acholeplasma laidlawii BN1-Na1(R) cells. Infectivity was destroyed by...
DNA isolated from Mycoplasmatales viruses MVL51 and MVGs51 was infectious when mixed with Acholeplasma laidlawii BN1-Na1(R) cells. Infectivity was destroyed by deoxyribonuclease but not by ribonuclease, Pronase, or specific antiserum to the virus. Host mycoplasma cells were only competent for transfection during late-log growth phase. The rates of the establishment of DNase insensitivity of viral DNA transfectants were similar to those of bacteriophage systems. The dose-response curve for transfection suggested that an average of six molecules of DNA must interact with a cell in order to produce one infectious center. Mycoplasmatales virus DNA exhibited a low efficiency of infection; one infectious center required 4 x 10(5) virus equivalents of DNA.
Topics: Bacteriophages; DNA, Viral; Deoxyribonucleases; Dose-Response Relationship, Drug; Immune Sera; Lysogeny; Mycoplasma; Pronase; Ribonucleases; Time Factors
PubMed: 4508332
DOI: 10.1073/pnas.69.11.3423 -
Journal of Clinical Microbiology Nov 1976A three-hour test has been developed to determine deoxyribonuclease activity of Enterobacteriaceae and staphylococci. The test is inexpensive and easy to perform. The...
A three-hour test has been developed to determine deoxyribonuclease activity of Enterobacteriaceae and staphylococci. The test is inexpensive and easy to perform. The rapid deoxyribonuclease test and the conventional method showed complete agreement with the strains tested.
Topics: Deoxyribonucleases; Enterobacteriaceae; Methods; Staphylococcus
PubMed: 993327
DOI: 10.1128/jcm.4.5.453-454.1976