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PubMed1988

Viral and cellular deoxyribonucleases are associated with herpes simplex virus replicative intermediates.

The presence of viral and cellular deoxyribonucleases was investigated in partially purified HSV replicative intermediates (nucleoprotein complexes) and nucelocapsids prepared from BHK-21 cell cultures infected with herpes simplex virus type 1. The 85kd virus-coded alkaline nuclease was detected by Western blotting experiments in infected cell extracts 7 h after infection, and was identified in viral DNA containing nucleoprotein complexes at 18 h after infection. With activity gel experiments, three cellular deoxyribonucleases were detected in mock-infected cells. Two of them were present in infected cell extracts, and one of them (50kd) was detected in nucleocapsids at 9 h P.I., and in nucleoprotein complexes at 18 h P.I. These data indicate a possible role of viral nuclease in the biosynthesis of late replicative intermediates, and suggest an active involvement of the nuclear matrix in viral replication.

Animals↗

PubMed1979

Immunofluorescence studies on deoxyribonuclease from mouse teratocarcinoma cells during cell differentiation.

Specific anti-DNase-I IgG have been used to detect deoxyribonuclease in teratocarcinoma cells by an indirect immunofluorescence method. All the cells studied show fluorescence staining. However, the patterns are quite different in embryonal carcinoma cells (amorphous cytoplasmic fluorescence and absence of nuclear staining) as compared to differentiated cell lines (diffuse, bright granular nuclear and fibrillar cytoplasmic fluorescence). It is possible by this method to distinguish different cell types derived from the same origin. Deoxyribonuclease from teratocarcinoma cells can therefore be considered as a marker of cell differentiation in this system.

Animals↗

PubMed1976

Protein inhibitor of acid deoxyribonucleases. Improved purification procedure and properties.

A method is described for the extensive purification of acid deoxyribonuclease (acid DNase) and its specific inhibitor from beef liver, the existence of which had been only supported by indirect evidence. By the use of insolubilized acid deoxyribonuclease, eight other proteins interacting with the enzyme have been detected. One of them (molecular weight, 59,000) was identified as responsible for phosphodiesterase activity which is often a contaminant of DNase preparations. Acid DNase (free of phosphodiesterase) and its inhibitor have been obtained as homogeneous proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of acid DNase and its inhibitor are, respectively, 26,500 and 21,500; those of other proteins range from 17,000 to 112,000. The properties of beef liver acid DNase are similar to those described for the enzymes extracted from other sources. The same alteration of DNase kinetics by this inhibitor, as that previously demonstrated with an impure protein has been confirmed; the sigmoidal shape observed at pH 5 for the plot of initial rate versus substrate concentration progressively disappears with increasing pH. We have also demonstrated that RNA, which inhibits the acid DNase through a competitive binding to the catalytic site, is able, like the substrate, to reverse the binding of inhibitor to the enzyme.

Animals↗

PubMed1979

A deoxyribonucleic acid binding protein from KB cells which inhibits deoxyribonuclease activity on single-stranded DNA.

A deoxyribonuclease inhibitor has been purified from KB cells by chromatography on single-stranded DNA-cellulose. Polyacrylamide gel electrophoresis showed the purified preparation to contain two major polypeptides in sodium dodecyl sulfate, with molecular weights of 72,000 and 65,000, but only one major band (with a molecular weight of approximately 140,000) after electrophoresis under nondenaturing conditions. The protein inhibits the hydrolysis of single-stranded DNA by KB DNase, DNase I, DNase II, and nuclease S1, but has no effect on the hydrolysis of double-stranded DNA by these enzymes. The inhibitor causes a reduction in the rate of hydrolysis of DNA by the deoxyribonuclease, probably by reducing the effective concentration of substrate.

Carrier Proteins↗

PubMed1980

Isolation of mutants of Caulobacter crescentus deficient in ATP-dependent deoxyribonuclease activity.

Caulobacter crescentus mutants sensitive to UV radiation, mitomycin C and methyl methane-sulfonate were isolated and tested for ATP-dependent deoxyribonuclease activity. Two mutants were identified of which one had less than 5 per cent of the wild-type level of the nuclease activity. This mutant in cross with another auxotrophic partner gave highly reproducible two-fold reduction in number of recombinants compared to a control cross. The suggested causes for reduced recombination frequency when one of the partners has residual ATP-dependent deoxyribonuclease activity are discussed.

Adenosine Triphosphate↗

PubMed1981

Detection of deoxyribonuclease activities of Mycoplasma pneumoniae.

Alkaline deoxyribonuclease activity was detected in culture of Mycoplasma pneumoniae. This enzyme required MgCl2 to stimulate its activity and preferred double-stranded to single-stranded DNA as substrate. By using polyacrylamide gel electrophoresis or isoelectric electrofocusing, two distinctive deoxyribonuclease activities were demonstrated both in cultured broth and cell homogenate.

Deoxyribonucleases↗

PubMed1982

Repair of mitochondrial DNA in Saccharomyces cerevisiae. Induction of cytoplasmic petite mutants in a nuclear mutant exhibiting thermosensitive mitochondrial deoxyribonuclease activity.

Four nuclear thermosensitive mutants have been obtained in which induction of up 100% cytoplasmic petite mutants (rho-) is observed upon cell incubation at 36 degrees C. For a given incubation time at 36 degrees C, the percentage of rho- is increased by preliminary gamma-ray irradiation. Under these conditions, the induction of rho- is a linear function of the irradiation dose. The retention of genetic information by rho- and of mitochondrial DNA synthesis in vivo and in vitro exclude that the mutants are deficient in the replication of mitochondrial DNA. The degradation of mitochondrial DNA labeled with [3H]dTTP in isolated mitochondria, has been monitored at 26 degrees C and at 36 degrees C after addition of 0.5% Triton X-100 in the presence or in the absence of ethidium bromide. In assays carried out at 26 degrees C, the degradation of mitochondrial DNA is similar in the parental strain and in the mutant gamma s rho 2. However, at 36 degrees C, the degradation of mitochondrial DNA is slower in the mutant. We have shown that a mitochondrial membrane deoxyribonuclease acting on double-stranded DNA at acid pH is thermosensitive in the mutant. Analysis of the meiotic segregants of a tetrad issued from the cross of the mutant with an isogenic parental strain shows co-segregation of rho- induction and of nuclease thermosensitivity in a 2:2 Mendelian pattern. These results suggest that a mitochondrial deoxyribonuclease is involved in the repair of damages caused to mitochondrial DNA by elevated temperature and by x-rays.

DNA Repair↗

PubMed2001

The cloning, genomic structure, localization, and expression of human deoxyribonuclease IIbeta.

Acidic endonuclease activity is present in all cells in the body and much of this can be attributed to the previously cloned and ubiquitously expressed deoxyribonuclease II (DNase II). Database analysis revealed the existence of expressed sequence tags and genomic segments coding for a protein with considerable homology to DNase II. This report describes the cloning of this cDNA, which we term deoxyribonuclease IIbeta (DNase IIbeta) and comparison of its expression to that of the originally cloned DNase II (now termed DNase IIalpha). The cDNA encodes a 357 amino acid protein. This protein exhibits extensive homology to DNase IIalpha including an amino-terminal signal peptide and a conserved active site, and has many of the regions of identity that are conserved in homologs in other mammals as well as C. elegans and Drosophila. The gene encoding DNase IIbeta has identical splice sites to DNase IIalpha. Human DNase IIbeta is highly expressed in the salivary gland, and at low levels in trachea, lung, prostate, lymph node, and testis, whereas DNase IIalpha is ubiquitously expressed in all tissues. The expression pattern of human DNase IIbeta suggests that it may function primarily as a secreted enzyme. Human saliva was found to contain DNase IIalpha, but after immunodepletion, considerable acid-active endonuclease remained which we presume is DNase IIbeta. We have localized the gene for human DNase IIbeta to chromosome 1p22.3 adjacent (and in opposing orientation) to the human uricase pseudogene. Interestingly, murine DNase IIbeta is highly expressed in the liver. Uricase is also highly expressed in mouse but not human liver and this may explain the difference in expression patterns between human and mouse DNase IIbeta.

Amino Acid Sequence↗

PubMed1985

Deoxyribonuclease IV from rat liver chromatin and the excision of apurinic sites from depurinated DNA.

Deoxyribonuclease IV, a 5'-3' exonuclease degrading double-stranded DNA from intra-strand nicks, has been purified from the chromatin of rat liver cells. The enzyme, which has an Mr of 58000, excises the apurinic (AP) sites from a depurinated DNA nicked 5' to these AP sites with the chromatin AP endonuclease. The excision is not the result of hydrolysis of the phosphodiester bond 3' to the AP sites since the excision product does not behave as deoxyribose 5-phosphate but as its 2,3-unsaturated derivative. This result suggests that, to remove the AP sites from the DNA nicked by an AP endonuclease, the chromatin deoxyribonuclease IV rather acts as a catalyst of beta-elimination.

Animals↗

PubMed1982

Inactivation of ATP-dependent deoxyribonuclease of Micrococcus luteus by 2,3-butanedione.

ATP-dependent deoxyribonuclease from Micrococcus luteus was purified to near homogeneity by a procedure involving gentle cell lysis, ammonium sulfate fractionation, TEAE-cellulose chromatography, Sephadex G-150 gel filtration and DNA-cellulose chromatography. Treatment of the enzyme with 2,3-butanedione, which binds specifically to arginyl residues, caused rapid loss of enzyme activities and the effect was enhanced by borate ion. The reaction obeyed first order kinetics with respect to the butanedione concentration, indicating that at least one functional arginyl residue is involved in the inactivation reaction. The enzyme was protected from inactivation by the presence of a low concentration of ATP, but not of ADP, AMP or adenosine. These results indicate that ATP-dependent deoxyribonuclease of Micrococcus luteus has functional arginyl residue(s) at an ATP-binding site.

Arginine↗

PubMed1969

Characterization of deoxyribonucleases induced by poxviruses.

Increases in deoxyribonuclease activity assayed at alkaline pH can be observed in poxvirus-infected cells when native or denatured deoxyribonucleic acid (DNA) is used as substrate. The deoxyribonuclease assayable with native DNA as substrate, induced in HeLa cells by cowpoxvirus or vaccinia virus WR, can be separated from the corresponding enzyme present in normal cells by chromatography on diethylaminoethyl cellulose. In addition, the two enzymes induced in the virus-infected cells differ from each other in their chromatographic properties. The two induced enzymes have been further characterized with respect to properties of enzymatic reaction.

Journal Article↗

PubMed2001

Over expression of inhibitor of caspase 3 activated deoxyribonuclease in human renal cell carcinoma cells enhances their resistance to cytotoxic chemotherapy in vivo.

PURPOSE: We determined whether inhibitor of caspase-3 activated deoxyribonuclease (ICAD) enhances resistance to apoptotic stimuli or inhibits DNA fragmentation by inactivating caspase-3 activated deoxyribonuclease. MATERIALS AND METHODS: The liposome mediated gene transfer method was used to introduce ICAD complementary DNA into ACHN cells and the expression of ICAD protein per clone was evaluated by Western blot analysis. Effects of cisplatin treatment on growth inhibition and apoptosis in the ACHN sublines were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, DNA fragmentation assay and Western blot analysis of poly(ADP-ribose) polymerase protein. The limiting dilution assay was also performed to examine the effect of cisplatin treatment under anchorage independent conditions. Furthermore, nude mice bearing the ACHN sublines were given intraperitoneal injection of 5 mg./kg. cisplatin 1 and 2 weeks after the implantation of tumor cells and changes in tumor volume was measured. RESULTS: ICAD transfected ACHN cells inhibited DNA degradation after cisplatin treatment but not control vector only transfected ACHN cells, whereas a similar degree of apoptotic cell death induced by cisplatin in ICAD and control vector only transfected ACHN cells was observed on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Western blot analysis of poly(ADP-ribose) polymerase protein. However, the limiting dilution assay revealed that ICAD transfected ACHN cells had high resistance to pretreatment with cisplatin compared with control vector only transfected ACHN cells. Moreover, although there was no significant difference in the in vivo growth of ACHN sublines, cisplatin treatment induced the elimination of control vector only transfected ACHN cell tumors. In contrast, most ICAD transfected ACHN cell tumors continued to grow after cisplatin treatment. CONCLUSIONS: These findings suggest that when ICAD is over expressed in tumor cells, it renders them highly resistant to therapy induced apoptosis, resulting in enhanced tumor progression.

Apoptosis Regulatory Proteins↗

PubMed1995

Genotyping of human deoxyribonuclease I polymorphism by the polymerase chain reaction.

We recently completely elucidated the molecular basis of genetic polymorphism in human deoxyribonuclease I and found it to be controlled by four codominant alleles, DNASE1*1, *2, *3 and *4. In this paper we describe a novel DNase I-genotyping system that could be used directly on DNA samples using the polymerase chain reaction (PCR) based on the three nucleotide substitutions underlying the protein polymorphism. The system consists of three independent reactions. Since the substitutions neither suppress nor create any known enzyme recognition site in the DNase I gene, two separate mismatched PCR followed by XhoI digestion methods were introduced to discriminate between the DNASE1*1 (or *3) and the DNASE1*2 (or *4) alleles, and to detect the DNASE1*4 allele. An amplification refractory mutation system was employed to detect DNASE1*3. A 100% correlation was found between the results of this genotyping method and those obtained by phenotyping using conventional isoelectric focusing. The high sensitivity and specificity of this genotyping method allows us to survey DNase I-polymorphism in small DNA samples.

Alleles↗

PubMed1981

Microfilaments interacting with heavy meromyosin and deoxyribonuclease I in cells of the ovarian follicle of a lizard.

Two types of filaments (microfilaments 4--6 nm in diameter, and intermediate filaments 7--10 nm in diameter) are common in the surface epithelial cells and theca fibroblasts of vitellogenic ovarian follicles of the lizard Anolis carolinensis. Heavy meromyosin (HMM), which forms complexes with actin filaments, interacts only with the microfilaments of theca fibroblasts. After myosin extraction of follicles no filaments disappeared, but when this treatment was followed by incubation with deoxyribonuclease I (DNA-ase I), which depolymerizes F-actin to G-actin, microfilaments disappeared from the theca fibroblasts. It is concluded that microfilaments in theca fibroblasts are actin-like and may contract to provide the mechanism of expulsion for the oocyte during ovulation. The intermediate filaments of the surface epithelial cells and theca fibroblasts may serve as a skeletal system for the large (up to 8 mm in diameter) vitellogenic follicle.

Animals↗

PubMed1981

A radial diffusion assay for plasma and serum deoxyribonuclease I.

A simple radial diffusion technique is described for assaying deoxyribonuclease I (DNase I) activity in whole human plasma and serum. Enzymatic activity was calculated from a standard curve constructed from the studies on the hydrolysis of calf thymus DNA by bovine pancreatic DNase of known concentrations. The assay was shown to be specific for nucleases which are active at neutral pH and require magnesium ions for activation. There was no significant difference in DNase I activity in plasma compared with that in serum from normal individuals (P greater than 0.05). The DNase I activity of 35 normal human sera was 26.1 +/- 9.2 ng/ml and ranged from 10.8-48.5 ng/ml. This technique may prove useful for the evaluation of DNase I activity in crude biological fluids and is not affected by the presence of enzymatic inhibitors and/or activators.

Adult↗

PubMed1981

Serum deoxyribonuclease I and clinical activity in systemic lupus erythematosus.

Serum deoxyribonuclease I (DNase I) activity in systemic lupus erythematosus (SLE) patients was shown to be lower than that of healthy laboratory personnel, rheumatoid arthritis, and scleroderma patients (P less than 0.001). The decrease in DNase I activity in SLE sera was not due to the effect of various autoantibodies or to heat labile DNase I inhibitor. A relationship between serum DNase I activity and active SLE was demonstrated. Patients with active lupus nephritis had the lowest levels of enzymatic activity.

Adult↗

PubMed1980

Effect of Mg2+ and Mn2+ on hydrolysis of calf thymus DNA by pancreatic deoxyribonuclease I.

Divalent cations are activators for DNA hydrolysis by pancreatic deoxyribonuclease I. Apparent Vm and Km changes have been studied in presence of Mn2+ or Mg2+. The activation process modifies both Vm and Km, their relationship with Mg2+ or Mn2+ being a linear one. Deoxyribonucleotides inhibit the DNA hydrolysis, whether Mg2+ or Mn2+ is the activator; the purine deoxyribonucleotides are more effective as inhibitors than the pyrimidine ones. The effect of some derivatives of adenine has been studied: the inhibition is maximum with 5'-dAMP and minimum with adenine or adenosine. A kinetic mechanism of enzymatic activation by Mn2+ or Mg2+ is discussed.

Adenine Nucleotides↗

PubMed1980

Conductimetric study of the binding of Mn2+ to bovine pancreatic deoxyribonuclease.

The binding of Mn2+ on bovine pancreatic deoxyribonuclease has been studied by a conductimetric method. At low ionic strengths, a high-affinity single binding site is demonstrated. The association constant value (K = 1.2 x 10(5) M-1 at pH 8) is high enough to conclude that, in standard experimental conditions for DNA hydrolysis, the reacting species is the DNAase-cation complex. Competitive binding studies in presence of Mg++ and Ca++ show that these cations do not bind on the Mn++ site.

Animals↗