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PubMed1997

The fifth allele of the human deoxyribonuclease I (DNase I) polymorphism.

The fifth allele, DNASE1*5, of human deoxyribonuclease I (DNase I) has been discovered. Polymerase chain reaction fragments containing exon 5 of the DNase I gene were screened for DNA polymorphism using single-strand conformation polymorphism (SSCP) analysis. DNAs from 114 unrelated Japanese and 81 German individuals were tested and a new variant was detected. By DNA sequencing analysis, this variant was found to be caused by a heterozygous G-A transition at nucleotide position 1227 that results in a Val to Met substitution at amino acid position 92 of the mature enzyme. The nucleotide substitution was also confirmed by mismatched polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis. Genotyping of the variant could be carried out by three independent reactions based on PCR amplification, and phenotyping by isoelectric focusing followed by immunostaining. The results supported the presence of the fifth codominant allele, DNASE1*5, which generates a new isozyme.

Alleles↗
PubMed1998

Detection of isozymes of deoxyribonucleases I and II on electrophoresed gels with picogram sensitivity using SYBR Green I.

A highly sensitive method for detecting deoxyribonucleases (DNases) I and II on an electrophoresed gel is described. A dried agarose film sheet containing DNA as a substrate and a buffer reagent was placed in contact with the gel surface after electrophoresis (DAFO method, Yasuda et al., Anal. Biochem. 1989, 183, 84-88). After an appropriate incubation period, the film sheet was peeled off and stained with SYBR-Green I (SG), and then the DNase isozyme bands were detected using a fluorescence image analyzer. We could detect pg levels of the DNases (DNase I, 2 pg; DNase II, 2pg), which represents a 32- to 128-fold increase in sensitivity compared with the original DAFO method using ethidium bromide (EB) as the fluorescent dye. A combination of this new detection method and isoelectric focusing electrophoresis in polyacrylamide gel allowed accurate DNase I typing from 1 microL human serum. This new technique has been named SG-DAFO, after its original dried agarose film overlay method using EB (EB-DAFO).

Benzothiazoles↗
PubMed2006

Deoxyribonuclease 1 aggravates acetaminophen-induced liver necrosis in male CD-1 mice.

An overdose of acetaminophen (APAP) (N-acetyl-p-aminophenol) leads to hepatocellular necrosis induced by its metabolite N-acetyl-p-benzoquinone-imine, which is generated during the metabolic phase of liver intoxication. It has been reported that DNA damage occurs during the toxic phase; however, the nucleases responsible for this effect are unknown. In this study, we analyzed the participation of the hepatic endonuclease deoxyribonuclease 1 (DNASE1) during APAP-induced hepatotoxicity by employing a Dnase1 knockout (KO) mouse model. Male CD-1 Dnase1 wild-type (WT) (Dnase1+/+) and KO (Dnase1-/-) mice were treated with 2 different doses of APAP. Hepatic histopathology was performed, and biochemical parameters for APAP metabolism and necrosis were investigated, including depletion of glutathione/glutathione-disulfide (GSH+GSSG), beta-nicotinamide adenine dinucleotide (NADH+NAD+), and adenosine triphosphate (ATP); release of aminotransferases and Dnase1; and occurrence of DNA fragmentation. As expected, an APAP overdose in WT mice led to massive hepatocellular necrosis characterized by the release of aminotransferases and depletion of hepatocellular GSH+GSSG, NADH+NAD+, and ATP. These metabolic events were accompanied by extensive DNA degradation. In contrast, Dnase1 KO mice were considerably less affected. In conclusion, whereas the innermost pericentral hepatocytes of both mouse strains underwent necrosis to the same extent independent of DNA damage, the progression of necrosis to more outwardly located cells was dependent on DNA damage and only occurred in WT mice. Dnase1 aggravates APAP-induced liver necrosis.

Acetaminophen↗
PubMed1999

Liver-specific and proliferation-induced deoxyribonuclease I hypersensitive sites in the mouse insulin-like growth factor binding protein-1 gene.

The insulin-like growth factor binding protein-1 (IGFBP-1) gene is highly expressed in fetal, perinatal, and regenerating liver. Up-regulation is transcriptionally mediated in regenerating liver and occurs in the first few minutes to hours after partial hepatectomy. In transgenic mice a 970-bp region from -776 to +151 of the IGFBP-1 promoter was sufficient for tissue-specific and induced expression of the gene in fetal and hepatectomized livers. However weak and/or poorly regulated expression in some transgenic lines suggested the existence of other regulatory regions. Here, genomic clones containing large regions 5' of the mouse IGFBP-1 gene sequence were isolated, subcloned, and sequenced. Deoxyribonuclease I (DNaseI) hypersensitivity analyses identified clusters of tissue-specific nuclease-sensitive sites in the promoter region, -100 to -300, -2,300, -3,100, and -5,000 along with other weak sites. After partial hepatectomy, enhanced sensitivity and/or novel sites were detected in the -100/-300, -5,000, and -3,100 regions, the promoter region remaining the most hypersensitive. A subset of these sites was present in fetal and perinatal livers. Novel tissue-specific sites that interacted with C/EBP and hepatic nuclear factor 3 (HNF3) transcription factors were identified in the -3,100 region. A hepatectomy-induced DNA binding complex containing the transcription factor USF1 was identified within the -100 to -300 region of the promoter. These results suggested that a complex array of tissue-specific and hepatic proliferation-induced transcription factors combine to regulate both the proximal promoter and more distal regulatory elements of the IGFBP-1 gene.

Animals↗
PubMed1991

Immunological characterization of the Epstein-Barr virus phosphoprotein PP58 and deoxyribonuclease expressed in the baculovirus expression system.

The open reading frames of the phosphoprotein pp58 (BMRFI) and the deoxyribonuclease (BGLF5) of the Epstein-Barr-virus (EBV) strain M-ABA were cloned in the baculovirus expression vectors pAc373 and pAc360 and expressed in the Spodoptera frugiperda (SF158) insect cells. The recombinant phosphoprotein pp58 expressed in SF158 cells was recognized by the anti-pp58 rabbit anti-sera which were generated by immunizing rabbits with a TrpE-BMRFI fusion protein expressed in E. coli. DNA-cellulose chromatography showed that the recombinant pp58 exhibited DNA-binding activities. Immunofluorescence, immunoblot and ELISA analysis indicated that sera from patients with nasopharyngeal carcinoma (NPC) contained antibodies against pp58. The recombinant EBV DNase expressed in SF158 cells was recognized by the anti-EBV DNase rabbit anti-sera which were generated by immunizing rabbits with a TrpE-C-terminal part of BGLF5 fusion protein expressed in E. coli. The anti-EBV DNase rabbit anti-sera recognized also a protein of about 52 kDa in the EBV-harboring human B-cell lines Raji, Jijoye, B95-8, M-ABA and BL74 induced by TPA and n-butyrate. The recombinant EBV DNase exhibited exonuclease and endonuclease activities, a requirement for magnesium, and a high pH optimum (8.0). Its enzyme activities could be inhibited by sera from NPC patients and anti-EBV DNase rabbit anti-sera. Comparable studies of Raji EBV-DNase and recombinant EBV-DNase implied that recombinant EBV-DNase could also be used in the enzyme activity assay for the detection of NPC. In contrast to the enzyme inhibition test, immunofluorescence and immunoblot analysis demonstrated that the recombinant EBV DNase exhibited only a weak immunological reaction with NPC sera.

Animals↗
PubMed1993

Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death).

Cell death by apoptosis occurs in a wide range of physiological events including repertoire selection of lymphocytes and during immune responses in vivo. A hallmark of apoptosis is the internucleosomal DNA degradation for which a Ca2+,Mg(2+)-dependent endonuclease has been postulated. This nuclease activity was extracted from both rat thymocyte and lymph node cell nuclei. When incubated with nuclei harbouring only limited amounts of endogenous nuclease activity, the ladder pattern of DNA fragments characteristic of apoptosis was induced. This extractable nucleolytic activity was immunoprecipitated with antibodies specific for rat deoxyribonuclease I (DNase I) and was inhibited by actin in complex with gelsolin segment 1, strongly pointing to the presence of a DNase I-type enzyme in the nuclear extracts. COS cells transiently transfected with the cDNA of rat parotid DNase I expressed the enzyme, and their nuclei were able to degrade their DNA into oligosome-sized fragments. PCR analysis of mRNA isolated from thymus, lymph node cells and kidney yielded a product identical in size to that from rat parotid DNase I. Immunohistochemical staining with antibodies to rat DNase I confirmed the presence of DNase I antigen in thymocytes and lymph node cells. The tissue distribution of DNase I is thus extended to tissues with no digestive function and to cells which are known to be susceptible to apoptosis. We propose that during apoptosis, an endonuclease indistinguishable from DNase I gains access to the nucleus due to the breakdown of the ER and the nuclear membrane.

Animals↗
PubMed1989

Enhanced deoxyribonuclease activity in human transformed cells and in Bloom's syndrome cells.

Human hereditary diseases such as xeroderma pigmentosum, Fanconi's anemia, ataxia telangiectasia, and Bloom's syndrome are characterized by a proneness for developing cancer associated with abnormalities in the processing of DNA damage. The molecular defects responsible for predisposing human tissues to cancer are still not well understood, despite the fact that a considerable amount of work has already been done on this problem. In this paper, we show that in human tumor cell lines, in cells transformed by DNA tumor viruses, and in cells derived from certain cancer-prone disorders, the level of activity of a 42-kDa deoxyribonuclease is many times higher than in diploid untransformed control cells. This suggests that this activity is linked to, or may play a role in, malignant transformation.

Animals↗
PubMed1994

Effects of administration of aerosolized recombinant human deoxyribonuclease on resting energy expenditure in patients with cystic fibrosis.

By improving pulmonary function in patients with cystic fibrosis (CF), recombinant human deoxyribonuclease (rhDNase) may affect resting energy expenditure (REE). To examine this hypothesis, we measured REE by indirect calorimetry in seven patients with CF before (day 0) and 2 weeks after (day 15) administration of aerosolized rhDNase. Baseline REE was higher in all patients than predicted for age, sex, and weight (mean +/- SEM 128 +/- 4.9%; range, 116-147%). After 2 weeks of aerosolized rhDNase, mean forced vital capacity (FVC) (in % of predicted values) improved significantly from 54.1 +/- 2.2 to 66.3 +/- 4.2% (mean improvement, 12.3%; 95% CI, 2.8, 21; P < 0.05) and REE decreased by 11.0% (95% CI 3.2, 17.5; P < 0.05). In addition, the larger the improvement in FVC in response to rhDNase the greater the decrease in energy expenditure (r - 0.88). The REE decreased in all patients who had an increase in FVC and remained unchanged in two patients who had no change in FVC. We conclude that patients with CF whose lung function improve in response to aerosolized rhDNase have an acute and proportionate reduction in their resting energy expenditure.

Adolescent↗
PubMed2006

Recombinant human deoxyribonuclease shortens ventilation time in young, mechanically ventilated children.

Recombinant human deoxyribonuclease I (dornase alfa) is currently used as an inhaled mucoactive agent in the treatment of cystic fibrosis. In a randomized, placebo-controlled, double-blind clinical study in 100 infants, we investigated whether the therapeutic use of dornase alfa can be extended to ventilated, fluid-restricted children to reduce reintubation rate, ventilation duration, pediatric intensive care unit (PICU) stay, and ventilation complications. While reintubation rates were similar for dornase alfa 7% vs. placebo 9% (odds ratio, 0.77; confidence interval, 0.11-4.9), the incidence of atelectasis (6 vs. 17, respectively; P-value 0.051), median ventilation time (2.2 vs. 3.4 days, respectively; P-value 0.043), median length of PICU stay (7 vs. 8 days, respectively; P-value 0.051), and mean costs (4,830 vs. 6,320, respectively) were lower in the dornase alfa group. No adverse effects were observed, even in critically ill patients. We found that dornase alfa was beneficial and safe. Our findings also indicate that dornase alfa is possibly of value from the first day of mechanical ventilation onward, particularly when longer ventilation (>3 days) is expected in fluid-restricted children after cardiac surgery.

Administration, Inhalation↗
PubMed1993

A fluorometric microplate-based assay of submicrogram monomeric actin by inhibition of deoxyribonuclease I.

Inhibition of bovine pancreatic deoxyribonuclease I (DNase I) has been a standard method for specific quantitation of monomeric (G-) actin. The aim of this work is to substantially enhance the sensitivity of this type of G-actin assay by using a fluorescent dye, thiazole orange (TO), which has a high optical absorption, moderate DNA affinity, and large fluorescence enhancement upon binding to DNA. The high fluorescence output and moderate affinity of TO's DNA complex produce a large fluorescence decrease when the complex is disrupted by DNase I, thus permitting a highly sensitive detection of G-actin by its inhibition on the fluorescence decrease. The results show that the dynamic responsiveness of a fluorometric G-actin assay can be improved by lowering the concentration of DNA and affinity of the labeling dye, as long as the fluorescence signal of the dye-DNA complex is sufficient for instrumental detection. With a DNA concentration of 6.0 micrograms/ml and TO dye labeling, as little as 20 ng of G-actin can be reliably detected in a fluorescence microplate scanner. This sensitivity also appears to be the utmost detection limit of any G-actin assay that is based on DNase I inhibition.

Actins↗
PubMed2000

Fluorometric determination of deoxyribonuclease I activity with PicoGreen.

A rapid and sensitive assay for the detection of deoxyribonuclease I (DNase I) activity is described. This method is based on the ability of PicoGreen dye to enhance its fluorescence when bound to double-stranded DNA. In the standard assay, reaction mixtures containing the DNase I sample and 0.2 microg of the substrate DNA were prepared in a fluorescence microtiter plate and incubated at 37 degrees C. At the end of the reaction, the diluted PicoGreen reagent was added to each well and fluorescence intensity was measured with a fluorescence plate reader. By this assay, it was possible to determine precisely as little as 5 pg of DNase I within an hour. Moreover, using a small amount of the substrate DNA, the method was shown to be suitable for the sensitive detection of DNase I inhibitor activity.

Animals↗
PubMed1995

Genomic organisation and expression of mouse deoxyribonuclease I.

Deoxyribonuclease I (DNase I) has recently been implicated in cell death by apoptosis, a process which is frequently accompanied by chromatin DNA degradation. Despite extensive studies on DNase I, its genomic organisation remained unknown. Here we report for the first time on the intron-exon structure of the DNase I gene. The coding region of mouse DNase I is composed of eight introns and eight exons, spanning 2315 base pairs. The deduced protein sequence is 91.5% identical to its rat counterpart, but does not carry the two mutations (Glu13 to D and V67 to I) responsible for the decrease in actin-binding of rat DNase I. The enzymatic activity of mouse DNase I is found in striated muscle, kidney, intestine, liver, lymphnodes, but not in the heart, spleen or pancreas.

Amino Acid Sequence↗
PubMed1999

Structural requirements of a human deoxyribonuclease II for the development of the active enzyme form, revealed by site-directed mutagenesis.

Using site-directed mutagenesis, we eliminated three potential N-glycosylation sites (N86, N212, and N266) of human deoxyribonuclease II (DNase II), conserved in mammalian enzymes, and a proteolytic processing site (Q46-R47), forming a propeptide subunit of the enzyme. We expressed a series of these mutant DNase II constructs in COS-7 and Hep G2 cells. Liberation of each glycosylation site at N86 and N266 and the cleavage site interfered dramatically with expression of the intracellular and secreted DNase II activities, irrespective of cell line transfected. A chimeric mutant in which the signal peptide of the DNase II was replaced with that of human DNase I had no intracellular or secreted enzyme activity. Therefore, a simultaneous attachment of a carbohydrate moiety to N86 and N266, cleavage of the propeptide from the single DNase II precursor, and the inherent signal peptide might be required for subcellular sorting and proteolytic maturation of the enzyme.

Amino Acid Substitution↗
PubMed1999

A new allele, DNASE1*6, of human deoxyribonuclease I polymorphism encodes an Arg to Cys substitution responsible for its instability.

A new allele, DNASE1*6, of human deoxyribonuclease I (DNase I) has been discovered by isoelectric focusing: its gene product has the most cathodic pI of the six electrophoretic variants. Results of DNA sequencing, mismatched PCR-restriction fragment length polymorphism, and transient transfection of the variant construct showed that the mutant was caused by a C-T transition at nucleotide position 1826, resulting in an Arg to Cys substitution at amino acid position 185 of the mature enzyme. The variant isoenzyme, expressed in COS-7 cells, was more labile than the other types. Instability and an increase in the pI value of the variant suggest that a structural alteration, perhaps due to aberrant formation of a disulfide bond, could occur in the enzyme.

Alleles↗
PubMed2000

Mammalian deoxyribonucleases I are classified into three types: pancreas, parotid, and pancreas-parotid (mixed), based on differences in their tissue concentrations.

Deoxyribonuclease I (DNase I) activities were measured in 14 different tissues from humans and 5 other mammals (bovine, pig, rabbit, rat, and mouse) by using the single radial enzyme diffusion (SRED) method, which is a sensitive and nonradioactive assay for nucleases. The results indicated that these species are classifiable into three groups on the basis of their different tissue distributions of DNase I. In human and pig, the pancreas showed the highest activity of DNase I; in rat and mouse, the parotid glands showed the highest activity; and in bovine and rabbit, both pancreas and parotid glands showed high activity. Therefore we designated human and pig DNase I as pancreas type, rat and mouse DNase I as parotid type, and bovine and rabbit DNase I as pancreas-parotid (or mixed) type. DNase I of the pancreas type was more sensitive to low pH than the other types. DNase I of pancreas type is secreted into the intestinal tract under neutral pH conditions, whereas the other types are secreted from the parotid gland and have to pass through the very acidic conditions in the stomach. Differences in the tissue distribution and acid sensitivity of mammalian DNases I may provide important information about their digestive function from the evolutionary perspective.

Acids↗
PubMed1997

Identification, localization, and expression of two novel human genes similar to deoxyribonuclease I.

Two novel cDNAs, DNAS1L2 and DNAS1L3, are predicted to encode proteins of 299 and 305 amino acids with 56 and 46% residue identity (71 and 63% similarity), respectively, to deoxyribonuclease I (DNase I). DNAS1L2 is located on a 16p13.3 cosmid, while DNAS1L3 maps to 3p14.3-p21.1 by fluorescence in situ hybridization and by PCR analysis of a radiation hybrid panel. Northern analysis revealed DNAS1L3 expression nearly exclusively in liver, while DNAS1L2 expression was detected in brain by RT-PCR. The previously defined DNL1L or DNAS1L1 is expressed highest in heart and skeletal muscle, while DNase I is expressed in the pancreas, parotid gland, and kidney. Thus, to date, four DNase I-like genes that show different tissue expression patterns are known. A comparison of DNAS1L1, DNAS1L2, and DNAS1L3 with the well-characterized DNase I suggests that the DNAS1L proteins are unlikely to be glycosylated or bind actin; however, catalytic and calcium- and DNA-binding residues are conserved, and potentially cleavable signal peptides are present among all these proteins. This analysis also identifies regions of high conservation among these proteins with no currently assigned function.

Amino Acid Sequence↗
PubMed1996

DNA-stacking interactions determine the sequence specificity of the deoxyribonuclease activity of 1,10-phenanthroline-copper ion.

Bis(1,10-phenanthroline)-copper(I) ion (OP2Cu+) binds reversibly to B-DNA and makes single-stranded cuts by oxidative attack on the deoxyribose moiety. The deoxyribonuclease activity is sequence-dependent yet not nucleotide-specific at the cutting site. OP2Cu+ sequence specificity was analysed in terms of local variations of DNA stability. Kinetic constants of strand cleavage were measured at sequence positions on the two strands and converted into activation free energies of the cleavage reaction. DNA unwinding free energies were calculated from the base sequence using B-DNA stacking parameters for calculations. The two free-energy variations were statistically compared for a series of DNA restriction fragments bearing the binding sites of regulatory proteins and representing a total of 345 DNA base positions. This study shows that the mean activation free energy of strand cleavage at a pair of opposing sugars across the DNA minor groove varies like the unwinding free energy of the DNA sequence delimited by opposing sugars (3 to 4 bp). A statistical equality between the two free-energy variations is demonstrated when considering the sum of the two cleavage events at the opposing sugars. Systematic deviations between the two free-energy distributions were observed at specific sequences, including polypurine-polypyrimidine tracts (AnTm/AmTn, CnTmCp/GpAmGn), alternating purine-pyrimidine tracts ((TA)n/(TA)n, (TG)n/(CA)n) and at certain G+C-rich triplets (GGC, GCC and CGC). The physical significance of these observations is discussed and a model of OP2Cu+ binding and cleavage specificity based on the free-energy equality is proposed.

Base Composition↗
PubMed1996

Site-directed mutagenesis of the catalytic residues of bovine pancreatic deoxyribonuclease I.

Bovine pancreatic deoxyribonuclease I (DNase I) is a well characterised endonuclease which cleaves double-stranded DNA to yield 5' phosphorylated polynucleotides. Co-crystal structures of DNase I with two different oligonucleotides have revealed the presence of several residues (R9, E78, H134, D168, D212 and H252) close to the scissile phosphate. The roles that these amino acids play in the catalytic mechanism have been investigated using site-directed mutagenesis. The following variants were used: R9A, E78T, H134Q, D168S, D212S and H252Q. The kinetics of all six mutants with both DNA and a small chromophoric substrate, thymidine-3',5'-di(p-nitrophenyl)-phosphate, were studied. Only R9A and E78T showed any significant turnover of the two substrates. D168S, H134Q, D212S and H252Q showed vanishingly low activities towards DNA and no detectable activity with thymidine-3',5'-di(p-nitrophenyl)-phosphate. These results demonstrate that H134, D168, D212 and H252 play a critical role in the catalytic mechanism. It is suggested that H134 and H252 (which are hydrogen-bonded to E78 and D212, respectively) provided general acid and general base catalysis. DNase I also requires Mg2+ and E39 has been identified as a ligand for this metal ion. We propose that D168 serves as a ligand for a second Mg2+, and thus DNase I, uses a two metal-ion hydrolytic mechanism. Both magnesium ions are used to supply electrophilic catalysis. Role assignment is based on the mutagenesis results, structural information, homologies between DNase I from different species and a comparison with exonuclease III. However, it is still not feasible to unequivocally assign a particular catalytic role to each amino acid/metal ion.

Animals↗