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Results for “Endodeoxyribonucleases”

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PubMed1993

Detection of Mg(2+)-dependent endonuclease activity in myeloid leukemia cell nuclei capable of producing internucleosomal DNA cleavage.

We detected Mg(2+)-dependent, Ca(2+)-independent endonuclease activity in non-apoptotic myeloid leukemia cell nuclei using autodigestion method which cleaved the chromatin of the autologous leukemia cells to an oligonucleosomal length pattern. Similar endonuclease activity could be successfully recovered in the protein extracts of the human leukemia cell nuclei. The extracts consistently elicited characteristic DNA cleavage of another leukemia cell (KG-1) nuclei as the target, the enzyme activity of which had been inactivated. We propose that this method is a useful tool for the study of endonucleases involved in apoptosis.

Animals↗

PubMed1994

Overexpression of activin-beta A subunit mRNA is associated with decreased activin type II receptor mRNA levels in the testes of alpha-inhibin deficient mice.

Activins and inhibins are polypeptides of the transforming growth factor-beta family that participate in differentiation and growth of diverse cell types, and are involved in endocrine/paracrine regulation of the hypothalamo-pituitary-gonadal axis. Mice with alpha-inhibin subunit gene deletion develop large testicular tumors. In these animals, a 200-fold increase in testicular expression of activin beta A subunit mRNA was detected using S1-nuclease protection analysis. Northern blot analysis demonstrated that a predominant mRNA form of approximately 6.5 kb and a second minor form of 4.5 kb were overexpressed in the testes of the alpha-inhibin deficient animals. Testicular expression of the type II activin receptor was decreased 3-fold in these mice. In contrast, hypothalamic beta A and type II activin receptor mRNA levels remained unaltered. alpha-Inhibin may play a role to suppress the expression of beta A mRNA in the mammalian testes. These results demonstrate that increased expression of activin is accompanied by a tissue specific reduction in the expression of its own receptor mRNA in vivo.

Activin Receptors↗

PubMed1995

Characterization of the DNA-binding activity of HIV-1 integrase using a filter binding assay.

Based on the selective binding of proteins and DNA to distinct filter materials a double-layered dot blot radio assay was developed to evaluate the binding of DNA to HIV-1 integrase. In this assay the DNA-binding was found to be independent of Mn2+ concentration, inhibited by concentrations of Mg2+ above 5 mM, abolished by zinc chelation and inhibited by monoclonal antibodies reacting with either the N-terminal or C-terminal regions of integrase. Atomic absorption spectroscopy revealed the molar ratio between integrase and zinc to be close to 1. It is concluded that both the N-terminal and the C-terminal regions of integrase are involved in DNA-binding and that the reported double-layered dot blot radio assay is well suited for further characterization of the integrase.

Base Sequence↗

PubMed1996

Folding-dependent in vitro protein splicing of the Saccharomyces cerevisiae VMA1 protozyme.

VMA1 translational product undergoes excision of a 50-kDa intervening segment (VDE: VMA1-derived endonuclease) and religation of the flanking regions to create a 69-kDa catalytic subunit of vacuolar membrane H+-ATPase. VDEs conjugated with polypeptides at both N- and C-terminal ends were expressed in Escherichia coli and examined for their ability to catalyze self-splicing. Processed VDE was found in soluble pools, while unspliced precursors accumulated in insoluble pools, forming inclusion bodies. We demonstrate in vitro protein splicing by refolding of the denatured precursor molecules. The processing reaction efficiently occurs with the purified precursor peptide. VDE bracketed by only 6 proximal and 4 distal amino acids is autocatalytically processed.

Amino Acid Sequence↗

PubMed1997

Correction of the DNA repair defect in Fanconi anemia complementation groups A and D cells.

We have previously isolated from Fanconi anemia, complementation groups A (FA-A) and D (FA-D) cells, a DNA endonuclease complex which is defective in its ability to incise DNA containing interstrand cross-links produced by psoralen plus UVA light. The repair capabilities of the FA complexes, compared with those of the corresponding normal complex, have now been examined using two types of complementation analysis. First, introduction of the normal complex, by electroporation, into 8-methoxypsoralen (8-MOP) plus UVA treated FA-A and FA-D cells resulted in correction of their repair defect, determined by measuring repair-related unscheduled DNA synthesis (UDS). The FA-A and FA-D complexes could similarly complement the repair defect in each others' cells, but not in their own. Second, mixing the normal with the FA-A and FA-D complexes, or the FA-A with the FA-D complex, in a cell-free system resulted in correction of the defect in ability of these FA complexes to incise damaged DNA. These results indicate that the normal complex contains the proteins needed to correct the DNA repair defect in FA-A and FA-D cells and that the FA-A and FA-D complexes contain the protein needed to complement the repair defect in each other.

Cell Line, Transformed↗

PubMed1997

Occurrence of DNase gamma-like apoptotic endonucleases in hematopoietic cells in Xenopus laevis and their relation to metamorphosis.

DNase gamma has been suggested to be an endonuclease responsible for thymic apoptosis in mammals. Using the frog, Xenopus laevis, we examined whether any hematopoietic cells other than thymocytes have DNase gamma activity. The activity gel assays and HeLa cell nuclear assays for DNase revealed that nuclei from red blood cells and cells in liver (an erythropoietic organ) contain DNase gamma-like activities (molecular mass of 38 and 36 kDa) indicated by their biochemical features (internucleosomal DNA cleavage, requirement of Ca2+ and Mg2+, and sensitivity to Zn2+). Distribution of these two (38 and 36 kDa) forms of enzyme was determined by cell fractionation analysis of liver cells: the 38 kDa form of enzyme was found in erythrocytes/erythroblasts and hepatocytes, whereas the 36 kDa form was in lymphocytes/macrophages. Interestingly, these two enzyme activities increased during metamorphic climax, at which time larval-type cells are converted to adult ones. It thus appears that these gamma-type DNases are involved in programmed cell death during metamorphosis.

Animals↗

PubMed1997

Action of mitochondrial endonuclease G on DNA damaged by L-ascorbic acid, peplomycin, and cis-diamminedichloroplatinum (II).

Mitochondrial DNA (mtDNA) suffers extensive damage from the environment, however, the enzymes involved in the repair of mtDNA are still unknown. Here, we partially purified mitochondrial endonuclease G (Endo G) from the bovine heart, and examined the action of Endo G on damaged DNA. Treatment of DNA with L-ascorbic acid or peplomycin, that introduces single-strand breaks through active oxygen radicals, greatly enhanced the susceptibility to nucleolytic attacks from Endo G. The enzyme cleaved at or near sites where single-strand breaks were present in the opposite strand. Cisplatin-mediated DNA damage, which causes intrastrand crosslinks between adjacent guanine residues, also facilitated Endo G digestion, indicating that the enzyme can recognize local distortions in the duplex DNA introduced by adducts. These nucleolytic properties of Endo G in vitro suggest its possible involvement in the maintenance of mtDNA by eliminating defective genomes from the multicopy pool.

Animals↗

PubMed1997

Anti-Ca2+, Mg2+-dependent endonuclease antibody detects specifically a class of chromatin-bound endonuclease.

A polyclonal antibody against purified bull seminal plasma Ca2+, Mg2+-dependent endonuclease was raised in a rabbit. The antibody specifically cross-reacted with chromatin-bound Ca2+, Mg2+-dependent endonucleases from bovine thymus, human placenta, and bovine, rat and mouse liver in addition to the bovine seminal enzyme. The antibody did not cross-react with other endonucleases examined, including the acid-endonucleases from bovine thymus and liver, porcine spleen DNase II, micrococcal nuclease, and bovine pancreas DNase I, a known Ca2+ and Mg2+ requiring endonuclease. The present results indicate that this antibody specifically recognizes a class of so-called Ca2+, Mg2+-dependent endonuclease, which is localized in cell nuclei of various tissues and is probably involved in chromatin degradation during apoptosis. The antibody will be used to study the functional role of this class of endonuclease.

Animals↗

PubMed1998

Chromosomal localization of a human deoxyribonuclease II gene (DNASE2) to 19p13.2-p13.1 using both the polymerase chain reaction and fluorescence in situ hybridization analysis.

Recently obtained information on the cDNA encoding human deoxyribonuclease II (DNase II) (T. Yasuda et al., 1998, J. Biol. Chem. 273, 2610-2616) has made it possible to demonstrate the precise position of the the human DNase II gene (DNASE2) on human chromosomes. Two different sets of oligonucleotide primers specific for human DNase II cDNA sequences were used to amplify unique DNA fragments in the human DNase II gene from a panel of human x rodent hybrid cell lines carrying different human chromosomes. Based on this analysis, DNASE2 was assigned to human chromosome 19. Furthermore, regional localization of the gene to 19p13.2-p13.1 was achieved by fluorescence in situ hybridization analysis using a full-length cDNA probe corresponding to the entire open reading frame.

Chromosome Mapping↗

PubMed1998

Cloning of cDNAs encoding porcine and human DNase II.

We report the molecular cloning of cDNAs encoding porcine and human DNase II and the genomic structure of the human DNase II gene. The full length cDNAs for porcine and human DNase II were isolated by polymerase chain reaction on the basis of amino acid sequences determined for the tryptic peptides of porcine liver DNase II. The porcine and human cDNAs contain 1095 and 1083 bp open reading frames, respectively, and encode 364 and 360 amino acid proteins with calculated molecular masses of 40,157 and 39,555, respectively. The amino acid sequencing of purified porcine DNase II reveals two N-termini with corresponding sequences present within the same open reading frame, suggesting proteolytic processing for the covalently bonded subunit structure of DNase II. Northern blot analysis demonstrated that a single transcript of 2.0 kb mRNA coding for DNase II is ubiquitously expressed in human tissues. A database search revealed that the human genomic sequence of chromosome 19p13.2 contains the DNase II gene. Characterization of the genomic sequence showed that the DNase II gene consists of six exons separated by five introns whose splice acceptor/donor sites agree with the GT/AG rule.

Amino Acid Sequence↗

PubMed1998

Endonucleolytic cleavage of RNA at 5' endogenous stem structures by human flap endonuclease 1.

Structure-specific nucleases called 5' flap endonucleases cleave unannealed 5' arms of template-primer DNA model substrates at the start of the duplex and are involved in Okazaki fragment processing during DNA synthesis. To determine the possible use of the enzymes in RNA structure analysis, the cleavage of synthetic and native RNAs was examined using flap endonuclease 1 (Fen1) of HeLa cells. RNAs are cleaved at about 20% of the rate of DNA model substrates, and most of the cleavage sites are within 200 nucleotides of the 5' end. Hydrolysis of MFA2 mRNA of yeast shows that the cleavages are at the start of five possible stem structures of a folded secondary structure predicted on the basis of both chemical and enzymatic structure probing. 16S ribosomal RNA of Escherichia coli is cleaved at several 5' stem structures of its phylogenetically predicted folded structure. This type of RNA cleavage specificity may be very useful in secondary structure analysis in the future and also may be used by cells for specific 5' end-geared RNA cleavages.

Base Sequence↗

PubMed1999

Generation of highly site-specific DNA double-strand breaks in human cells by the homing endonucleases I-PpoI and I-CreI.

We have determined the ability of two well-characterized eukaryotic homing endonucleases, I-PpoI from the myxomycete Physarum polycephalum and I-CreI from the green alga Chlamydomonas reinhardtii, to generate site-specific DNA double-strand breaks in human cells. These 18-kDa proteins cleave highly conserved 15- or 24-bp rDNA homing sites in their respective hosts to generate homogeneous 4-base, 3' ends that initiate target intron transposition or "homing." We show that both endonucleases can be expressed in human cells and can generate site-specific DNA double-strand breaks in 28S rDNA and homing site plasmids. These endonuclease-induced breaks can be repaired in vivo, although break repair is mutagenic with the frequent generation of short deletions or insertions. I-PpoI and I-CreI should be useful for analyzing DNA double-strand break repair in human cells and rDNA.

Base 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

Cloning of a cDNA encoding a rat DNase II-like acid DNase.

DNase II is a well-known deoxyribonuclease (DNase) that catalyzes the hydrolysis of DNA into oligonucleotides under acidic conditions. We have identified a novel DNase that shows homology to DNase II, named DLAD, from a search of an expressed sequence tag data base. The full-length cDNA for rat DLAD cloned by polymerase chain reaction encodes a 356-amino acid polypeptide containing a putative N-terminal signal peptide and 5 potential N-glycosylation sites; there is a predicted catalytic domain resemblance to rat DNase II. The predicted DLAD translation product shares 32.9% identity with DNase II. Interestingly, expression of the DRAD mRNA is highly restricted to the liver. A Myc-His tagged recombinant DLAD recovered mainly from the cytoplasm of transfected HeLa S3 cells has a divalent cation-independent DNase activity. The DLAD activity prefers acidic conditions to neutral. The recombinant protein expressed in HeLa S3 cells inhibits the expression of GFP- and lac Z-expression vectors, suggesting that DLAD may play a role in elimination of exogenous DNA. Identification of the full-length cDNA for DLAD would lead to an understanding of the physiology of this DNase II-like molecule.

Amino Acid Sequence↗

PubMed2000

Identification of functional elements in the bidirectional promoter of the mouse Nthl1 and Tsc2 genes.

The gene of mammalian endonuclease III homologs (NTHL1/Nthl1), a DNA glycosylase/AP lyase involved in base excision repair, lies immediately adjacent to one of the tuberous sclerosis disease-determining genes, TSC2/Tsc2, in a head-to-head orientation. To clarify the regulation of these divergent genes, we studied the promoter activities of these transcription units by luciferase assay using HeLa cells. We found that the short spacer sequence of 63 base pairs (bp) between the mouse Nthl1 and Tsc2 genes shows bidirectional promoter activity essential for the transcription of both genes. The 63-bp sequence is well conserved among several mammalian species and contains two Ets-transcription factor binding sites (EBSs) in opposite directions. An Ets-family protein in the HeLa nuclear extract specifically bound to either EBSs. Mutation of the core motif of the EBS demonstrated that EBS positively regulates transcription of both mNthl1 and mTsc2 genes. These EBSs had an additive effect on transcription, and each EBS functioned equally in both directions.

Animals↗

PubMed2000

Characterization of two DNase gamma-specific monoclonal antibodies and the in situ detection of DNase gamma in the nuclei of apoptotic rat thymocytes.

Two novel monoclonal antibodies (mAbs), hg302 and hg303, raised against a synthetic peptide corresponding to the basic domain of human DNase gamma, are characterized in detail. In Western blot analysis, hg303 recognizes both wild type and C-terminal Myc-His-tagged human DNase gamma, but does not cross-react with human DNase I family members, DNase I, DNase X, or DNAS1L2. On the other hand, dot blot analysis reveals the fine specificity of hg302; it recognizes human, mouse, and rat DNase gamma, but not other DNase I family DNases under non-denaturing conditions. Furthermore, hg302 efficiently immunoprecipitates wild type, but not C-terminal Myc-His-tagged, human DNase gamma from cell lysates. In immunohistochemical analysis, hg302 strongly recognizes DNase gamma in the nuclei of X-ray irradiation-induced apoptotic, but not normal rat thymocytes. The specific detection of DNase gamma in apoptotic nuclei is confirmed by indirect-immunofluorescence analysis using TNF-alpha-induced apoptotic HeLa S3 cells transfected with DNase gamma. These results, together with the observations that DNase gamma is present in normal thymocytes and its activity is unchanged during the apoptosis, suggest that some molecular change(s), which triggers the activation of DNase gamma, occurs in response to apoptotic stimuli in the basic domain, and hg302 specifically recognizes the activated DNase gamma in immunohistochemical analysis.

Amino Acid Sequence↗

PubMed2001

Archaeoglobus fulgidus RNase HII in DNA replication: enzymological functions and activity regulation via metal cofactors.

RNA primer removal during DNA replication is dependent on ribonucleotide- and structure-specific RNase H and FEN-1 nuclease activities. A specific RNase H involved in this reaction has long been sought. RNase HII is the only open reading frame in Archaeoglobus fulgidus genome, while multiple RNases H exist in eukaryotic cells. Data presented here show that RNase HII from A. fulgidus (aRNase HII) specifically recognizes RNA-DNA junctions and generates products suited for the FEN-1 nuclease, indicating its role in DNA replication. Biochemical characterization of aRNase HII activity in the presence of various divalent metal ions reveals a broad metal tolerance with a preference for Mg(2+) and Mn(2+). Combined mutagenesis, biochemical competitions, and metal-dependent activity assays further clarify the functions of the identified amino acid residues in substrate binding or catalysis, respectively. These experiments also reveal that Asp129 form a second-metal binding site, and thus contribute to activity attenuation.

Amino Acid Sequence↗

PubMed2001

Isolation and characterization of the DLAD/Dlad genes, which lie head-to-head with the genes for urate oxidase.

We previously found that a novel DNA endonuclease named DLAD (DNase II-Like Acid DNase) is specifically expressed in murine liver. Here, we describe the isolation and characterization of the human DLAD and mouse Dlad genes. Both DLAD and Dlad consist of 6 exons. DLAD encodes a 361 amino acid protein sharing 34.6% amino acid identity with human DNase II. Although a recombinant protein for the putative human DLAD has a divalent cation-independent acid DNase activity, expression of the DLAD mRNA containing the entire open reading frame was not detected in any human tissues tested, except for lung, in which a short 1.1 kb transcript lacking the first two exons is expressed. Interestingly, sequence analysis of Dlad showed that the 1st exon of the urate oxidase gene, Uox, is located on the opposite strand in its 5'-flanking region. The head-to-head organization of DLAD and UOX is conserved in the human genomic sequence. Promoter analysis revealed that the intergenic region between Dlad and Uox has promoter activity for both the Dlad and Uox directions, however, the corresponding human genomic fragment has promoter activity only for DLAD. It is known that murine Uox is expressed only in the liver, whereas human UOX has been inactivated as a pseudogene. On the basis of these results, the expression of DLAD/Dlad and UOX/Uox is suggested to be coordinated by a common regulatory mechanism(s), and the balance between the two enzymes is thought to be important for maintaining the purine nucleotide pool in the liver.

5' Flanking Region↗