Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nature of the endonucleases responsible for DNA fragmentation in apoptosis has not yet been clearly defined. The intracellular acidity has been known to greatly affect apoptosis probably by affecting the activity of the endonucleases. In this study, the implication of pH in the apoptosis was investigated through the use of human HL-60 leukemia cells. The cells were incubated in media with different pH ranging from 3.5 to 7.5 for 4 hrs and the mode of cell death was investigated. The trypan blue exclusion assay showed that close to 25% and 90% of the cells were dead when incubated in pH 6.4 and pH 5.0 media, respectively. The agarose gel electrophoresis of DNA demonstrated that significant DNA fragmentation occurred in the HL-60 cells incubated in the pH 6.2-6.4 media for 4 hr indicating cell death by apoptosis. The electron microscopy study also demonstrated that many of the cells incubated in the pH 6.4 medium were in the process of apoptosis while the cells maintained in the pH 5.0 medium were dying by necrosis. The intracellular pH (pHi) of HL-60 cells was 6.6-6.9 when the extracellular pH (pHe) was 6.2-6.4. These results demonstrated that DNase I which has a maximal endonuclease activity near pH 7.0 may be responsible for the apoptosis accompanied by DNA fragmentation in HL-60 cells in the pH 6.4 medium. This observation is at variance with the previous reports that DNase II mediate the DNA fragmentation in apoptosis. The cell death at extremely low pH (pH 5.0) appeared to be due mainly to necrosis.
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PMID:Effects of intracellular pH on apoptosis in HL-60 human leukemia cells. 859 48

DNA fragmentation is a common biochemical hallmark of apoptosis. It is catalyzed by endogenous Ca2+, Mg(2+)-dependent endonuclease(s). Although the exact identity of the apoptotic endonuclease is still a matter of debate, a number of candidate nucleases have been proposed like NUC18, DNase II and DNase I. Relatively large amounts of nucleases are also expressed by mycoplasmas, cell wall-less bacteria of the class Mollicutes, which are found as contaminants in up to 45% of the continuous cell lines in current use. In order to clarify the effect of these pathogens on the investigation of apoptosis in cell culture systems, we looked for biochemical markers (DNA fragmentation, nuclease expression) and morphological changes characteristic of apoptosis (cell shrinkage, chromatin condensation, apoptotic bodies) in Mycoplasma hyorhinis-free and -infected cultures of the human pancreatic adenocarcinoma cell line PaTu 8902 and of mouse NIH 3T3 fibroblasts. For that purpose we employed cells cultured under standard conditions and cells exposed to the protein synthesis inhibitor cycloheximide, which is known to induce apoptosis in various cell systems. After exposure to cycloheximide only the mycoplasma-positive cells exhibited internucleosomal DNA degradation. In contrast, nuclease activities in the molecular range of 47 to 54 kDa were detected in cell homogenates and culture supernatants of infected cultures of both control and cycloheximide-treated cells, whereas mycoplasma-free cultures were nuclease-negative. The expression of the nucleases and the cycloheximide-induced DNA fragmentation were suppressed by the prokaryote-specific protein synthesis inhibitor chloramphenicol. Moreover, partially purified nucleases from supernatants of infected cells were able to cleave the DNA of isolated substrate nuclei at internucleosomal sites. These data indicate that DNA ladder formation in cell culture systems can also be caused by mycoplasmal nucleases which apparently penetrate the host cells after cycloheximide treatment or more generally after cellular stress. Therefore, internucleosomal DNA fragmentation in established cell lines has to be regarded with care, unless mycoplasmal infection can be excluded, or the existence of endogenous endonucleases can be proven. The presence of endonucleolytic activities of about 47 to 54 kDa molecular mass has now to be regarded as highly indicative of contaminations with M. hyorhinis. In contrast, the expression of an apoptotic morphology was not restricted to infected cells; in both mycoplasma-free and -contaminated cultures, cells with condensed chromatin were observed after staining with the DNA binding dye Hoechst 33342. Electron microscopic studies revealed that most of the cells containing compacted DNA were phagocytosed by unaffected fellow cells. Presumably because of the relatively long exposure (72 h) to cycloheximide we also observed secondary necrosis as indicated by the parallel occurrence of morphological characteristics of apoptosis (chromatin condensation) and necrosis (loss of membrane integrity and organelle swelling).
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PMID:Internucleosomal DNA fragmentation in cultured cells under conditions reported to induce apoptosis may be caused by mycoplasma endonucleases. 888 84

Xib, a gene recently reported to reside on the q28 region of the human X chromosome [Pergolizzi et al. (1996) Gene 168, 267-270], contains an open reading frame homologous to those of the DNase I family enzymes. The full open reading frame of this gene has been fused to the E. coli gene of the maltose binding protein and expressed in bacteria as a chimeric protein. The partially purified chimeric protein is enzymatically active. It introduces single and double stranded breaks into supercoiled DNA, at 30 degrees C in the absence of divalent cations and at a pH optimum of 5.2. To our knowledge this enzyme represents the first cloned human endonuclease with characteristics similar to those of acidic DNase II.
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PMID:Functional characterization of a human DNase-like protein encoded by a gene positioned in Xq28. 909 69

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.
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PMID:Anti-Ca2+, Mg2+-dependent endonuclease antibody detects specifically a class of chromatin-bound endonuclease. 924 Apr 53

One approach to discriminate among specific DNases in apoptosis is to use inhibitors specific for each endonuclease. Zn2+ is known to inhibit Ca(2+)- and Mg(2+)-dependent endonuclease enzymatic activities during apoptosis. Acidic DNases were thought to be insensitive to Zn2+. In this paper, we analyse the effects of Zn2+ on activity of DNase II, either purified or in nuclei from lens fiber cells. These cells follow a physiological nuclear degeneration with DNase II accumulation in their nuclei. We show that Zn2+ is able to inhibit also this acidic endonuclease at a concentration of 1-6 mM. At a higher concentration of Zn2+, DNA is extensively degraded during the assay, masking the inhibition of the enzyme. This DNA degradation in the presence of Zn2+ has led to an overestimation of the activity of DNase II in studies of apoptosis. Hence, Zn2+ cannot be used to specifically identify one endonuclease among the different DNases involved in nuclear degradation during programmed cell death.
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PMID:On the use of Zn2+ to discriminate endonucleases activated during apoptosis. 935 93

The most widely recognized biochemical change associated with the majority of apoptotic systems is the degradation of genomic DNA. Among the enzymes that may participate in this cleavage, the acidic cation-independent DNase II is a likely candidate since it is activated in many apoptotic cells. To better understand its role, we purified and sequenced a DNase II extracted from porcine spleen. Protein sequencing of random peptides demonstrated that this enzyme is derived from a ubiquitous serpin, the leukocyte elastase inhibitor (LEI), by an acidic-dependent posttranslational modification or by digestion with elastase. We call this novel enzyme L-DNase II. In vitro experiments with purified recombinant LEI show that the native form has no effect on purified nuclei whereas its posttranslationally activated form induces pycnosis and DNA degradation. Antibodies directed against L-DNase II showed, in different cell lines, an increased expression and a nuclear translocation of this enzyme during apoptosis. Since the appearance of the endonuclease activity results in a loss of the anti-protease properties of LEI, the transformation from LEI to L-DNase II may act as a switch of protease and nuclease pathways, each of which is activated during apoptosis.
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PMID:L-DNase II, a molecule that links proteases and endonucleases in apoptosis, derives from the ubiquitous serpin leukocyte elastase inhibitor. 958 2

We have previously implicated deoxyribonuclease II (DNase II) as an endonuclease responsible for DNA digestion during apoptosis. The full-length human cDNA has now been cloned. The cDNA contains an open reading frame of 1078 bases coding for a 40-kDa protein. This protein is 10 kDa larger than commercially supplied enzyme, which has been proteolytically cleaved at an internal aspartate residue. The gene is located at chromosome 19p13.2, and has no significant homology to other human proteins, but has >30% identity to three predicted genes in Caenorhabditis elegans. To determine whether overexpression of DNase II induces apoptosis in Chinese hamster ovary cells, the cDNA was cotransfected with a plasmid encoding green fluorescent protein. Within 24 h, a significant proportion of green fluorescent protein-positive cells contained condensed chromatin, whereas vector-only controls remained viable. Considering that DNase II is normally active only at low pH, it was surprising that transfection induced chromatin condensation. To confirm that transfection was not activating another endonuclease, cells were incubated with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)-fluoromethylketone; this failed to inhibit chromatin condensation induced by DNase II. These results demonstrate that DNase II acts downstream of caspase activation and that it may be activated by an as yet unknown mechanism to induce DNA digestion during apoptosis.
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PMID:The cloning and expression of human deoxyribonuclease II. A possible role in apoptosis. 981 84

We cloned and partially characterized a human endonuclease (Xib) which shows sequence homologies to pancreatic DNase I but an enzymatic activity closer to DNase II. We report on the structural differences found between Xib and other recently cloned human DNases. Fluores cence microscopy analysis of transiently transfected cells with Xib::pEGFP constructs indicate that the protein is located in the cytoplasm and possibly anchored to a membrane, as deduced from a hydrophobic amino acid stretch present at the C-terminal end. Xib is overexpressed in muscle and cardiac tissues and is alternately spliced in several normal and neoplastic cells. In situ hybridization studies using human cardiac and muscle biopsies indicate accumulation of Xib transcript in the vacuoles of muscle cells from patients affected by vacuolar myopathy as acid maltase deficiency; however, no point mutations were detected in their DNA.
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PMID:Molecular characterization of a novel endonuclease (Xib) and possible involvement in lysosomal glycogen storage disorders. 1656 3

In this report, we describe the molecular cloning and characterization of DLAD, a novel mammalian deoxy-ribonuclease homologous to DNase II. The full length cDNA for mouse DLAD has been cloned by polymerase chain reaction. The cDNA contains a 1065 bp open reading frame (ORF) encoding a 354 amino acid protein with a calculated molecular mass of 40 767. The predicted protein for DLAD shares 34.4% identity with DNase II. DLAD is also homologous to three predicted proteins, C07B5.5, F09G8.2 and K04H4.6, from the nematode Caenorhabditis elegans. Furthermore, the third ORF of the fowlpox virus genome is found to encode a DLAD homologue showing 37. 1% identity at the amino acid level. Northern blot analysis reveals that expression of the DLAD mRNA is highly restricted to the liver. DLAD mainly exists as a cytoplasmic protein with divalent cation-independent endonuclease activity and cleaves DNA to produce 3'-phosphoryl/5'-hydroxyl ends. It is active under a wide range of pH with maximum activity at pH 5.2. Among known DNase inhibitors tested, aurintricarboxylic acid and Zn(2+)are found to be effective inhibitors of the DLAD activity.
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PMID:DLAD, a novel mammalian divalent cation-independent endonuclease with homology to DNase II. 1049 74

The Caenorhabditis elegans nuc-1 gene has previously been implicated in programmed cell death due to the presence of persistent undegraded apoptotic DNA in nuc-1 mutant animals. In this report, we describe the cloning and characterization of nuc-1, which encodes an acidic nuclease with significant sequence similarity to mammalian DNase II. Database searches performed with human DNase II protein sequence revealed a significant similarity with the predicted C. elegans C07B5.5 ORF. Subsequent analysis of crude C. elegans protein extracts revealed that wild-type animals contained a potent endonuclease activity with a cleavage preference similar to DNase II, while nuc-1 mutant worms demonstrated a marked reduction in this nuclease activity. Sequence analysis of C07B5.5 DNA and mRNA also revealed that nuc-1(e1392), but not wild-type animals contained a nonsense mutation within the CO7B5.5 coding region. Furthermore, nuc-1 transgenic lines carrying the wild-type C07B5.5 locus demonstrated a complete complementation of the nuc-1 mutant phenotype. Our results therefore provide compelling evidence that the C07B5.5 gene encodes the NUC-1 apoptotic nuclease and that this nuclease is related in sequence and activity to DNase II.
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PMID:The C. elegans apoptotic nuclease NUC-1 is related in sequence and activity to mammalian DNase II. 1090 46


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