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)

We report the overproduction of the non-specific endonuclease domain of the bacterial toxin colicin E9 and its preliminary characterisation in vitro. The enzymatic colicins (61 kDa) are normally released from producing cells in a complex with their cognate inhibitors, known as the immunity proteins (9.5 kDa). Tryptic digestion of the purified ColE9 complex was found to generate two major components, a monomer derived from the N-terminal and central regions of the toxin and a heterodimer comprising the catalytically active C-terminal domain of the colicin bound to its intact immunity protein, Im9. N-terminal amino acid sequencing, in conjunction with electrospray mass spectrometry, shows that preparations of the DNase domain isolated by this method are heterogeneous, thus making subsequent mechanistic and structural analysis difficult. This problem was circumvented by selectively overexpressing the C-terminal 15-kDa nuclease domain of colicin E9 in tandem with its cognate inhibitor in Escherichia coli. This tandem overexpression strategy allowed high-level production of a 25-kDa protein complex comprising the C-terminal DNase domain of colicin E9 tightly bound to its specific inhibitor Im9, thus masking the anticipated toxicity of the nuclease. The DNase domain was then separated from Im9 under denaturing conditions, refolded by removal of the denaturant and the renatured protein shown to possess both endonuclease and Im9 binding activity. These results describe a novel method for the overproduction of a nuclease in bacteria by co-expressing its specific inhibitor and lay the foundations for a full mechanistic, biophysical and structural characterization of the isolated DNase domain of the colicin E9 toxin.
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PMID:Tandem overproduction and characterisation of the nuclease domain of colicin E9 and its cognate inhibitor protein Im9. 812 2

A quantitative endonuclease assay, which relies on the introduction of single and double strand breaks into supercoiled plasmid DNA, was used to study the activity of the extracellular nuclease of Serratia marcescens SM6 in buffer and in groundwater. The parallel enzyme concentration-dependent production of relaxed and linear plasmid molecules suggests that the nuclease produces single and double strand breaks in duplex DNA. Bovine serum albumin stimulated the nuclease activity towards DNA and RNA and increased the stability of the enzyme against thermal inactivation. The DNase activity at 4 degrees C and 50 degrees C was almost half of that at the optimum temperature (37 degrees C). The nuclease was active in groundwater, although the specific activity was lower than in buffer. In a groundwater aquifer microcosm, mineral-adsorbed transforming DNA was substantially less accessible to the nuclease than was dissolved DNA. The data suggest that the extracellular nuclease of Serratia marcescens may contribute to DNA turnover in the environment and that adsorption of DNA to minerals provides protection against the nuclease.
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PMID:The extracellular nuclease of Serratia marcescens: studies on the activity in vitro and effect on transforming DNA in a groundwater aquifer microcosm. 814 44

We have discovered a mitochondrial, site-specific DNase in Saccharomyces cerevisiae with properties like that of a Type II restriction endonuclease. The enzyme, termed SceIII, cleaves the palindromic sequence 5'GCCGGC, to give 3' ends recessed by 4 bases. SceIII is the first restriction-like endonuclease to be described in yeast mitochondria.
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PMID:Evidence for a site-specific endonuclease in yeast mitochondria which recognizes the sequence 5'GCCGGC. 819 76

The protein encoded by the archaeal intron in the 23S rRNA gene of the hyperthermophile Desulfurococcus mobilis is a double-strand DNase that, like group I intron homing endonucleases, is capable of cleaving an intronless allele of the gene. This enzyme, I-Dmo I, is unusual among the intron endonucleases in that it is thermostable and is expressed only from linear and cyclized intron species and not from the precursor RNA. However, in analogy to its eukaryotic counterparts, but unlike the bacteriophage enzymes, I-Dmo I makes a staggered double-strand cut that generates 4-nt 3' extensions. Additionally, although the archaeal and group I introns have entirely different structural properties and splicing pathways, I-Dmo I shares sequence similarity, in the form of the LAGLI-DADG motif, with group I intron endonucleases of eukaryotes. These observations support the independent evolutionary origin of endonucleases and intron core elements and are consistent with the invasive potential of endonuclease genes.
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PMID:A site-specific endonuclease encoded by a typical archaeal intron. 839 Jun 59

Apoptosis is a mechanism for eliminating unwanted cells from the cell community of multicellular organisms. Abnormalities in the regulation of apoptosis may play a part in the aetiology of cancer, autoimmune diseases, AIDS, degenerative nerve diseases and malformation. On of the hallmarks of apoptosis is the enzymatic cleavage of genomic DNA into nucleosomal oligomers. The identification of an endonuclease responsible for apoptosis might help to explain how this cell suicide is regulated and why DNA is cleaved. Here, we found that gamma type of DNase was retained in apoptotic rat thymocyte nuclei. The mode of DNA cleavage, 3'-hydroxyl (OH)/5'-phosphoryl (P) ends, by homogeneously purified DNase gamma (Mr = 33 kDa) and its Zn2+ sensitivity match those observed in apoptosis in thymocytes induced by irradiation or glucocorticoid treatment, indicating that this endonuclease is a central component of the thymic apoptosis machinery.
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PMID:[Apoptosis: its molecular mechanisms and biological roles]. 857 68

We have used random mutagenesis to identify putative active-site residues in the C-terminal cytotoxic endonuclease domain of the bacterial toxin colicin E9. Six single-site mutations in the DNase domain were isolated which destroyed the toxic action of the colicin. DNA sequencing identified the mutations as Gly460Asp, Arg544Gly, Glu548Gly, Thr571Ile, His575Tyr and His579Tyr. All six wild-type residues are highly conserved in the DNase domains of both the E group colicins and the closely related pyocins. Site-directed mutagenesis was then used to substitute the wild-type amino acid residue at each of these positions for an alanine residue in order to distinguish important from unimportant sites. Two of the six alanine-mutant colicins (Gly460Ala and His579Ala) exhibited significant in vivo activity, unlike the original mutation of these residues, and were therefore not characterised further. The Thr571Ala mutant colicin, although not inactive, was significantly less active than the control. The other three alanine mutants (Arg544Ala, Glu548Ala and His575Ala remained completely inactive in the in vivo tests. Each 15 kDa alanine-mutant DNase domain was overexpressed and purified using a tandem-expression strategy which relies on the enzyme being able to bind to the natural inhibitor, Im9. Tryptophan emission spectra of the alanine mutants showed significant alterations in the emission maxima of all but the His575Ala mutant, suggesting changes in the tertiary structure of these mutant proteins. Activity measurements, using the spectrophotometric Kunitz assay, indicated that the Thr571Ala mutant was partially active as an endonuclease but the remaining alanine mutants were all completely inactive. All four mutant proteins, however, retained their ability to bind DNA in a gel shift assay, suggesting the mutations affect catalytic rather than substrate-binding residues. Searching the sequence databases for possible homology to other DNA-binding proteins revealed a significant match between residues 464 to 487 of the E9 DNase domain and helix IV of the POU domain of eukaryotic transcription factors.
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PMID:Identification of putative active-site residues in the DNase domain of colicin E9 by random mutagenesis. 870 51

Drosophila ribosomal protein PO was overexpressed in Escherichia coli to allow for its purification, biochemical characterization and to generate polyclonal antibodies for Western analysis. Biochemical tests were originally performed to see if overexpressed PO contained DNase activity similar to that recently reported for the apurinic/apyrimidinic (AP) lyase activity associated with Drosophila ribosomal protein S3. The overexpressed ribosomal protein was subsequently found to act on AP DNA, producing scissions that were in this case 5' of a baseless site instead of 3', as has been observed for S3. As a means of confirming that the source of AP endonuclease activity was in fact due to PO, glutathione S-transferase (GST) fusions containing a Factor Xa cleavage site between GST and PO were constructed, overexpressed in an E.coli strain defective for the major 5'-acting AP endonucleases and the fusions purified using glutathione-agarose affinity column chromatography. Isolated fractions containing purified GST-PO fusion proteins were subsequently found to have authentic AP endonuclease activity. Moreover, glutathione-agarose was able to deplete AP endonuclease activity from GST-PO fusion protein preparations, whereas the resin was ineffective in lowering DNA repair activity for PO that had been liberated from the fusion construct by Factor Xa cleavage. These results suggested that PO was a multifunctional protein with possible roles in DNA repair beyond its known participation in protein translation. In support of this notion, tests were performed that show that GST-PO, but not GST, was able to rescue an E.coli mutant lacking the major 5'-acting AP endonucleases from sensitivity to an alkylating agent. We furthermore show that GST-PO can be located in both the nucleus and ribosomes. Its nuclear location can be further traced to the nuclear matrix, thus placing PO in a subcellular location where it could act as a DNA repair protein. Other roles beyond DNA repair seem possible, however, since GST-PO also exhibited significant nuclease activity for both single- and double-stranded DNA.
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PMID:Drosophila ribosomal protein PO contains apurinic/apyrimidinic endonuclease activity. 893 86

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.
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PMID:Occurrence of DNase gamma-like apoptotic endonucleases in hematopoietic cells in Xenopus laevis and their relation to metamorphosis. 907 Feb 68

The major nuclease from Mycoplasma penetrans has been purified to homogeneity. The enzyme seems to be present as a membrane-associated precursor of 50 kDa and as a peripheral membrane monomeric polypeptide of 40 kDa that is easily removed by washing of cells with isotonic buffers and in the aqueous phase upon Triton partitioning of Triton X-114-solubilized protein. The 40-kDa nuclease was extracted from M. penetrans cells by Triton X-114 and phase fractionation and was further purified by chromatography on Superdex 75 and chelating Sepharose (Zn2+ form) columns. By gel filtration, the apparent molecular mass was 40 kDa. The purified enzyme exhibits both a nicking activity on superhelical and linear double-stranded DNA and a nuclease activity on RNA and single-stranded DNA. No exonuclease activity was found for this enzyme. This nuclease required both Mg2+ (optimum, 5 mM) and Ca2+ (optimum, 2 mM) for activity and exhibited a pH optimum between pH 7 and 8 for DNase activity. It was inhibited by Zn2+, Mn2+, heparin, sodium dodecyl sulfate, and chelator agents such EDTA and EGTA, but no effect was observed with ATP, 2-mercaptoethanol, N-ethylmaleimide, dithiothreitol, nonionic detergents, phenylmethylsulfonyl fluoride, and iodoacetamide. Nuclease activity was inhibited by diethylpyrocarbonate at both pH 6 and 8 and by pepstatin, suggesting the involvement of a histidine and an aspartate in the active site. When added to human lymphoblast nuclei, the purified M. penetrans endonuclease induced internucleosomal fragmentation of the chomatin into oligonucleosomal fragments. On the basis of this result, and taking into account the fact that M. penetrans has the capacity to invade eucaryotic cells, one can suggest, but not assert, that produced Ca2+/Mg2+-dependent endonuclease may alter the nucleic acid metabolism of host cells by DNA and/or RNA degradation and may act as a potential pathogenic determinant.
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PMID:Purification and characterization of Mycoplasma penetrans Ca2+/Mg2+-dependent endonuclease. 907 6

A sensitive method for the assay of Ca2+, Mg(2+)-dependent endonuclease was developed. The assay procedure is composed of two parts: (i) microscale endonuclease digestion of highly polymerized calf thymus DNA and (ii) the quantification of DNA breaks by measuring the activation of poly(ADP-ribose) polymerase, which is known to be activated proportionally to the number of nicks and ends of DNA added in the reaction mixture. This method was approximately 10(5)-fold more sensitive than a conventional DNase assay detecting acid-soluble DNA formation and, thus, the activity of 20 to 100 fg of purified bull seminal Ca2+, Mg(2+)-dependent endonuclease could be reliably measured. Ca2+ and Mg2+ requirements and the response to histone H2B of the endonuclease were also demonstrated by this method. Using this method, the assay of a very small amounts of Ca2+, Mg(2+)-dependent endonuclease in crude extracts of calf thymus chromatin was possible. This method may be applied to other types of endonucleases by modifying the mixture for endonuclease reaction.
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PMID:Assay method for femtogram order of Ca2+, Mg(2+)-dependent endonuclease. 917 8


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