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)

Two group I introns (CpSSU.1 and CpSSU.2) that each potentially encode a protein with two copies of the LAGLI-DADG motif were identified in the Chlamydomonas pallidostigmatica chloroplast small subunit rRNA gene. They both belong to subgroup IA3 and represent novel insertion positions in this gene (sites 508 and 793 in the Escherichia coli 16S rRNA). The proteins encoded by the two introns were synthesized in vitro and tested for their ability to cleave the homing site of their respective introns. Only the CpSSU.1-encoded protein (I-CpaII) was found to display specific DNA endonuclease activity. At 0.1 mM MgCl2, I-CpaII nicks only the bottom (transcribed) DNA strand, but at concentrations ranging from 0.5 to 5.0 mM, it cleaves both DNA strands (leaving a 4 nucleotide single-stranded extension with 3'-OH overhangs) while preferentially nicking the bottom strand. The rate of cleavage of the top strand increases with increasing concentration of MgCl2. The preliminary data derived from these endonuclease assays suggest that the mode of DNA cleavage by I-CpaII is directed by the availability of Mg2+ and the affinity of different binding sites for this cation.
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PMID:The site-specific DNA endonuclease encoded by a group I intron in the Chlamydomonas pallidostigmatica chloroplast small subunit rRNA gene introduces a single-strand break at low concentrations of Mg2+. 763 Jul 30

A common strongly ordered multi-step-pattern of endogenous DNA degradation was induced in rat liver nuclei and intact thymocytes, prepared in the presence of chelating agents and incubated in the presence of CaCl2 and/or MgCl2. It consisted of sequential generation of 0.3 Mbp, then 0.05 Mbp DNA fragments and finally of oligo- and mononucleosomal DNA. Oligonucleosomal DNA was generated when the genome had already been disintegrated into 0.05 Mbp DNA fragments. ZnCl2 completely inhibited advanced genome cleavage to oligo- and mononucleosomal DNA without affecting the initial generation of large DNA fragments. Therefore, the endonucleolytic activity which produce large DNA fragments is different from Ca2+/Mg2+ endonuclease. The similar pattern of DNA degradation was observed in thymocytes treated with dexamethasone and with the topoisomerase II inhibitor VM-26, the agents known to induce apoptosis. The effect of VM-26 strongly suggests the involvement of topoisomerase II in generation of large DNA fragments. Multi-level organization and regulation of the chromatin structure determine the stepwise process of genome degradation. Detachment of chromatin from the nuclear matrix attachment regions may be one of the possible mechanisms of switching off the genome function and triggering the multi-step process of endogenous chromatin degradation thus leading to cell death in terminal differentiation or stress-induced apoptosis.
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PMID:Comparative study of induction of endogenous DNA degradation in rat liver nuclei and intact thymocytes. 774 35

Streptococcus thermophilus strain CNRZ 455 produces a type II restriction endonuclease designated Sth455I. This enzyme was isolated from cell extracts by anionic and cationic exchange chromatography. This yielded an enzyme preparation free of non-specific nucleases. The optimal reaction conditions for Sth455I are: MgCl2, 30 mM; pH range, 8-9; incubation temperature, 37-42 degrees C; and a high NaCl concentration, 100-200 mM. The results of single- and double-digestion experiments indicates that Sth455I is an isoschizomer of BstNI and EcoRII showing different sensitivity to methylation. The enzyme exhibits restriction activity on the DNA of three bacteriophages of S. thermophilus and no activity on the phage lytic for strain CNRZ 455. The restriction/modification system associated with this strain is discussed.
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PMID:Restriction/modification in Streptococcus thermophilus: isolation and characterization of a type II restriction endonuclease Sth455I. 776 29

A restriction endonuclease, Aor13HI, an isoschizomer of BspMII, was purified to homogeneity from cell extracts of Acidiphilium organovorum strain 13H. The enzyme has a molecular mass of 60,000 daltons and consists of two subunits identical in molecular mass of 30,000 daltons. Aor13HI endonuclease, like BspMII, recognizes the palindromic six-base sequence 5'-TCCGGA-3', and cleaves between the T and C to produce a four-base 5' extension. Aor13HI is not inhibited by dam-dependent methylation. The isoelectric point of the enzyme is 5.7. Aor13HI activity was maximum at pH 7.5, 100 mM KCl, 7.5-10 mM MgCl2, and 55 degrees C. The enzyme was stable up to 60 degrees C. The N-terminal amino acid sequence (30 residues) of Aor13HI did not show any similarity with the sequence of other restriction endonucleases reported.
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PMID:Restriction endonuclease Aor13HI from Acidiphilium organovorum 13H, a new isoschizomer of BspMII: purification and characterization. 776 67

We have used the intrinsic tryptophan fluorescence of the EcoRV restriction endonuclease to monitor changes in protein conformation during binding and cleavage of a duplex oligodeoxynucleotide substrate. Appropriate conditions for single-turnover reactions were first determined by steady-state kinetics. When single turnovers were monitored by stopped-flow fluorescence, the mixing together of EcoRV, oligonucleotide and MgCl2 resulted in a rapid increase in tryptophan fluorescence followed by a slow decrease. Further analysis by order-of-mixing and quench experiments showed that the transient increase in fluorescence was due to a conformational change coupled to DNA binding, while the subsequent decay was concomitant with phosphodiester hydrolysis. The rate of the latter step varied with the concentration of Mg2+ ions, but another Mg(2+)-dependent transition was observed upon the addition of MgCl2 to a preformed enzyme-DNA complex. These results lead to a reaction scheme in which one Mg2+ binds to the active site prior to phosphodiester hydrolysis but a second Mg2+ is then needed to carry out the hydrolytic reaction. This scheme is correlated to the crystal structures of the EcoRV endonuclease and its complexes with DNA and Mg2+ ions.
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PMID:Rapid reaction analysis of the catalytic cycle of the EcoRV restriction endonuclease. 781 66

Physical and chemical properties, as well as polypeptide structure of DNAse from Mycoplasma fermentans PG-18, have been determined. The enzyme in a native form exists probably as a decamere (10X34 kD) and manifests maximal activity at weak alkaline pH range. The temperature optimum of the enzyme is --37 degrees C. DNAse appears to be Mg2+-dependent and has its maximal activity at 10 mM MgCl2. EDTA completely inhibits DNAse activity. The given DNAse has been determined to cleave a phosphodiether bond in 3'-position of deoxyribose and to have both exo- and endonuclease activity, since it has hydrolized both native linear doublestranded DNA and closed-circle plasmid DNA.
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PMID:[The physicochemical properties of the DNAse of Mycoplasma fermentans PG-18]. 802 91

During genetic crosses between the interfertile green algae Chlamydomonas eugametos and Chlamydomonas moewusii, the I-CeuI endonuclease encoded by the fifth group I intron (CeLSU.5) in the C. eugametos chloroplast large subunit rRNA gene mediates the mobility of this intron by introducing a double-strand break near the insertion site of the intron in the corresponding C. moewusii intronless allele. To characterize the biochemical properties of this endonuclease, we have purified I-CeuI and determined the optimal reaction conditions for cleavage. I-CeuI activity is maximal at 50 degrees C, pH 10.0, 2.5 mM MgCl2 and in the absence of NaCl. Unlike the well-characterized I-SceI endonuclease, I-CeuI remains stable following preincubation in the absence of substrate. We discuss the role that homing endonucleases may have played in the evolution of Chlamydomonas chloroplast group I introns.
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PMID:The I-CeuI endonuclease: purification and potential role in the evolution of Chlamydomonas group I introns. 814 39

The L-21 ScaI ribozyme (E) derived from the self-splicing group I intron of Tetrahymena pre-rRNA catalyzes an RNA endonuclease reaction analogous to the first step in self-splicing: CCCUCUAAAAA (S) + G-->CCCUCU+GAAAAA. We show herein that the pH dependence for the single-turnover reaction E.S+G-->products follows a pH dependence with pKapp = 6.9 (10 mM MgCl2, 50 degrees C). This result was surprising because the titratable groups of RNA have pKa values of < approximately 4 or > approximately 9. Thus, two models were considered: (i) the ribozyme structure perturbs a pKa such that the pKapp of 6.9 corresponds to an actual titration or (ii) the pKapp is a kinetic pKa, reflecting a change in the rate-limiting step rather than an actual titration. Oligonucleotide substrates with -H (deoxyribose), -F (2'-fluoro-2'-deoxyribose), and -OH (ribose) substitutions at the 2' position of the U residue at the cleavage site [U(-1)] vary considerably in their intrinsic reactivities. In the ribozyme reaction these substrates reacted at very different rates at low pH, but approached the same limiting reaction rate at high pH. Similarly, substitution of the pro-RP nonbridging oxygen atom of the reactive phosphoryl group by sulfur lowers the intrinsic reactivity of the oligonucleotide substrate. In the ribozyme reaction, this "thio effect" was 2.3 below pH 6.9, whereas the thio substitution had no effect on the rate above pH 6.9.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparison of pH dependencies of the Tetrahymena ribozyme reactions with RNA 2'-substituted and phosphorothioate substrates reveals a rate-limiting conformational step. 817 3

The Cfr9I restriction endonuclease recognizes and cleaves duplex DNA sequence C decreases CCGGG. The binding of restriction endonuclease Cfr9I to DNA was examined in the absence of Mg2+ using gel-mobility-shift and nitrocellulose-filter-binding assays. It was shown that restriction endonuclease Cfr9I bound DNA fragments either containing or lacking the canonical recognition sequence with equal affinity. These results suggest that the specificity of restriction endonuclease Cfr9I is expressed during the catalytic step. The cleavage of supercoiled pUC18 DNA by restriction endonuclease Cfr9I showed that at low concentrations of MgCl2, only with open-circular DNA, nicks appeared in one strand at the recognition sequence, while the cleavage of the second strand was very slow. At higher concentrations of MgCl2 the enzyme cleaves either one or both strands of the DNA. Under these conditions the supercoiled DNA was converted to open-circular and linear forms simultaneously rather than consecutively. It was shown that open-circular DNA was a poor substrate for restriction endonuclease Cfr9I. These results suggested that both Mg2+ and intact recognition sequence are required to drive the enzyme into correct conformation to ensure DNA cleavage.
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PMID:Catalytic and binding properties of restriction endonuclease Cfr9I. 822 80

New site-specific endonucleases BspBS31I, BstBS32I, BspIS41, BstTS5I, BspTS514I were isolated from five thermophilic soil bacteria Bacillus sp. BS31, B. stearothermophilus BS32, Bacillus sp. IS4, B. stearothermophilus TS5, Bacillus sp. TS514. The enzymes are isoschizomers of the restriction endonuclease BbvII. Endonuclease BspTS514I was obtained pure from interfering contaminations by two consecutive chromatographies on blue agarose and hydroxyapatite. The enzyme exhibits a maximal activity at 55 degrees C in 10 mM tris-HCl (pH 9.2), 10 mM MgCl2 and 50 mM NaCl.
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PMID:[Isolation and properties of site-specific endonuclease BspTS514I from the thermophilic bacteria Bacillus species TS514]. 828 20


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