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 describe the partial purification of an endonuclease from calf thymus that nicks phage PM2 DNA irradiated with UV doses producing only a few pyrimidine dimers per molecule. It has much less activity on DNA that has been subjected to enzymatic photoreactivation after UV irradiation. The calf thymus endonuclease is different from other mammalian UV-endonucleases so far described in that it seems to be dimer specific. The enzyme is stimulated by Mg2+ and is inactive in the presence of EDTA. It binds to UV-irradiated DNA-Sepharose from which it is released by low concentrations of KCl. Gel filtration data indicate that the endonuclease may belong to a high molecular weight protein or protein complex. The enzyme is very labile and freezing increases its lability.
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PMID:UV-endonuclease from calf thymus with specificity toward pyrimidine dimers in DNA. 4 Feb 30

An in vitro complementation assay has been used for partial purification of uvrA+, uvrB+, and uvrC+ gene products from Escherichia coli. The uvrB+ and uvrC+ products cochromatograph on DEAE-cellulose and are completely resolved from the uvrA+ product, which has been further purified by phosphocellulose chromatography of the nonadsorbed protein fraction from the DEAE-cellulose. Neither the uvrB+/uvrC+ nor the uvrA+ product shows appreciable endonuclease activity on UV-irradiated DNA when tested separately. However, these factors complement each other to yield and ATP-dependent endonuclease activity specific for UV-irradiated DNA. Gel filtration experiments with the partially purified proteins indicate that the functional uvrA+ gene product has a molecular weight of 100,000. The uvrB+ gene product has an apparent molecular weight of 70,000, but it is presently unclear if this is the size of the uvrB+ product alone or the size of a complex of the uvrB+ and uvrC+ gene products.
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PMID:Reconstitution of an Escherichia coli repair endonuclease activity from the separated uvrA+ and uvrB+/uvrC+ gene products. 35 11

A new restriction endonuclease has been isolated from Bacillus sphaericus R. The purification procedure includes Bio-Gel filtration, (NH4)2SO4 fractionation and phosphocellulose chromatography. After the phosphocellulose step the enzyme preparation is free of non-specific nucleases. Bsp cleaves double-stranded DNA with the same specificity as Bacillus subtilis (Bsu) and Haemophilus aegyptius (HaeIII) restriction endonucleases, as concluded from digests and double-digests of phiX174 replicative form DNA with Bsu and Bsp. The 5'-terminal nucleotide of the cleavage products was shown to be C. Bacillus sphaericus R produces Bsp in extremely large quantities and the enzyme can be easily purified in high yield.
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PMID:A new sequence-specific endonuclease (Bsp) from Bacillus sphaericus. 59 Jul 42

Mitochondrial DNA from several mammalian species has been digested with a site-specific restriction endonuclease (HaeIII) from Haemophilus aegyptius. A quantitative analysis of the resulting specific fragments indicates that the mtDNA of any individual mammal is predominantly a single molecular clone. Gel analysis of specific cleavage products has proven quite sensitive in detecting differences in mtDNA: mtDNAs from the more distantly related mammals studied (e.g., donkey and dog) are found to have few bands in common and very closely related mammals (e.g., donkey and horse) share only about 50% of their bands. This procedure has detected several intraspecies mtDNA differences. Six distinct human patterns have been found, with one pattern usually differing from another in two or three bands. mtDNAs from different organs of single individuals have also been analyzed, and no differences have been found.
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PMID:Specific cleavage analysis of mammalian mitochondrial DNA. 106 Jan 30

Circular dichroic spectra of T7 RNA polymerase show minima at 222 nm ([theta]m=-7.9 X 10(3) deg cm2/dmol) and 208 nm ([theta]m =-7.55 X 10(3) deg cm2/dmol) and a maximum at 193 nm ([theta]m = 1.2 X 10(4) deg cm2/dmol). The small mean residue ellipticity above 200 nm indicates that the secondary structure contains approximately 12% alpha helix. The secondary structure is unaltered by high salt, glycerol, -SH reagents, nitration of tyrosyl residues, and chelating agents. Binding of the native enzyme to [32P]T7 DNA has been measured by the retention of the protein-[32P]DNA complexes on nitrocellulose filters. At 37degrees T7 RNA polymerase binds to its promoters in the absence of NTP's. Binding and catalytic activity are both abolished at 0degree. Binding of the initiating [gamma-32P]GTP can also be detected by the filter binding assay. Native T7 RNA polymerase is inactivated by reaction with 1 mol of 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) or 1 mol of [14C]iodoacetamide. The latter reaction is blocked by Nbs2 suggesting that a single -SH group is required for activity. Alkylation of the -SH group does not alter binding of the enzyme to the DNA template, but modifies the binding of GTP to the enzyme. Nitration of approximately4 surface tyrosyl residues of the protein prevents binding to T7 DNA. The restriction endonuclease, Hpa II, cuts T7 DNA into approximately40 fragments and reduces total RNA synthesis by T7 RNA polymerase by 70%. Fragmentation of the DNA template by Hpa II does not alter the rate of RNA chain initiation by T7 polymerase, and restriction fragments accounting for approximately25% of the T7 DNA still bind tightly to the enzyme. Thus the T7 RNA polymerase promoters remain intact on the restriction fragments. Gel electrophoresis of the transcription products, using restriction fragments as templates, show that of the seven in vitro transcripts produced by T7 RNA polymerase from whole T7 DNA, only the smallest (representing the last 1.5% of the genome) is transcribed from Hpa II fragments. The remaining transcripts are replaced by six new and much shorter mRNA's. The DNA fragments containing the promoters for these mRNA's have been removed from the fragment mix by binding them to the enzyme and retaining the complexes on nitrocellulose filters.
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PMID:T7 RNA polymerase: conformation, functional groups, and promotor binding. 110 55

The PCR was used to alter transcriptional and translational signals surrounding the Flavobacterium okeanokoites restriction endonuclease (fokIR) gene, so as to achieve high expression in Escherichia coli. By changing the ribosome-binding site sequence preceding the fokIR gene to match the consensus E. coli signal and by placing a positive retroregulator stem-loop sequence downstream of the gene, Fok I yield was increased to 5-8% of total cellular protein. Fok I was purified to homogeneity with phosphocellulose, DEAE-Sephadex, and gel chromatography, yielding 50 mg of pure Fok I endonuclease per liter of culture medium. The recognition and cleavage domains of Fok I were analyzed by trypsin digestion. Fok I in the absence of a DNA substrate cleaves into a 58-kDa carboxyl-terminal and 8-kDa amino-terminal fragment. The 58-kDa fragment does not bind the DNA substrate. Fok I in the presence of a DNA substrate cleaves into a 41-kDa amino-terminal fragment and a 25-kDa carboxyl-terminal fragment. On further digestion, the 41-kDa fragment degrades into 30-kDa amino-terminal and 11-kDa carboxyl-terminal fragments. The cleaved fragments both bind DNA substrates, as does the 41-kDa fragment. Gel-mobility-shift assays indicate that all the protein contacts necessary for the sequence-specific recognition of DNA substrates are encoded within the 41-kDa fragment. Thus, the 41-kDa amino-terminal fragment constitutes the Fok I recognition domain. The 25-kDa fragment, purified by using a DEAE-Sephadex column, cleaves nonspecifically both methylated (pACYCfokIM) and nonmethylated (pTZ19R) DNA substrates in the presence of MgCl2. Thus, the 25-kDa carboxyl-terminal fragment constitutes the Fok I cleavage domain.
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PMID:Functional domains in Fok I restriction endonuclease. 158 61

RNase L activated by 2-5A (a series of 2'-5'-linked adenylic oligoribonucleotides) is a key enzyme of the interferon system. To study RNase L (endonuclease L) in intact cells independently of intracellular 2-5A and of its activity, we have developed polyclonal antibodies against RNase L. RNase L from mouse spleen was purified on a column of 2-5A-Sepharose and used to immunize rabbits in co-injection with polyadenylic-polyuridylic acid as adjuvant. Antibodies were purified by chromatography on Affi-Gel blue and 2-5A-Sepharose-immobilized RNase L. These polyclonal antibodies immunoprecipitate the 80- and 40-kDa forms of RNase L in mouse spleen. In Western blot, only the 80-kDa form of RNase L is recognized by these antibodies. These purified antibodies were used to localize RNase L in the cytoplasm of intact mouse NIH 3T3 cells by immunofluorescence. The cytoplasmic localization of RNase L was confirmed by its 2-5A binding activity after cellular fractionation.
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PMID:Polyclonal antibodies against RNase L. Subcellular localization of this enzyme in mouse cells. 170 39

To investigate the potentials of DNA methylation and H1 histone in regulating the action of DNA binding proteins, well ordered complexes were formed by slow salt gradient dialysis of mixtures of H1 histone with either methylated or nonmethylated DNA. The sites methylated in the plasmids were CCGG. Methylation of cytosine in this site protects the DNA against HpaII endonuclease but not against MspI. However, when the methylated DNA was complexed to H1, it was protected against MspI. The protection was only effective for a subset of the MspI restriction sites. The protection of DNA afforded by the combination of H1 binding and DNA methylation did not apply to EcoRI, PstI, or BamHI sites and so did not seem to be due to aggregation of the DNA by H1 histone. Gel retardation assays indicated that the affinity of H1 for methylated DNA was not detectably different from its affinity for nonmethylated DNA. Probably methylated DNA when bound to H1 is in a conformation that is resistant to MspI endonuclease. Such conformational changes induced by DNA methylation and H1 binding might affect the action of other DNA binding proteins, perhaps in chromatin as well as in H1.DNA complexes.
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PMID:The combination of DNA methylation and H1 histone binding inhibits the action of a restriction nuclease on plasmid DNA. 170 75

Four-arm DNA branched junctions are stable analogues of Holliday recombinational intermediates. A number of four-arm DNA junctions synthesized from oligonucleotides have now been studied. Gel mobility or chemical footprinting experiments on several immobile four-arm junctions indicate that in the presence of Mg2+, they assume a preferred conformation consisting of two helical domains, each formed by stacking a particular pair of arms on each other. We show here that a junction we designate as J1c that has the same chemical composition as one we have previously studied in detail, J1, but is formed from the four strands complementary to those of the latter, exhibits the reverse stacking preference. The pattern of self-protection of the strands of J1c exposed to Fe(II).EDTA-induced scission reveals that twofold symmetry is preserved, but the opposite pair of strands preferentially cross over. Moreover, the Fe(II).EDTA scission profiles of J1c indicate that this junction exhibits a weaker bias as to which strands cross over than is observed in J1. The preference for the dominant species in J1 is 1.3 times greater than in J1c at 4 degrees C and in the presence of 10 mM Mg2+, based on chemical reactivity data. This is confirmed by a cleavage experiment using the resolvase enzyme, endonuclease I, from bacteriophage T7. This difference could reflect either sequence-dependent differences in the equilibrium among isomers, or in the structure of these junctions. Chemical footprinting experiments using the probes MPE.Fe(II) and (OP)2Cu(I) show that the high-affinity ligand binding site in immobile junctions is determined by junction geometry.
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PMID:Conformational preference and ligand binding properties of DNA junctions are determined by sequence at the branch. 190 7

Glucocorticoids stimulate apoptosis in rat thymocytes that is characterized by internucleosomal DNA degradation. We have previously identified an 18-kDa calcium-dependent nuclease whose activity is associated with this DNA degradation. The existence of this nuclease has been challenged by Alnemri and Litwack (1989) J. Biol. Chem. 264, 4104-4111, who suggest that the nuclease we observed was histone H2B. We report here a modified nuclease assay which uses [32P] DNA as a substrate that has enabled the purification and characterization of the 18-kDa nuclease (NUC18). Using Bio-Rex 70 chromatography in conjunction with this assay, we show that NUC18 can be separated from histone H2B. Enzymatically active NUC18, purified to apparent homogeneity, failed to react with two different anti-histone H2B antibodies. NUC18 was inactive in the absence of calcium and known inhibitors of apoptosis, i.e. zinc and aurintricarboxylic acid inhibit its activity. Although NUC18 activity was detected in nuclear extracts of thymocytes of both control and glucocorticoid-treated thymocytes, these activities were distinct. Gel filtration analysis revealed that NUC18 was present as a high molecular weight complex (greater than 100 kDa) in both groups of cells, whereas it also existed as a low molecular weight form in glucocorticoid-treated cells. Thus, NUC18 remains a candidate for the endonuclease responsible for the DNA degradation component of the apoptotic process.
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PMID:Identification, purification, and characterization of a calcium-dependent endonuclease (NUC18) from apoptotic rat thymocytes. NUC18 is not histone H2B. 191 79


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