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)

A method is described for measuring equilibrium constants of DNA-protein interactions using gel chromatography. This technique has been used to study the sequence-specific interaction of the HinfI restriction endonuclease with DNA. HinfI has a monomeric molecular weight of 31000 and exists as a dimer in its active form. The protein binds to supercoiled DNA molecules containing its recognition site with an apparent free energy of -13.9 kcal/mol of sites. This interaction is highly salt sensitive and causes a release of 3.4 ion pairs. The affinity of the nuclease for its recognition site is largely independent of both pH (6.5-8.5) and temperature (7-35 degrees C) and was not affected by variations in the degenerate middle position of the site. Linear DNA fragments containing the HinfI recognition site were bound as tightly as supercoiled molecules. Binding to nonspecific DNA sites or to methylated DNA sites was approximately 6 orders of magnitude weaker. In general, enzyme activity and binding affinity paralleled each other.
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PMID:Measurement of DNA-protein equilibria using gel chromatography: application to the HinfI restriction endonuclease. 299 May 40

Two of four siblings expressed the salt-losing form of congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH) and had identical human lymphocyte antigen (HLA) and complement C4 (fourth component of complement) types (HLA-A3,C4,B35,C4A3,C4BQO,DR1/A2,C-,B18,C4A3, C4BQO,DR6). The father and one unaffected sibling were heterozygous carriers of CAH, as determined by a 30-min iv ACTH stimulation test and HLA typing. In addition, the iv ACTH stimulation test revealed that the mother and the other unaffected sibling also carried an allele for an attenuated form of CAH. Restriction endonuclease digests of genomic DNA obtained from members of this family and from normal unrelated subjects were hybridized with cDNA probes encoding human 21-hydroxylase and C4. With the 21-hydroxylase probe, Southern blots prepared from control DNA samples revealed two major restriction fragments in each of four restriction endonuclease digests; TaqI produced major bands at 3.7 and 3.2 kilobases (kb), KpnI at 4.0 and 2.9 kb, EcoRI at 18 and 13 kb, and BglII at 15 and 12.5 kb. Southern blots prepared from DNA of the two patients lacked the 3.7-kb TaqI and 2.9-kb KpnI fragments, but had increased hybridization intensity (relative to control DNA samples) in the 3.2-kb TaqI and 4.0-kb KpnI fragments. By contrast, blots with EcoRI or BglII had two large hybridization fragments not different from control DNA samples. These data indicate the presence of two different 21-hydroxylase genes. Additional mapping studies revealed that the two genes had the restriction pattern of the inactive 21-hydroxylase gene. When genomic DNA that had been isolated from all members of this family and from normal subjects was hybridized with the human C4 cDNA probe, the restriction fragment hybridization patterns for all four endonuclease digests were similar in the two groups. Hence, our results suggest that the 21-hydroxylase deficiency of our patients is due to conversion of the active 21-hydroxylase gene to the inactive gene. This gene conversion was associated with absence of functional C4B protein, without any detectable alterations in the restriction fragment pattern of the C4 genes.
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PMID:Gene conversion in salt-losing congenital adrenal hyperplasia with absent complement C4B protein. 300 62

The frequencies of chromosomal aberrations induced by the restriction endonuclease Alu I (recognition site AG/CT) can be elevated to a similar extent by additional treatments with a single-strand-specific endonuclease from Neurospora crassa (EC 3.1.30.1), or with ammonium sulfate in which the Neurospora endonuclease is suspended. These data indicate that Alu I does not produce DNA single-strand breaks in the chromatin of living cells, which can be recognized by the Neurospora endonuclease. The salt may induce conformational changes in the chromatin which make more recognition sites available for Alu I. Experiments with recovery times between the treatments with Alu I and the salt indicate that Alu I can act in the nucleus for at least 40 min.
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PMID:Elevation of Alu I-induced frequencies of chromosomal aberrations in Chinese hamster ovary cells by Neurospora crassa endonuclease and by ammonium sulfate. 301 98

Evidence is presented that endonuclease digestion of isolated, unfixed chromosomes results in the production of banding patterns similar to those produced by digestion of fixed, air-dried chromosomes. Mouse L cell chromosomes were isolated under acidic or relatively neutral pH conditions, exposed in situ (as wet mounts on glass slides) or in vitro (in suspension) to micrococcal nuclease, Alu I or Eco RI, treated with a buffered salt solution, and stained with Giemsa. After any of these endonuclease treatments in situ, the centromeric regions of the chromosomes were intensely stained, characteristic of the C-banding observed in fixed chromosomes exposed to the same treatments. Although the fixed chromosomes were morphologically well-preserved after endonuclease digestion, the morphology of chromosomes digested in situ was variable, ranging from normal to swollen to highly distorted chromosomes. In the latter, the endonucleases induced dispersion of non-C-band chromatin; however, C-bands were still apparent as condensed, differentially-stained regions. Exposure of isolated chromosomes to Alu I in vitro also resulted in well-defined C-banding and led to the extraction of about 70% of the chromosomal DNA. From these results, the mechanism of endonuclease-induced C-banding appears to involve the dispersion and extraction of digested chromatin.
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PMID:Endonuclease banding of isolated mammalian metaphase chromosomes. 301 70

BamHI endonuclease and methylase were found to exhibit a kinetic preference for linear pBR322 DNA substrates containing the recognition site in a central location. The Km values for substrates having the recognition site in a terminal location were approximately 3-fold greater than those with a centrally located site. This phenomenon may be partially due to facilitated transfer of the enzymes to the recognition site over nonspecific flanking sequences. The exploitation of facilitated transfer by these enzymes has been inferred from studies demonstrating kinetic preferences for longer DNA substrates. The reaction rates of the endonuclease were 9-fold greater with full-length pBR322 DNA than with a 74-base pair derivative. The methylase exhibits a kinetic preference for longer substrates but only under conditions of comparatively higher DNA concentrations. In addition, the methylase has the property of increasing long chain preference with increasing salt concentrations up to 120 mM. Increasing salt concentrations decreased the endonuclease's preference for longer substrates. Nonspecific inhibition studies revealed qualitative and quantitative differences between the two enzymes under catalytic conditions. These studies suggest that BamHI endonuclease and methylase interact with nonspecific DNA in different ways.
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PMID:Differences in the kinetic properties of BamHI endonuclease and methylase with linear DNA substrates. 301 63

Protection of restriction endonuclease cleavage sites by Escherichia coli RNA polymerase bound to the replicative form I of bacteriophage S13 DNA has been used to identify a number of regions of RNA polymerase binding. Digestion with HincII, AluI, HinfI, or HaeIII, under conditions optimized for "open" complex formation, revealed 12 regions of RNA polymerase binding. Based on differential salt sensitivities, five of the regions were classified as strong or tight binding sites. These were located before genes A (two sites), B, and D and at the 5' end of gene F. The seven regions which exhibited weaker binding were located at the 5' end of gene C (two sites), in the middle of gene D, just before and at the 3' end of gene F, at the 5' end of gene G, and in the middle of gene H. The sites before genes B and D coincide with sites previously identified as promoters in bacteriophage phi X174. One of the sites before gene A, that at nucleotides 5175-5211, represents a new putative promoter site in bacteriophage S13 and phi X174 located before the previously identified A gene promoter at nucleotides 10-45.
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PMID:Localization of Escherichia coli RNA polymerase-binding sites on bacteriophage S13 replicative form I DNA by protection of restriction enzyme cleavage sites. 303 27

Poly(ADP-ribose) polymerase activity was measured in a crude nuclear fraction isolated from HeLa cells. It was found that the addition of ammonium sulfate or other salts to the standard incubation medium inhibited the formation of poly(ADP-ribose). Through the use of alkaline sucrose density gradients it was also noted that this same increase in ionic strength inhibited the in vitro breakdown of the HeLa DNA. Additional experiments with alkaline sucrose density gradients and deoxyribonuclease I showed that the in vitro activity of poly(ADP-ribose) polymerase is largely dependent upon DNA fragmentation but that DNA fragmentation at least in vitro is not dependent upon the formation of poly(ADP-ribose). These observations imply that this nuclear enzyme is not extremely sensitive to changes in the ionic strength of the reaction media but is affected indirectly, supposedly through changes in the endonuclease activity of the HeLa nuclei. If this proves to be true, then the addition of salt to the incubation medium for poly(ADP-ribose) polymerase could prove to be a valuable tool for the study of ADP-ribosylation reactions.
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PMID:Increased ionic strength: effects on DNA fragmentation and poly(ADP-ribose) polymerase activity in HeLa nuclei. 309 21

A nucleic acid affinity matrix containing a short oligodeoxynucleotide ligand has been prepared as an example of a material which can be used for the rapid and effective isolation of sequence specific DNA binding proteins. Two complementary oligodeoxynucleotides have been employed, one of which contains a small 5'-spacer arm with a terminal thiol group. Using this terminal thiol group, the ligand can be covalently coupled to Tresyl-activated Sepharose 4B or Epoxy-activated Sepharose 6B via a thioether linkage. This approach allows the specific attachment of the nucleic acid ligand via its 5'-terminus to the insoluble matrix. The double stranded affinity material was obtained by annealing of the complementary DNA fragment. As an example, we have used an eicosomer affinity column containing the sequence d(GAATTC) for the isolation of the Eco RI restriction endonuclease. Using a single column, the enzyme could be isolated by eluting the column with a single step or multistep gradient of increasing salt concentration. The enzyme was purified to 75%-85% homogeneity with yields of 0.1 mg to 0.2 mg from 0.5 g of cell paste.
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PMID:An oligodeoxynucleotide affinity column for the isolation of sequence specific DNA binding proteins. 319

A deoxyribonuclease was partially purified from the free-living nematode Caenorhabditis elegans. The DNase functioned as an endonuclease and introduced both single-strand nicks and double-strand breaks into DNA. The enzyme hydrolyzed double-stranded DNA seven times more rapidly than single-stranded DNA. DNase activity was not affected by the addition of divalent cations below 1 mM but was inhibited at higher ionic concentrations. In addition, the enzyme was not inhibited in the presence of 10 mM EDTA. The enzyme was inhibited by salt concentrations greater than 20 mM. Three independent mutations in the nuc-1 gene were shown to reduce nuclease activity to less than 1% of that seen in wild-type organisms.
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PMID:An endonuclease from Caenorhabditis elegans: partial purification and characterization. 322 46

The final step in aldosterone biosynthesis, an oxidation at position 18 of 18-hydroxycorticosterone, is catalyzed by an enzymatic activity termed corticosterone methyl oxidase II (CMO II). This activity is mediated in vitro by P450c11 (steroid 11-hydroxylase), a cytochrome P-450 enzyme that also catalyzes the preceding two steps of 11-hydroxylation and 18-hydroxylation. CMO II deficiency, an inherited defect in the 18-oxidation step, impairs aldosterone biosynthesis and thus leads to a clinical syndrome of salt wasting. To test the hypothesis that CMO II deficiency results from a mutation affecting the structural gene for P450c11, we examined 11 affected and 21 unaffected members of six families with this disorder. After DNA samples were digested with the restriction endonuclease MspI (thereby cutting the DNA at specific sites) and hybridized with a P450c11 DNA probe, a unique DNA fragment in the P450c11 structural gene was detected in subjects with the deficiency. The DNA fragment and the disease trait were inherited together in each family, demonstrating that CMO II deficiency is caused by a mutation in or very near the structural gene for P450c11 on chromosome 8. We conclude that the metabolic diseases of CMO II and 11-hydroxylase deficiency, which have distinct clinical symptoms, may be caused by different mutations in the single gene for a multifunctional enzyme.
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PMID:An inherited defect in aldosterone biosynthesis caused by a mutation in or near the gene for steroid 11-hydroxylase. 326 27


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