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)

Sin4p is a component of a mediator complex associated with the C-terminal domain of RNA polymerase II and SIN4 is required for proper regulation of several genes in yeast, including the HO endonuclease gene, glucose repressible genes and MATa cell-specific genes. Previous studies indicated that SIN4 may influence transcription through changes in the organization of chromatin. We have examined a specific chromatin structure associated with MATa cell-specific repression in sin4 MATalpha cells to determine if SIN4 is required for nucleosome positioning. Although the loss of SIN4 has no effect on nucleosome location, we find that the sensitivity of bulk chromatin from sin4 cells to micrococcal nuclease digestion is strikingly increased relative to chromatin from isogenic wild-type cells. The nuclease hypersensitivity of chromatin from sin4 cells is not related to gross alterations in histone gene expression or to bulk increases in histone modification. Our experiments suggest that SIN4 directly or indirectly regulates a global aspect of chromatin accessibility, providing a molecular basis for phenotypic similarities between sin4 mutations and mutations in histones.
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PMID:Global alterations in chromatin accessibility associated with loss of SIN4 function. 909 35

We examine the generality of transcription factor-mediated chromatin remodeling by monitoring changes in chromatin structure in a yeast (Saccharomyces cerevisiae) episome outside of the context of a natural promoter. The episome has a well defined chromatin structure and a binding site for the transcription factor GAL4 but lacks a nearby functional TATA element or transcription start site, so that changes in chromatin structure are unlikely to be caused by transcription. To separate changes caused by binding and by activation domains, we use both GAL4 and a chimeric, hormone-dependent activator consisting of the GAL4 DNA-binding domain, an estrogen receptor (ER) hormone-binding domain, and a VP16 activation domain (Louvion, J.-F., Havaux-Copf, B. and Picard, D. (1993) Gene (Amst.) 131, 129-134). Both GAL4 and GAL4.ER.VP16 show very little perturbation of chromatin structure in their nonactivating configurations. Substantial additional perturbation occurs upon activation. This additional perturbation is marked by changes in micrococcal nuclease cleavage patterns, restriction endonuclease accessibility, and DNA topology and is not seen with the nonactivating derivative GAL4.ER. Remodeling by GAL4.ER.VP16 is detectable within 15 min following hormone addition and is complete within 45 min, suggesting that replication is not required. We conclude that activation domains can exert a major influence on chromatin remodeling by increasing binding affinity and/or by recruitment of other chromatin remodeling activities and that this remodeling can occur outside the context of a bona fide promoter.
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PMID:Chromatin remodeling by transcriptional activation domains in a yeast episome. 911 Oct 67

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

The methods available for analysis of the chromatin of Schizosaccharomyces pombe are time consuming (>8 h) and/or result in some degradation of the chromatin. Here we report an optimised method for the preparation of spheroplasts and the isolation of nuclei which takes <25 min and is suitable for analysis of chromatin structure by micrococcal nuclease, restriction endonuclease or by immunoprecipitation.
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PMID:Isolation of nuclei for chromatin analysis in fission yeast. 935 88

The Holliday junction is a key intermediate in genetic recombination. Here, we examine the effect of a nucleosome core on movement of the Holliday junction in vitro by spontaneous branch migration. Histone octamers consisting of H2A, H2B, H3, and H4 are reconstituted onto DNA duplexes containing an artificial nucleosome-positioning sequence consisting of a tandem array of an alternating AT-GC sequence motif. Characterization of the reconstituted branch migration substrates by micrococcal nuclease mapping and exonuclease III and hydroxyl radical footprinting reveal that 70% of the reconstituted octamers are positioned near the center of the substrate and the remaining 30% are located at the distal end, although in both cases some translational degeneracy is observed. Branch migration assays with the octamer-containing substrates reveal that the Holliday junction cannot migrate spontaneously through DNA organized into a nucleosomal core unless DNA-histone interactions are completely disrupted. Similar results are obtained with branch migration substrates containing an octamer positioned on a naturally occurring sequence derived from the yeast GLN3 locus. Digestion of Holliday junctions with T7 endonuclease I establishes that the junction is not trapped by the octamer but can branch migrate in regions free of histone octamers. Our findings suggest that migration of Holliday junctions during recombination and the recombinational repair of DNA damage requires proteins not only to accelerate the intrinsic rate of branch migration but also to facilitate the passage of the Holliday junction through a nucleosome.
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PMID:A histone octamer blocks branch migration of a Holliday junction. 937 46

Although the product of the UL12 gene of herpes simplex virus type 1 (HSV-1) has been shown to possess both exonuclease and endonuclease activities in vitro, and deletion of most of the gene within the viral genome results in inefficient production and maturation of infectious virions, the function of the deoxyribonuclease (DNase) activity per se in virus replication remains unclear. In order to correlate the in vitro and in vivo activities of the protein encoded by UL12, mutant proteins were tested for nuclease activity in vitro by a novel hypersensitivity cleavage assay and for their ability to complement the replication of a DNase null mutant, AN-1. Rabbit reticulocyte lysates programmed with wild-type UL12 RNA cleaved at the same sites cleaved by purified HSV-1 DNase, but distinct from those cleaved by DNase 1 or micrococcal nuclease. All mutants which lacked DNase activity in vitro also failed to complement the replication of AN-1 in nonpermissive cells. Likewise, all mutants which contained HSV-1 DNase activity, as detected by the hypersensitivity cleavage assay, were capable of complementing the replication of the DNase null mutant, though to varying extents. Of particular note was the d1-126 mutant protein, which, despite having the same specific activity as the wild-type enzyme in vitro, complemented the replication of AN-1 significantly less than the wild-type protein. The results suggest that DNase activity per se is required for efficient replication of HSV-1 in vivo. However, residues, including the N-terminal 126 amino acids, which are dispensable for enzymatic activity in vitro may facilitate the accessibility or activity of the protein in vivo.
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PMID:Structure-function analysis of the herpes simplex virus type 1 UL12 gene: correlation of deoxyribonuclease activity in vitro with replication function. 952 34

We describe physicochemical and enzymatic properties of 5' bridging phosphorothioester linkages at specific sites in DNA oligonucleotides. The susceptibility to hydrolysis at various pH values is examined and no measurable hydrolysis is observed at pH 5-9 after 4 days at 25 degrees C. The abilities of three 3'- and 5'-exonuclease enzymes to hydrolyze the DNA past this linkage are examined and it is found that the linkage causes significant pauses at the sulfur linkage for T4 DNA polymerase and calf spleen phosphodiesterase, but not for snake venom phosphodiesterase. Restriction endonuclease (Nsi I) cleavage is also attempted at a 5'-thioester junction and strong resistance to cleavage is observed. Also tested is the ability of polymerase enzymes to utilize templates containing single 5'-S-thioester linkages; both Klenow DNA polymerase and T7 RNA polymerase are found to synthesize complementary strands successfully without any apparent pause at the sulfur linkage. Finally, the thermal stabilities of duplexes containing such linkages are measured; results show that T m values are lowered by a small amount (2 degrees C) when one or two thioester linkages are present in an otherwise unmodified duplex. The chemical stability and surprisingly small perturbation by the 5' bridging sulfur make it a good candidate as a physical and mechanistic probe for specific protein or metal interactions involving this position in DNA.
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PMID:Chemical and enzymatic properties of bridging 5'-S-phosphorothioester linkages in DNA. 962 13

Ribonuclease P activity from infusoria Tetrahymena pyriformis has been isolated and purified more than 1000-fold over cytosol crude extract. Purified tRNA 5' endonuclease processes in vitro heterologous substrates, precursors of the human tRNA(Tyr) and Drosophila melanogaster tRNA(Leu), exactly at the 5' end of the mature molecules. The activity was abolished by micrococcal nuclease and protease treatment indicating that both RNA and protein components are essential for its activity. The most abundant polypeptides in the purified enzyme fractions have molecular masses of about 100, 44 and 35 kDa. The enzyme requires divalent cations for its activity and shows optimal activity in the presence of the low concentrations of the monovalent salts. Substrate structural requirements for the purified enzyme were analyzed with different tRNA precursor models. The analysis of the derivatives of tRNA(Leu) precursors with altered aminoacyl stem structures reveals that end of the stem is important for substrate 5' end processing with purified enzyme.
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PMID:Partial characterization of the ribonuclease P from Tetrahymena pyriformis. 981 Apr 66

The 5-methyl group of thymidine residues protrudes into the major groove of double helical DNA. The structural influence of this exocyclic substituent has been examined using a PCR-made 160 bp fragment in which thymidine residues were replaced with uridine residues. We show that the dT-->dU substitution and the consequent deletion of the methyl group affects the cleavage of DNA by deoxyribonuclease I and micrococcal nuclease. Analysis of the DNase I cleavage sites, in terms of di and trinucleotides, indicates that homopolymeric tracts of d(AT) become significantly more susceptible to DNase I cleavage when uridine is substituted for thymidine residues. The results indicate that removal of the thymidine methyl groups from the major groove at AT tracts induces structural perturbations that transmit into the opposite minor groove, where they can be detected by endonuclease probing. In contrast, DNase I footprinting experiments with different mono and bis-intercalating drugs reveal that dT-->dU substitution does not markedly affect sequence-specific drug-DNA recognition in the minor or major groove of the double helix. The consequences of demethylation of thymidine residues are discussed in terms of changes in the minor groove width connected to variations in the flexibility of DNA and the intrinsic curvature associated with AT tracts. The study identifies the methyl group of thymine as an important molecular determinant controlling the width of the minor groove and/or the flexibility of the DNA.
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PMID:Demethylation of thymine residues affects DNA cleavage by endonucleases but not sequence recognition by drugs. 1044 37

Here we report the co-factor requirements for DNA fragmentation factor (DFF) endonuclease and characterize its cleavage sites on naked DNA and chromatin substrates. The endonuclease exhibits a pH optimum of 7.5, requires Mg(2+), not Ca(2+), and is inhibited by Zn(2+). The enzyme generates blunt ends or ends with 1-base 5'-overhangs possessing 5'-phosphate and 3'-hydroxyl groups and is specific for double- and not single-stranded DNA or RNA. DFF endonuclease has a moderately greater sequence preference than micrococcal nuclease or DNase I, and the sites attacked possess a dyad axis of symmetry with respect to purine and pyrimidine content. Using HeLa cell nuclei or chromatin reconstituted on a 5 S rRNA gene tandem array, we prove that the enzyme attacks chromatin in the internucleosomal linker, generating oligonucleosomal DNA ladders sharper than those created by micrococcal nuclease. Histone H1, high mobility group-1, and topoisomerase II activate DFF endonuclease activity on naked DNA substrates but much less so on chromatin substrates. We conclude that DFF is a useful reagent for chromatin research.
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PMID:Cleavage preferences of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease) on naked DNA and chromatin substrates. 1071 48


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