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)

The thyroglobulin 33-S mRNA was isolated from sheep thyroid total polysomes. The 33-S RNA, twice purified on a 1% sodium dodecylsulfate/sucrose gradient, was 30-fold enriched in thyroglobulin messenger activity and was estimated as 50% pure by its messenger activity and 80% pure by the electrophoretic profile. It was used as template for complementary DNA synthesis and hybridized up to 85% of the DNA copy with pseudo-first-order kinetics. Back-hybridization kinetics showed that the purified mRNA corresponds to a major kinetic component with a base sequence complexity of 10000 nucleotides as determined by comparison to globin mRNA. Cross-reactivity of [3H]cDNA with liver RNA is less than 10%. Restriction endonuclease digestion of [3H]cDNA yielded a discrete band pattern. The distribution of thyroglobulin mRNA among free polysomes, membrane-bound polysomes and extrapolysomal pools was analyzed using hybridization to the specific [3H]cDNA probe. Free particles were recovered in the supernatant and membrane-bound particles in the pellet after a brief centrifugation of detergent-free homogenate (5 min at 27000 x g: procedure A; 12 min at 130000 x g: procedure B) with precautions taken to avoid cross-contamination. Using procedure A, 80% of thyroglobulin mRNA sequences were found in the membrane-bound fraction. Using procedure B, where contamination of free particles by membrane-bound particles was avoided by high-speed initial centrifugation and further isolation through a discontinuous sucrose gradient, 95-98% of thyroglobulin mRNA sequences were recovered in membrane-bound polysomes. In total polysomes, 89% of thyroglobulin mRNA sequences were in the polysomal area and shifted to ribosomal subunits after EDTA treatment.
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PMID:Subcytoplasmic distribution of thyroglobulin mRNA in normal sheep thyroid. 737 33

We have investigated the Ca2+ dependency of DNA degradation into nucleosome-sized fragments in intact chromaffin-like PC12 cells and PC12 nuclear fractions. In intact cells we were unable to trigger DNA fragmentation by inducing either transient or sustained elevations of cytoplasmic Ca2+ ([Ca2+]i) with the Ca2+ ionophore ionomycin. On the contrary, DNA fragmentation was induced in intact cells by the intracellular Zn2+ chelator NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN). To characterize further PC12 cell endonuclease activity, we then investigated digestion by purified PC12 cell fractions of exogenously added plasmids. In nuclear fractions two endonuclease activities were identified: an acidic (pH 5.0) endonuclease activity that was fully Ca2+- and Mg(2+)-independent; and a neutral (pH 7.6) endonuclease activity that was Ca(2+)-independent but Mg(2+)-dependent. Both endonuclease activities were inhibited by Zn2+. Nuclear membrane permeabilization greatly enhanced plasmid digestion at pH 7.6, but not at pH 5.0. This suggests that neutral endonuclease was located in a membrane-bound compartment, whereas acidic endonuclease was freely accessible to the substrate even in the presence of an intact nuclear membrane. In intact nuclei, digestion of genomic DNA could not be triggered by increasing the bivalent cation composition of the medium. On the contrary, in hypotonic medium we observed a large spontaneous nucleolytic DNA degradation that was increased by Zn2+ chelation. However, an acidic pH shift was a potent stimulus for DNA fragmentation in isotonic as well as hypotonic medium.
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PMID:Ionic regulation of endonuclease activity in PC12 cells. 748 21

The membrane-bound form of guanylate cyclase represents a biologically active atrial natriuretic factor receptor (GC/ANF-R). We have constructed genomic map of murine GC-A/ANF-R gene using 17 different restriction endonucleases. The restriction mapping results indicated that murine GC-A/ANF-R gene is approximately 20 kb single copy with multiple smaller exons and bigger introns. The Kpn I and Sfu I restriction digests produced 27 kb and 35 kb fragments, respectively, which hybridized with 5'- and 3'-flanking cDNA probes. Both of these fragments should cover the entire murine GC-A/ANF-R genomic sequences. The southern blot hybridization of genomic DNA from human, rat and mouse, using murine 5'-flanking cDNA probe indicated the presence of higher variant sequences in the 5'-flanking region of GC-A/ANF-R gene among different species. The noncoding 5'-flanking probe (350 bp) hybridized only to mouse genomic DNA but not to the human or rat DNA. These sequence variations located in the noncoding 5'-flanking region of GC-A/ANF-R gene may explain the divergent evolutionary development among different species. This is the first demonstration of the restriction endonuclease digestion and genomic mapping of murine GC-A/ANF-R gene which should be valuable to the understanding of its regulation and function.
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PMID:Genomic restriction endonuclease analysis and mapping of murine guanylate cyclase-A/atrial natriuretic factor receptor gene. 790 87

We have previously demonstrated the induction of the early response transcription factor NGFI-A in castration-induced regression of the rat ventral prostate. We have developed an in vitro model to investigate the role of kinase signal transduction and early transcriptional regulation in apoptosis of androgen-sensitive prostate cells. Cell death was induced in the androgen-sensitive human prostate line, LNCaP, by addition of the protein kinase activator, 12-tetradecanoylphorbol 13-acetate (TPA). TPA-induced death of LNCaP cells was asynchronous and exhibited morphological and ultrastructural features indicative of apoptosis. Specifically, cytoplasmic contraction, condensation of nuclear chromatin, and formation of membrane-bound "apoptotic bodies" were observed. Additionally, the characteristic endonuclease-generated DNA ladder, commonly associated with apoptosis, was observed in TPA-treated LNCaP cultures. TPA-induced LNCaP apoptosis was preceded by rapid yet transient induction of the early response transcription factors NGFI-A and c-fos. In dose-response experiments, NGFI-A and c-fos gene activation parallels the induction of death in LNCaP cells. TPA-induced expression of NGFI-A and c-fos and death of LNCaP cultures were blocked by pretreatment with staurosporine, a potent inhibitor of several protein kinases. Based on these studies, we suggest that activation of a TPA-inducible kinase(s) mediates apoptosis of androgen-sensitive prostate cells by means of an intracellular pathway that may involve the transient activation of the early response transcription factors NGFI-A and c-fos.
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PMID:Phorbol ester-induced apoptosis is accompanied by NGFI-A and c-fos activation in androgen-sensitive prostate cancer cells. 794 88

Microsporidia are obligate intracellular protozoan parasites that can cause opportunistic infections in AIDS patients. Species from five genera of microsporidia are presently known to infect man. One species, Septata intestinalis originally was detected in stool specimens of individuals with chronic diarrhea and subsequently was found to disseminate to the kidneys, lungs, and nasal sinuses. This organism has since been reclassified as Encephalitozoon and in this study, we report the culture of Encephalitozoon intestinalis from a bronchoalveolar lavage specimen and a nasal mucus aspirate of two AIDS patients living in the USA. The bronchoalveolar and nasal microsporidian isolates grew in several continuous cell lines including RK-13, MDCK, HT-29, Caco-2, Vero, and I047. Transmission electron microscopy of the clinical and cell culture specimens revealed that the new isolates appeared to be E. intestinalis based on morphology and growth of organisms in septated membrane-bound parasitophorous vacuoles. The new E. intestinalis isolates were characterized and compared with the first isolated E. intestinalis that was cultured from stool to confirm their identity and to determine if there existed any minor differences, as seen in the closely related Encephalitozoon cuniculi strains. By the methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis staining for proteins and carbohydrates, Western blot immunodetection, and polymerase chain reaction-based methods with restriction endonuclease digestion, double-stranded DNA heteroduplex mobility shift analysis, and DNA sequencing of the ribosomal DNA intergenic spacer region, the new isolates were identical to each other and to the reference isolate of E. intestinalis. In addition, with any of these methods, the E. intestinalis organisms could be distinguished from the three E. cuniculi strains, Encephalitozoon hellem, and Vittaforma corneae, which is important for diagnostics, therapeutic strategies, and epidemiology.
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PMID:Characterization of Encephalitozoon (Septata) intestinalis isolates cultured from nasal mucosa and bronchoalveolar lavage fluids of two AIDS patients. 856 8

Target sequence-specific DNA binding regions of the restriction endonuclease EcoRII were identified by screening a membrane-bound EcoRII-derived peptide scan with an EcoRII recognition site (CCWGG) oligonucleotide duplex. Dodecapeptides overlapping by nine amino acids and representing the complete protein were prepared by spot synthesis. Two separate DNA binding regions, amino acids 88-102 and amino acids 256-273, which share the consensus motif KXRXXK, emerged. Screening 570 single substitution analogues obtained by exchanging every residue of both binding sites for all other amino acids demonstrated that replacing basic residues in the consensus motifs significantly reduced DNA binding. EcoRII mutant enzymes generated by substituting alanine or glutamic acid for the consensus lysine residues in DNA binding site I expressed attenuated DNA binding, whereas corresponding substitutions in DNA binding site II caused impaired cleavage, but enzyme secondary structure was unaffected. Furthermore, Glu96, which is part of a potential catalytic motif and also locates to DNA binding site I, was demonstrated to be critical for DNA cleavage and binding. Homology studies of DNA binding site II revealed strong local homology to SsoII (recognition sequence, CCNGG) and patterns of sequence conservation, suggesting the existence of functionally related DNA binding sites in diverse restriction endonucleases with recognition sequences containing terminal C:G or G:C pairs.
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PMID:Regions of endonuclease EcoRII involved in DNA target recognition identified by membrane-bound peptide repertoires. 998 71

Biological polyadenylation, first recognized as an enzymatic activity, remained an orphan enzyme until poly A sequences were found on the 3' ends of eukarvotic mRNAs. Their presence in bacteria viruses and later in archeae (ref. 338) established their universality. The lack of compelling evidence for a specific function limited attention to their cellular formation. Eventually the newer techniques of molecular biology and development of accurate nuclear processing extracts showed 3' end formation to be a two-step process. Pre-mRNA was first cleaved endonucleolytically at a specific site that was followed by sequential addition of AMPs from ATP to the 3' hydroxyl group at the end of mRNA. The site of cleavage was specified by a conserved hexanucleotide, AAUAAA, from 10 to 30 nt upstream of this 3' end. Extensive purification of these two activities showed that more than 10 polypeptides were needed for mRNA 3' end formation. Most of these were in complexes involved in the cleavage step. Two of the best characterized are CstF and CPSF, while two other remain partially purified but essential. Oddly, the specific proteins involved in phosphodiester bond hydrolysis have yet to be identified. The polyadenylation step occurs within the complex of poly A polymerase and poly A-binding protein, PABII, that controls poly A length. That the cleavage complex, CPSF, is also required for this step attests to a tight coupling of the two steps of 3' and formation. The reaction reconstituted from these RNA-free purified factors correctly processes pre-mRNAs. Meaningful analysis of the role of poly A in mRNA metabolism or function was possible once quantities of these proteins most often over-expressed from cDNA clones became available. The large number needed for two simple reactions of an endonuclease, a polymerase and a sequence recognition factor, pointed to 3' end formation as a regulated process. Polyadenylation itself had appeared to require regulation in cases where two poly A sites were alternatively processed to produce mRNA coding for two different proteins. The 64-KDa subunit of CstF is now known to be a regulator of poly A site choice between two sites in the immunoglobulin heavy chain of B cells. In resting cells the site used favors the mRNA for a membrane-bound protein. Upon differentiation to plasma cells, an upstream site is used the produce a secreted form of the heavy chain. Poly A site choice in the calcitonin pre-mRNA involves splicing factors at a pseudo splice site in an intron downstream of the active poly site that interacts with cleavage factors for most tissues. The molecular basis for choice of the alternate site in neuronal tissue is unknown. Proteins needed for mRNA 3' end formation also participate in other RNA-processing reactions: cleavage factors bind to the C-terminal domain of RNA polymerase during transcription; splicing of 3' terminal exons is stimulated port of by cleavage factors that bind to splicing factors at 3' splice sites. nuclear ex mRNAs is linked to cleavage factors and requires the poly A II-binding protein. Most striking is the long-sought evidence for a role for poly A in translation in yeast where it provides the surface on which the poly A-binding protein assembles the factors needed for the initiation of translation. This adaptability of eukaryotic cells to use a sequence of low information content extends to bacteria where poly A serves as a site for assembly of an mRNA degradation complex in E. coli. Vaccinia virus creates mRNA poly A tails by a streamlined mechanism independent of cleavage that requires only two proteins that recognize unique poly A signals. Thus, in spite of 40 years of study of poly A sequences, this growing multiplicity of uses and even mechanisms of formation seem destined to continue.
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PMID:A history of poly A sequences: from formation to factors to function. 1210 57

The replication checkpoint kinase Cds1 preserves genome integrity by stabilizing stalled replication forks. Cds1 targets substrates through its FHA domain. The Cds1 FHA domain interacts with Mus81, a subunit of the Mus81-Eme1 structure-specific endonuclease. We report here that Mus81 and Rhp51 are required for generating deletion mutations in fission yeast replication mutants that experience replication stress. A mutation in the Mus81 FHA-binding motif eliminates its Cds1-binding and Cds1-dependent phosphorylation. Furthermore, this mutation exacerbates the deletion mutator phenotype of a replication mutant, and induces a hyper-recombination phenotype in hydroxyurea-treated cells. In unperturbed cells, Mus81 associates with chromatin throughout S phase. In replication mutants grown at semipermissive temperature, Mus81 undergoes minor Cds1-dependent phosphorylation, remains chromatin-associated, generates deletion mutations, and maintains cell growth. Upon S-phase arrest by acute hydroxyurea treatment, Mus81 is not required for cell viability but is essential for recovery from replication fork collapse. Moreover, Mus81 undergoes extensive Cds1-dependent phosphorylation and dissociates from chromatin in hydroxyurea-arrested cells, thereby preventing it from cleaving stalled replication forks that could lead to fork breakage and chromosomal rearrangement. These results provide novel insights into how Cds1 regulates Mus81 accordingly when cells experience different replication stress to preserve genome integrity.
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PMID:Replication checkpoint kinase Cds1 regulates Mus81 to preserve genome integrity during replication stress. 1580 65

The approximately 30-kb coronavirus (+)RNA genome is replicated and transcribed by a membrane-bound replicase complex made up of 16 viral nonstructural proteins (nsp) with multiple enzymatic activities. The complex includes an RNA endonuclease, NendoU, that is conserved among nidoviruses but no other RNA virus, making it a genetic marker of this virus order. NendoU (nsp15) is a Mn(2+)-dependent, uridylate-specific enzyme, which leaves 2'-3'-cyclic phosphates 5' to the cleaved bond. Neither biochemical nor sequence homology criteria allow a classification of nsp15 into existing endonuclease families. Here, we report the crystal structure of the severe acute respiratory syndrome coronavirus nsp15 at 2.6-A resolution. Nsp15 exhibits a unique fold and assembles into a toric hexamer with six potentially active, peripheric catalytic sites. The structure and the spatial arrangement of the catalytic residues into an RNase A-like active site define a separate endonuclease family, endoU, and represent another spectacular example of convergent evolution toward an enzymatic function that is critically involved in the coronavirus replication cycle.
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PMID:Crystal structure and mechanistic determinants of SARS coronavirus nonstructural protein 15 define an endoribonuclease family. 1688 30

Poly-ADP-ribosylation is a unique post-translational modification that controls various nuclear events such as repair of DNA single-strand breaks. Recently, the protein containing the poly-ADP-ribose (pADPr)-binding zinc-finger (PBZ) domain was shown to be a novel AP endonuclease and involved in a cell cycle checkpoint. Here, we determined the three-dimensional structure of the PBZ domain from Drosophila melanogaster CG1218-PA using NMR spectroscopy. The domain folds into a C2H2-type zinc-finger structure in an S configuration, containing a characteristic loop between the zinc-coordinating cysteine and histidine residues. This is distinct from the structure of other C2H2-type zinc fingers. NMR signal changes that occur when pADPr binds to the PBZ domains from CG1218-PA and human checkpoint with FHA (forkhead-associated) and ring finger (CHFR) and mutagenesis suggest that a surface relatively well conserved among PBZ domains may serve as a major interface with pADPr.
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PMID:Solution structure of a zinc-finger domain that binds to poly-ADP-ribose. 2008 64


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