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 biochemical role of poly(ADP-ribosyl)ation on internucleosomal DNA fragmentation associated with apoptosis was investigated in HL 60 human premyelocytic leukemia cells. It was found that UV light and chemotherapeutic drugs including adriamycin, mitomycin C, and cisplatin increased poly(ADP-ribosyl)ation of nuclear proteins, particularly histone H1. A poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide, prevented both internucleosomal DNA fragmentation and histone H1 poly(ADP-ribosyl)ation in cells treated with the apoptosis inducers. When nuclear chromatin was made accessible to the exogenous nuclease in a permeabilized cell system, chromatin of UV-treated cells was more susceptible to micrococcal nuclease than the chromatin of control cells. Suppression of histone H1 poly(ADP-ribosyl)ation by 3-aminobenzamide reduced the micrococcal nuclease digestibility of internucleosomal chromatin in UV-treated cells. These results suggest that the poly(ADP-ribosyl)ation of histone H1 correlates with the internucleosomal DNA fragmentation during apoptosis mediated by DNA damaging agents. This suggestion is supported by the finding that xeroderma pigmentosum cells which are defective in introducing incision at the site of DNA damage, failed to induce DNA fragmentation as well as histone H1 poly(ADP-ribosyl)ation after UV irradiation. We propose that poly(ADP-ribosyl)ation of histone H1 protein in the early stage of apoptosis facilitates internucleosomal DNA fragmentation by increasing the susceptibility of chromatin to cellular endonuclease.
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PMID:Poly(ADP-ribosyl)ation of histone H1 correlates with internucleosomal DNA fragmentation during apoptosis. 862 64

Is the pathway of protein folding determined by the relative stability of folding intermediates, or by the relative height of the activation barriers leading to these intermediates? This is a fundamental question for resolving the Levinthal paradox, which stated that protein folding by a random search mechanism would require a time too long to be plausible. To answer this question, we have studied the guanidinium chloride (GdmCl)-induced folding/unfolding of staphylococcal nuclease [(SNase, formerly EC 3.1.4.7; now called microbial nuclease or endonuclease, EC 3.1.31.1] by stopped-flow circular dichroism (CD) and differential scanning microcalorimetry (DSC). The data show that while the equilibrium transition is a quasi-two-state process, kinetics in the 2-ms to 500-s time range are triphasic. Data support the sequential mechanism for SNase folding: U3 <--> U2 <--> U1 <--> N0, where U1, U2, and U3 are substates of the unfolded protein and N0 is the native state. Analysis of the relative population of the U1, U2, and U3 species in 2.0 M GdmCl gives delta-G values for the U3 --> U2 reaction of +0.1 kcal/mol and for the U2 --> U1 reaction of -0.49 kcal/mol. The delta-G value for the U1 --> N0 reaction is calculated to be -4.5 kcal/mol from DSC data. The activation energy, enthalpy, and entropy for each kinetic step are also determined. These results allow us to make the following four conclusions. (i) Although the U1, U2, and U3 states are nearly isoenergetic, no random walk occurs among them during the folding. The pathway of folding is unique and sequential. In other words, the relative stability of the folding intermediates does not dictate the folding pathway. Instead, the folding is a descent toward the global free-energy minimum of the native state via the least activation path in the vast energy landscape. Barrier avoidance leads the way, and barrier height limits the rate. Thus, the Levinthal paradox is not applicable to the protein-folding problem. (ii) The main folding reaction (U1 --> N0), in which the peptide chain acquires most of its free energy (via van der Waals' contacts, hydrogen bonding, and electrostatic interactions), is a highly concerted process. These energy-acquiring events take place in a single kinetic phase. (iii) U1 appears to be a compact unfolded species; the rate of conversion of U2 to U1 depends on the viscosity of solution. (iv) All four relaxation times reported here depend on GdmCl concentrations: it is likely that none involve the cis/trans isomerization of prolines. Finally, a mechanism is presented in which formation of sheet-like chain conformations and a hydrophobic condensation event precede the main-chain folding reaction.
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PMID:Least activation path for protein folding: investigation of staphylococcal nuclease folding by stopped-flow circular dichroism. 863 10

Transcription of the osteocalcin gene, which encodes a 10 kDa bone-specific protein, is controlled by modularly organized basal regulatory sequences and hormone-responsive enhancer elements. We have previously shown that in the ROS 17/2.8 rat osteosarcoma cell line, which continuously expresses the osteocalcin gene, key regulatory elements reside in two DNase I hypersensitive sites that are fucntionally correlated with transcriptional activity. We now report that a specific nucleosomal organization supports this constitutive expression in ROS 17/2.8 cells, and that chromatin remodeling directly correlates with the developmentally regulated transcriptional activation of the osteocalcin gene during differentiation of normal diploid rat osteoblasts. By combining DNase I, micrococcal nuclease, and specific restriction endonuclease digestion analysis, we observed that the presence of DNAse I hypersensitive sites (-170 to -70 and -600 to -400) and a selective nucleosome positioning over the OC gene promoter are directly associated with developmental stage-specific transcriptional activation in bone-derived cells.
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PMID:Changes in chromatin structure support constitutive and developmentally regulated transcription of the bone-specific osteocalcin gene in osteoblastic cells. 866 2

The relationship between chromatin structure and endonuclease sensitivity was investigated. The cells used in this study were a) human myelogenous leukemic cell lines (HL-60, ML-I, U-937, THP-I) (Group I), which produced internucleosomal DNA cleavage, and b) human T-cell leukemia (MOLT-4), erythroleukemia (K562), glioblastoma (T98G, U87MG) and glioma (KG-1-C) cell lines (Group II), which produced no internucleosomal DNA cleavage, upon treatment with various apoptosis-inducing agents. When the nuclei, isolated from these cells were digested with micrococcal nuclease, chromatin DNA was cleaved into oligonucleosomal units. Although sensitivity to micrococcal nuclease considerably differed from cell to cell, Group I cells were generally more sensitive to micrococcal nuclease digestion than Group II cells. Similar sensitivity to DNase I was observed in both groups of cells. Acid-urea polyacrylamide gel electrophoresis of histone fractions from control and apoptosing HL-60 cells (induced either by hydrogen peroxide or UV irradiation) revealed no significant change in the relative composition of five major histones, indicating the absence of selective degradation of histone HI, but rather the nonspecific degradation of many nuclear proteins. These data suggest a difference in a chromatin structure between Group I and II cells, which might result in the selective production of internucleosomal DNA cleavage only in Group I cells.
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PMID:Chromatin structure and endonuclease sensitivity in human leukemic cell lines. 870 41

The hyperthermophilic archaeon Sulfolobus acidocaldarius uses a novel RNA-containing endonuclease to excise and mature 16S rRNA from the precursor (pre) rRNA transcript. A cell-free processing system has been developed using an in vitro transcribed RNA substrate containing the entire 144 nucleotide 5' external transcribed spacer (5'ETS) and the first 72 nucleotides of 16S rRNA. The cell-free extract cleaves in the 5'ETS at positions -99, -31, and +1 (i.e., the 5'ETS-16S junction). These positions are at or near the positions cleaved in vivo during processing of the pre rRNA transcript. The processing activity has been purified between 100 and 200-fold and appears to contain five or six polypeptide components and perhaps as many as 10 different small RNA components. Using combined reverse transcription-PCR amplification, full or partial cDNA copies of two of the RNA components have been obtained. One of the RNAs exhibits sequence and structural similarities to eukaryotic U3 snoRNA. The processing activity has been shown to be inactivated by micrococcal nuclease. It can be reactivated by reconstituting using bulk RNA from S.acidocaldarius but not bulk RNA from distantly related organisms. The activity is also abolished by RNase H digestion in the presence of oligonucleotides complementary to the U3-like RNA. These results demonstrate that the U3-like RNA is an essential component of the pre rRNA processing RNP endonuclease. Furthermore, this RNP endonuclease is not a derived eukaryotic feature, instead its existence predates the divergence of archaea and eukaryotes.
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PMID:Preribosomal RNA processing in archaea: characterization of the RNP endonuclease mediated processing of precursor 16S rRNA in the thermoacidophile Sulfolobus acidocaldarius. 872 97

Estrogen-mediated accumulation of the avian apolipoprotein (apo) II mRNA is in part due to its stabilization. To identify the biochemical activity responsible for this effect, radiolabeled, capped, and polyadenylated apoII mRNA was incubated in vitro in liver cytosolic extracts from roosters who received either estrogen (estrogen-treated extract) or the vehicle (control extract) parenterally. The mRNA was very stable in estrogen-treated extract but was rapidly degraded in control extract. The RNA was degraded predominantly by endonuclease rather than exonuclease activity. The addition of the estrogen-treated extract to the control extract prevented the degradation of the mRNA in trans. This biochemical activity was heat labile and was also destroyed by proteinase K but not by micrococcal nuclease, indicating that estrogen treatment resulted in the expression of a protein in the liver that stabilized the apoII mRNA by inhibiting its nucleolytic degradation. This mRNA stabilization factor was labile around 60 degrees C, whereas the RNase remained stable up to 80 degrees C. Studies on mRNA protein interaction showed that both control and estrogen-treated extracts contain mRNA-binding (mRNP) proteins that bind apoII mRNA. An increased binding to apoII mRNA by a subset of these proteins was observed with estrogen-treated extract as compared with the control extract. This activity, although it afforded complete protection from nucleolytic degradation to apoII and apo A1 mRNAs, appeared to provide less protection to mRNAs encoding chicken serum albumin and vitellogenin, suggesting differential stabilization of mRNAs. These studies indicate that a cytosolic mRNA-stabilization factor, providing apoII mRNA complete protection from nucleolytic degradation, is expressed in the avian liver upon estrogen treatment. This appears to be the first time that a biochemical activity responsible for hormone-mediated stabilization of mRNAs and estrogen induction of mRNA binding by specific mRNPs have been identified and partially characterized in vitro.
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PMID:In vitro characterization of an estrogen-regulated mRNA stabilizing activity in the avian liver. 877 38

A membrane-independent morphogenetic viral signal peptide is identified within bacteriophage T4 internal protein III (IPIII). Utilizing a phagederived expression-packaging-processing system, which packages foreign proteins fused with IPIII into the phage capsid, a synthetic cleavage site introduced at the C terminus of IPIII, is demonstrated to be functional and permits processing of fusion proteins. IPIII, which possesses a native P21 cleavage site at its N terminus, is altered to possess a second P21 cleavage site at its C terminus where cleavage occurs by means of the scaffold proteinase P21 within the capsid. The altered IPIII was inserted into an expression vector to permit the creation of fusion proteins with staphylococcal nuclease, EcoRI endonuclease, beta-globin, and luciferase. Western immunoblot analysis of packaged T4eG326 indicates that the IPIII:fusion-proteins are packaged into phage and proteolytically processed, thus the synthetic P21 cleavage site positioned at the C terminus of IPIII is demonstrated to be functional, and 20 to 200 protein molecules are packaged per capsid. Truncation experiments identified the minimal portion of IPIII required to achieve targeting into the phage capsid as a ten amino acid residue from the N terminus, which includes the N-terminal methionine residue and the proteinase P21 cleavage site, designated the CTS (capsid targeting sequence). The addition of the CTS to a fragment of luciferase permits the protein to be packaged and processed, which demonstrates that the CTS is by itself sufficient to target foreign protein to the capsid. The imputed dual function of the CTS is supported by site-directed PCR mutagenesis, which reveals two functionally separate domains of the CTS for targeting and processing. The CTS appears to function in a core-related targeting mechanism that directs a polymorphic set of proteins into the T-even capsid or scaffold. Although structure formation is often assumed to involve extended protein interfaces, the analysis shows that a limited but specific sequence, the CTS, drives the interaction required to achieve targeting.
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PMID:Capsid targeting sequence targets foreign proteins into bacteriophage T4 and permits proteolytic processing. 878 Jul 80

We have investigated the endonuclease activity of the influenza A virus RNA polymerase in an in vitro assay with an artificial influenza-like mRNA containing a cap structure at its 5' terminus, followed by a 10 nt beta-globin mRNA sequence, and the 5' and 3' conserved termini of a truncated nucleoprotein (NP) cRNA influenza sequence. Results showed that partially purified virion ribonucleoprotein complexes (RNPs) and micrococcal nuclease treated RNPs cleaved the artificial influenza-like mRNA substrate specifically at positions near the 5' terminus to generate capped 14 and 15 nucleotide long RNA fragments which subsequently served as primers to initiate transcription. The endonuclease activity was completely blocked by addition of cap analog and competitively inhibited by added globin mRNA. Furthermore, an in vitro reconstituted influenza RNA transcription reaction containing a truncated NP vRNA as template, micrococcal nuclease treated RNPs and globin mRNA as primer, synthesized capped and uncapped full length (+) sense products. Enzyme kinetics showed that capped RNA was made earlier in the reaction; it reached a peak at 120 min and then declined. However, uncapped cRNA synthesis appeared later and remained as the dominant product later in the reaction. The nature of these products was confirmed by ribonuclease protection assays and by primer extension.
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PMID:Influenza A virus RNA-dependent RNA polymerase cleaves influenza mRNA in vitro. 880 82

The extracellular nuclease from Serratia marcescens is a non-specific endonuclease that hydrolyzes double-stranded and single-stranded DNA and RNA with high specific activity. Steady-state and presteady-state kinetic cleavage experiments were performed with natural and synthetic DNA and RNA substrates to understand the mechanism of action of the Serratia nuclease. Most of the natural substrates are cleaved with similar Kcat and K(m) values, the Kcat/K(m) ratios being comparable to that of staphylococcal nuclease. Substrates with extreme structural features, like poly(dA).poly(dT) or poly(dG).poly(dC), are cleaved by the Serratia nuclease with a 50 times higher or 10 times lower K(m), respectively, as salmon testis DNA. Neither with natural DNA or RNA nor synthetic oligodeoxynucleotide substrates did we observe substrate inhibition for the Serratia nuclease as reported recently. Experiments with short oligodeoxynucleotides confirmed previous results that for moderately good cleavage activity the substrate should contain at least five phosphate residues. Shorter substrates are still cleaved by the Serratia nuclease, albeit at a rate reduced by a factor of more than 100. Cleavage experiments with oligodeoxynucleotides substituted by a single phosphorothioate group showed that the negative charge of the pro-Rp-oxygen of the phosphate group 3' adjacent to the scissile phosphodiester bond is essential for cleavage, as only the Rp-phosphorothioate supports cleavage at the 5' adjacent phosphodiester bond. Furthermore, the modified bond itself is only cleaved in the Rp-diastereomer, albeit 1000 times more slowly than the corresponding unmodified phosphodiester bond, which offers the possibility to determine the stereochemical outcome of cleavage. Pre-steady-state cleavage experiments demonstrate that it is not dissociation of products but association of enzyme and substrate or the cleavage of the phosphodiester bond that is the rate-limiting step of the reaction. Finally, it is shown that Serratia nuclease accepts thymidine 3',5'-bis(p-nitrophenyl)phosphate as a substrate and cleaves it at its 5'-end to produce nitrophenol and thymidine 3'-(p-nitrophenylphosphate) 5-phosphate. The rate of cleavage of this artificial substrate, however, is 6-7 orders of magnitude smaller than the rate of cleavage of macromolecular DNA or RNA.
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PMID:Kinetic analysis of the cleavage of natural and synthetic substrates by the Serratia nuclease. 891 58

Previously, we reported that histone H1 binding to nucleosome cores results in the repression of binding of the basic helix-loop-helix upstream stimulatory factor (USF) (Juan, L.-J., Utley, R. T., Adams, C. C., Vettese-Dadey, M., and Workman, J. L. (1994) EMBO J. 13, 6031-6040). We have tested whether this inhibition resulted from H1-mediated changes in nucleosome positioning (Ura, K., Hayes, J. J., and Wolffe, A. P. (1995) EMBO J. 14, 3752-3765) forcing the USF recognition sequence into less accessible locations within the nucleosome. Nucleosome boundaries were determined by assays combining micrococcal nuclease and restriction endonuclease digestion. A unique pair of boundaries were observed, indicating a single nucleosome translational position. This nucleosome position did not change on H1 or USF binding. Thus, H1 repression of USF binding was independent of nucleosome mobility, indicating an alternative mechanism of H1 repression. H1 repressed USF binding at a site 35 base pairs into the nucleosome core more effectively than at a site near the "linker" DNA, suggesting that inhibition by H1 was not simply due to steric occlusion. Instead, these data are consistent with a model by which H1 binding reduces transient dynamic exposure of the DNA from the histone octamer surface (Polach, K. L., and Widom, J. (1995) J. Mol. Biol. 254, 130-149).
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PMID:H1-mediated repression of transcription factor binding to a stably positioned nucleosome. 901 16


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