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)

Human NK cells (with CD3-/56+ phenotype) acquired features characteristic of apoptosis after incubation with autologous monocytes, as revealed by apoptotic nuclear morphology, degradation of DNA into oligonucleosomal fragments, and reduced nuclear interchalation of propidium iodide. In contrast, T cells (CD3+/56-) remained non-apoptotic. The monocyte-induced apoptosis in NK cells was prevented by catalase, a scavenger of hydrogen peroxide; whereas superoxide dismutase (a scavenger of superoxide anion), hydroxyl radical scavengers such as mannitol and deferoxamine, or the hypochlorus acid scavenger taurine did not prevent apoptosis. Sodium azide, a myeloperoxidase inhibitor, substantially reduced the monocyte-induced apoptosis in NK cells. Exogenous hydrogen peroxide, at concentrations exceeding 1 microns, induced apoptosis in both NK and T cells. Apoptosis induced by hydrogen peroxide occurred independently of synthesis of protein or mRNA and was blocked by the endonuclease inhibitor aurin tricarboxylic acid. Furthermore, oxidatively induced apoptosis in NK cells was inhibited by herbimycin A, indicating that apoptosis was dependent on protein kinases. Two to five times more hydrogen peroxide was required to induce apoptosis in T cells compared with NK cells. Similarly, NK cells were considerably more susceptible to apoptosis induced by the topoisomerase II inhibitor etoposide or by gamma-irradiation than were T cells. We conclude that monocyte-derived reactive oxygen metabolites kill NK cells by apoptosis and that NK cells are unusually sensitive to oxidatively as well as non-oxidatively induced apoptosis.
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PMID:Induction of apoptosis in NK cells by monocyte-derived reactive oxygen metabolites. 859 91

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

The resonance Raman spectra of 4-thiothymidine [4ST] have been recorded (a) in the free deoxynucleoside form, (b) when incorporated into the single stranded oligodeoxynucleotide d(AG[4ST]-TC), and (c) within the double-stranded self-complementary dodecamer d(GACGA[4ST]ATCGTC). Vibrational mode assignments of almost all the major Raman bands observed in each spectra have been made, mainly by comparison with the published assignments of related nucleosides and nucleotides. Differences between the spectra were observed, particularly when [4ST] and d(AG[4ST]TC) were compared to d(GACGA[4ST]ATCGTC). This is explained in terms of the variations in structure between single-and double-stranded DNA. Good quality spectra were obtained at nucleotide/oligonucleotide concentrations of between 100 and 500 microM and this coupled with an apparatus that uses small volumes (100 microL) allowed measurement of the spectrum of d(GACGA[4ST]ATCGTC) bound to the EcoRV endonuclease. This well characterised nuclease, for which crystal structures are available, recognizes d(GATAT) sequences. When this is replaced with d(GA[4ST]ATC), a poor substrate results but turnover can be prevented during data accumulation by omission of the essential cation Mg2+. Large shifts in several of the Raman bands were observed, and these have been related to the environment of the [4ST] base in the protein-bound oligonucleotide as deduced from the crystal structure. The wavenumber for the C = S stretch vibration in free d(GACGA[4ST]ATCGTC) has been used to calculate the strength of the Watson-Crick hydrogen bond between the sulphur atom in [4ST] and the 6-NH2 group on its partner dA. On binding to the enzyme, the shift in the wavenumber of the C = S stretch indicates this Watson-Crick hydrogen bond is weakened, in good agreement with X-ray structures. The advantage of using [4ST] as a resonance Raman probe is that it absorbs at 340 nm, a wavelength where other nucleic acid and protein absorbance is minimal. Thus the spectra obtained are very simple and consist of signals that arise predominantly from the thiobase alone, and this facilitates data interpretation.
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PMID:Resonance Raman spectroscopy of 4-thiothymidine and oligodeoxynucleotides containing this base both free in solution and bound to the restriction endonuclease EcoRV. 867 35

The contact between EcoRI endonuclease and the "primary clamp" phosphate of its recognition site pGAATTC is absolutely required for recognition of the canonical and all variant DNA sites. We have probed this contact using oligonucleotides containing single stereospecific (Rp)- or (Sp)- phosphorothioates (Ps). At the GAApTTC position, where the endonuclease interacts with only one phosphoryl oxygen at the central DNA kink, Rp-Ps inhibits and Sp-Ps stimulates binding and cleavage [Lesser et al. (1992) J. Biol. Chem. 267, 24810-24818]: in contrast, at the pGAATTC position both diastereomers inhibit binding. For single-strand substitution, the penalty in binding free energy (delta delta G0bind) is slightly greater for Sp-Ps (+ 0.9 kcal/mol) than for Rp-Ps (+ 0.7 kcal/mol). Binding penalties are approximately additive for double-strand substitution (Rp,Rp-Ps or Sp,Sp-Ps). Neither Ps diastereomer in one DNA strand affects the first-order rate constants for cleavage in the unmodified DNA strand, and only Sp-Ps inhibits the cleavage rate constant (3-fold) in the modified DNA strand. Thus, the second-order cleavage rate (including binding and catalysis) is inhibited 14-fold by Sp-Ps and 45-fold by Sp,Sp-Ps. In the canonical complex, the phosphate at pGAATTC is completely surrounded by protein and each nonbridging phosphoryl oxygen receives two hydrogen bonds from the endonuclease, such that in either orientation the increased bond length of P-S- inhibits binding. However, the pro-Sp oxygen interacts with residues that are connected (by proximity or inter-side-chain hydrogen bonding) to side chains with essential roles in catalysis, so cleavage is preferentially inhibited when these side chains are slightly displaced by the Sp-Ps diastereomer.
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PMID:Chiral phosphorothioates as probes of protein interactions with individual DNA phosphoryl oxygens: essential interactions of EcoRI endonuclease with the phosphate at pGAATTC. 868 20

One, two or four copies of the 'helix-hairpin-helix' (HhH) DNA-binding motif are predicted to occur in 14 homologous families of proteins. The predicted DNA-binding function of this motif is shown to be consistent with the crystallographic structure of rat polymerase beta, complexed with DNA template-primer [Pelletier, H., Sawaya, M.R., Kumar, A., Wilson, S.H. and Kraut, J. (1994) Science 264, 1891-1903] and with biochemical data. Five crystal structures of predicted HhH motifs are currently known: two from rat pol beta and one each in endonuclease III, AlkA and the 5' nuclease domain of Taq pol I. These motifs are more structurally similar to each other than to any other structure in current databases, including helix-turn-helix motifs. The clustering of the five HhH structures separately from other bi-helical structures in searches indicates that all members of the 14 families of proteins described herein possess similar HhH structures. By analogy with the rat pol beta structure, it is suggested that each of these HhH motifs bind DNA in a non-sequence-specific manner, via the formation of hydrogen bonds between protein backbone nitrogens and DNA phosphate groups. This type of interaction contrasts with the sequence-specific interactions of other motifs, including helix-turn-helix structures. Additional evidence is provided that alphaherpesvirus virion host shutoff proteins are members of the polymerase I 5'-nuclease and FEN1-like endonuclease gene family, and that a novel HhH-containing DNA-binding domain occurs in the kinesin-like molecule nod, and in other proteins such as cnjB, emb-5 and SPT6.
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PMID:The helix-hairpin-helix DNA-binding motif: a structural basis for non-sequence-specific recognition of DNA. 869 86

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

Hypoxia is considered to result in a necrotic form of cell injury. We have recently demonstrated a role of endonuclease activation, generally considered a feature of apoptosis, to be almost entirely responsible for DNA damage in hypoxic injury to renal tubular epithelial cells. The role of reactive oxygen metabolites in endonuclease-induced DNA damage and cell death in chemical hypoxic injury has not been previously examined. LLC-PK1 cells exposed to chemical hypoxia with antimycin A resulted in enhanced generation of intracellular reactive oxygen species as measured by oxidation of a sensitive fluorescent probe, 2',7'-dichlorofluorescin diacetate. Superoxide dismutase, a scavenger of superoxide radical, significantly reduced the fluorescence induced by antimycin A and provided significant protection against chemical hypoxia-induced DNA strand breaks (as measured by the alkaline unwinding assay). Pyruvate, a scavenger of hydrogen peroxide, provided significant protection against chemical hypoxia-induced DNA strand breaks and DNA fragmentation (as measured by agarose gel electrophoresis). The interaction between superoxide anion and hydrogen peroxide in the presence of a metal catalyst leads to generation of other oxidant species such as hydroxyl radical. Hydroxyl radical scavengers, dimethylthiourea, salicylate, and sodium benzoate, and two metal chelators, deferoxamine and 1,10-phenanthroline, also provided marked protection against DNA strand breaks and DNA fragmentation. These scavengers of reactive oxygen metabolites and metal chelators provided significant protection against cell death as measured by trypan blue exclusion and lactate dehydrogenase release. Taken together, these data indicate that reactive oxygen species play an important role in the endonuclease activation and consequent DNA damage, as well as cell death in chemical hypoxic injury to renal tubular epithelial cells.
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PMID:Role of reactive oxygen metabolites in DNA damage and cell death in chemical hypoxic injury to LLC-PK1 cells. 876 Feb 63

The Serratia endonuclease is an extracellularly secreted enzyme capable of cleaving both single- and double-stranded forms of DNA and RNA. It is the first member of a large class of related and usually dimeric endonucleases for which a structure is known. Using X-ray crystallography, the structure of monomer of this enzyme was reported by us previously (Miller MD et al., 1994, Nature Struct Biol 1:461-468). We now confirm the dimeric nature of this enzyme through light-scattering experiments and identify the physiologic dimer interface through crystal packing analysis. This dimerization occurs through an isologous twofold interaction localized to the carboxy-terminal subdomain of the enzyme. The dimer is a prolate ellipsoid with dimensions 30 A x 35 A x 90 A. The dimer interface is flat and contains four salt links, several hydrogen bonds, and nonpolar interactions. Buried water is prominent in this interface and it includes an unusual "cubic" water cluster. The position of the two active sites in the dimer suggests that they can act independently in their cleavage of DNA, but have a geometrical advantage in attacking substrate relative to the monomer.
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PMID:Identification of the Serratia endonuclease dimer: structural basis and implications for catalysis. 877 Nov 93

Linear diffusion along DNA is a mechanism of enhancing the association rates of proteins to their specific recognition sites on DNA. It has been demonstrated for several proteins in vitro, but to date in no case in vivo. Here we show that the restriction endonuclease EcoRV slides along the DNA, scanning approximately 1000 bp in one binding event. This process is critically dependent on contacts between amino acid residues of the protein and the backbone of the DNA. The disruption of single hydrogen bonds and, in particular, the alteration of electrostatic interactions between amino acid side chains of the protein and phosphate groups of the DNA interfere with or abolish effective sliding. The efficiency of linear diffusion is dependent on salt concentration, having a maximum at 50 mM NaCl. These results suggest that a nonspecific and mobile binding mode capable of linear diffusion is dependent on a subtle balance of forces governing the interaction of the enzyme and the DNA. A strong correlation between the ability of EcoRV mutants to slide along the DNA in vitro and to protect Escherichia coli cells from phage infection demonstrates that linear diffusion occurs in vivo and is essential for effective phage restriction.
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PMID:Linear diffusion of the restriction endonuclease EcoRV on DNA is essential for the in vivo function of the enzyme. 889 Jan 84

Synthetic oligodeoxynucleotides with single methyl phosphonate (mp) substitutions were used for an analysis of the contribution of phosphate contacts to the recognition of the cleavage site by the restriction endonuclease EcoRV. Only in the last position within the recognition sequence, is the methyl phosphonate substitution tolerated by the enzyme. The wild-type enzyme cleaves the Sp diastereomer of the oligodeoxynucleotide GACGATATmpCGTC and the unmodified sequence with equal rates, whereas the Rp diastereomer is cleaved much more slowly. Inspection of the crystal structure of an EcoRV-DNA complex revealed that the non-bridging oxygen atoms of the phosphodiester bond between the T and C bases are in hydrogen bonding distance of the hydroxyl group of the amino acid Thr94. We therefore tried to engineer a variant of EcoRV that would prefer a methyl phosphonate linkage over a normal phosphodiester bond and produced mutants with amino acid exchanges at position 94. One of them, Thr94Val, shows a dramatically reduced activity towards the unmodified DNA and does not accept the Rp diastereomer, but cleaves the Sp diastereomer with the same rate as wild-type EcoRV. Its selectivity, i.e. the ratio of cleavage rates determined for the unmodified and modified substrates, differs by three orders of magnitude from that of the wild-type enzyme.
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PMID:EcoRV-T94V: a mutant restriction endonuclease with an altered substrate specificity towards modified oligodeoxynucleotides. 896 53


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