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)

Substrate specificities of FPG protein (also known as formamidopyrimidine DNA glycosylase) and 8-hydroxyguanine endonuclease were compared by using defined duplex oligodeoxynucleotides containing single residues of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA), and 2,6-diamino-4-hydroxy-5-(N-methyl)formamidopyrimidine (Me-Fapy). Duplexes containing 8-oxodG positioned opposite dC, dG, or dT were cleaved, whereas single-stranded DNA and duplexes containing 8-oxodG.dA or 8-oxodA positioned opposite any of the four DNA bases were relatively resistant. Both enzymes cut duplexes containing 8-oxoG.dC 3' and 5' to the modified base but failed to cleave duplex DNA containing synthetic abasic sites, mismatches containing dG, or unmodified DNA. 8-Oxoguanine, identified by HPLC-electrochemical detection techniques, was released during the enzymatic reaction. Apparent Km values for FPG protein acting on duplex substrates containing a single Me-Fapy or 8-oxodG residue positioned opposite dC were 41 and 8 nM, respectively, and those for 8-hydroxyguanine endonuclease were 30 and 13 nM, respectively. Comparison of the properties of the two enzyme activities suggest that they are identical. In view of the widespread distribution of 8-oxodG in cellular DNA, the demonstrated miscoding and mutagenic properties of this lesion, and the existence of a bacterial gene coding for FPG protein, we propose that 8-oxodG DNA is the primary physiological substrate for a constituent glycosylase found in bacteria and mammalian cells.
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PMID:8-oxoguanine (8-hydroxyguanine) DNA glycosylase and its substrate specificity. 205 52

An improved procedure for the preparation of 3-deaza-2'-deoxyadenosine (d3CA) is described which is suitable for the synthesis of gram quantities of this analogue. Using phosphoramidite chemistry d3CA has been incorporated into the Eco RV restiction endonuclease recognition sequence (underlined) present in the self-complementary dodecamer d(GACGATATCGTC). The modified oligonucleotides have been thoroughly characterised by nucleoside composition analysis, circular dichroism and thermal melting studies. Studies with Eco RV show that incorporation of d3CA into either the central or outer dA-dT base-pair results in a substantial reduction in the rate of cleavage. The two-step conversion of d3CA to 3-deaza-2'-deoxyadenosine-5'-O-triphosphate (d3CATP) via the 5'-O-tosylate is also described. d3CATP is not a substrate in the poly[d(AT)].poly[d(AT)] primed polymerisation for either E. coli DNA polymerase I or Micrococcus luteus DNA polymerase. In a more detailed kinetic analysis d3CATP was shown to be a competitive inhibitor of E. coli DNA polymerase I with respect to dATP.
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PMID:Molecular recognition in the minor groove of the DNA helix. Studies on the synthesis of oligonucleotides and polynucleotides containing 3-deaza-2'-deoxyadenosine. Interaction of the oligonucleotides with the restriction endonuclease EcoRV. 239 41

To investigate the relative importance of each of the ribose 3'-hydroxyl groups of 2-5A (ppp5' A2'p5'A2'-p5' A) in determining binding to and activation of the 2-5A-dependent endonuclease (RNase L), the 3'-hydroxyl functionality of each adenosine moiety of 2-5A trimer triphosphate was sequentially replaced by hydrogen. The analog in which the 5'-terminal adenosine was replaced by 3'-deoxyadenosine (viz. ppp5'(3'dA)-2'p5' A2'p5' A) was bound to RNase L as well as 2-5A itself and was only 3 times less potent than 2-5A as an activator of RNase L. On the other hand, when the second adenosine unit was replaced by 3'-deoxyadenosine (viz. ppp5' A2'p5'(3'dA)2'p5' A), binding to RNase L was decreased by a factor of eight relative to 2-5A trimer and, even more dramatically, there was a 500-1000-fold drop in ability to activate the 2-5A-dependent endonuclease. Finally, when the 3'-hydroxyl substituent was converted to hydrogen in the 2'-terminal residue of 2-5A, a significant increase in both binding and activation ability occurred. We conclude that only the 3'-hydroxyl group of the second (from the terminus) nucleotide residue of 2-5A is needed for effective activation of RNase L.
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PMID:Only one 3'-hydroxyl group of ppp5' A2'p5'A2'p5' A (2-5A) is required for activation of the 2-5A-dependent endonuclease. 333 33

Analogs of the triphosphate 2'-5'-linked adenylate trimer (ppp5'A2'p5'A2'p5'A, called 2-5A) which contain 3'-deoxyadenosine (cordycepin) instead of adenosine either in positions one and two, or in all three positions, are 10-100-fold less potent than is parent 2-5A in inhibition of protein synthesis in intact cells, when utilizing calcium co-precipitation techniques to introduce the 5'-triphosphate oligonucleotides into the cells. That the inhibition of protein synthesis was a consequence of activation of the 2-5A-dependent endonuclease by the 3'-deoxyadenosine analogs of 2-5A was demonstrated in obtaining the ribosomal RNA cleavage pattern that is characteristic of endonuclease activation by parent 2-5A. Additional results (i.e. lack of activity by the dimer species ppp5'(3'dA)2'p5'-(3'dA) or the monomer 3'dA) as well as kinetic analysis both in intact cells and in cell-free extracts provided further evidence that the inhibition of protein synthesis observed with these 3'-deoxyadenosine 2-5A analogs was not due to their degradation to the antimetabolite monomer unit 3'-deoxyadenosine.
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PMID:Cordycepin analogs of ppp5'A2'p5'A2'p5'A (2-5A) inhibit protein synthesis through activation of the 2-5A-dependent endonuclease. 397 41

Cordycepin-5'-triphosphate (3'-deoxyadenosine-5'-triphosphate) can be incorporated into the 3'-ends of DNA fragments using terminal deoxynucleotidyl transferase from calf thymus (Bollum, 1974). Because cordycepin-5'-monophosphate lacks a 3'-OH group, only a single residue is incorporated. Furthermore, DNA molecules that contain cordycepin-5'-monophosphate at their 3'-ends become resistant to hydrolysis by exonucleases that require free 3'-OH ends. As an alternative to 5'-end labeling of complementary DNA strands, we have used [32P]cordycepin-5'-triphosphate labeling of 3'-ends to confirm the nucleotide sequence of a HhaI-endonuclease-generated pTU4-plasmid DNA fragment that contains several hot spots for insertions of the transposable genetic element Tn3. 3'-End labeling with [32P] cordycepin-5'-triphosphate has also proved useful in determining the sequence of the pTU4 DNA in the vicinity of a strategically located SstII endonuclease cleavage site in the replication region of the plasmid.
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PMID:3'-end labeling of DNA with [alpha-32P]cordycepin-5'-triphosphate. 624 22

Cordycepin (3'-deoxyadenosine) analogues of 2-5A were prepared by dicyclohexylcarbodiimide-induced polymerization of cordycepin 5'-monophosphate. A series of oligomers of the general formula p5'(3'dA)-2'[p5'(3'dA)]n (n = 1-5) was obtained. Cordycepin trimer 5'-monophosphate (n = 2) and cordycepin tetramer 5'-monophosphate (n = 3) were converted to the corresponding 5'-di- and triphosphates via the phosphorimidazolide method. Confirmation of assigned structures was provided through enzyme digestions, 1H and 31P NMR, and comparison with material which was synthesized by a completely independent route. Neither the cordycepin trimer triphosphate, ppp5'(3'dA)-2'p5'(3'dA)2'p5'(3'dA), nor the cordycepin tetramer triphosphate, ppp5'(3'dA)2'p5'(3'dA)2'p5'(3'dA)2'p5'-(3'dA), were inhibitors of translation in cell-free extracts of mouse L-cells programmed with encephalomyocarditis virus. In rabbit reticulocyte lysates, the cordycepin trimer triphosphate was devoid of activity, whereas the cordycepin tetramer triphosphate had approximately 1/100th the activity of 2-5A tetramer triphosphate, ppp5'A2'p5'A2'p5'A2'p5'A. Even though the cordycepin analogues were unable to activate the 2-5A-dependent endoribonuclease, they were able to bind to the endonuclease, thereby antagonizing the translational inhibitory effects of 2-5A. Thus, replacement of the 3'-hydroxyl groups of all the ribose moieties of 2-5A results in an analogue which can bind to the 2-5A-dependent endoribonuclease but is incapable of activating the enzyme. The 3'-hydroxyl groups of 2-5A, therefore, are critical for activation of the 2-5A-dependent endonuclease.
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PMID:Cordycepin analogues of 2-5A and its derivatives. Chemical synthesis and biological activity. 629 9

2-chloroadenosine induced DNA fragmentation and cell death in human thymocytes primarily by Ca(2+)-dependent mechanisms. Incubation of human thymocytes with 2-chlorodeoxyadenosine (5-1000 nM) also induced cell death (apoptosis) which was dependent on macromolecule synthesis and involved activation of an endonuclease which was inhibited by Zn2+. The effect of 2-chlorodeoxyadenosine was prevented by addition of dipyridamole, a strong nucleoside transport inhibitor, or of deoxycytidine, previously shown to compete for uptake by deoxycytidine kinase. 2-Chlorodeoxyadenosine-induced apoptosis did not involve increases in the cytosolic Ca2+ concentration, but required the presence of intracellular Ca2+. It was not inhibited by activators of protein kinase C previously shown to inhibit Ca(2+)-dependent cell death. Addition of 2-chlorodeoxyadenosine induced an increase in the amount of p53 in human thymocytes, while 2-chloroadenosine had no effect. These data suggest that 2-chloroadenosine and 2-chlorodeoxyadenosine induce cell death in human thymocytes via different signalling pathways.
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PMID:The 2-chlorodeoxyadenosine-induced cell death signalling pathway in human thymocytes is different from that induced by 2-chloroadenosine. 748 99

The DNA-binding properties of the EcoRV restriction endonuclease and modification methyltransferase with their recognition sequence (GATATC) were analyzed using the electrophoretic band-shift assay. It has previously been observed that the endonuclease does not bind specifically to GATATC sequences in the absence of the essential cofactor Mg2+. To investigate any possible roles for Mg2+ in promoting specific DNA binding, a set of hydrolysis-resistant oligonucleotide substrates were synthesized that contained either phosphate (phosphorothioate, 3'-S-phosphorothiolate), sugar (4'-thiothymidine), or base (7-deaza-2'-deoxyadenosine) modifications. However, it was found that none of these were specifically bound by the endonuclease in either the absence or the presence of Mg2+. In contrast, the methylase bound to GATATC sequences much more strongly than to nonspecific sites, and it was possible to observe the formation of enzyme--DNA complexes by gel retardation. Binding to GATATC sequences was increased by the addition of sinefungin, a nonreactive analogue of the essential cofactor S-adenosyl-L-methionine (AdoMet). Presumably this also occurs with AdoMet although methylation and turnover prevented its direct observation. In the presence of sinefungin the strongest binding was observed with hemimethylated EcoRV sequences (Kd = 11-13 nM), and unmethylated DNA was bound less well (Kd = 46 nM). Specific, albeit weaker binding was also seen with the dimethylated product (Kd = 143 nM). A difference in electrophoretic mobility was observed between enzyme-substrate and enzyme-product complexes suggestive of structural differences between them. The Kapp value found for sinefungin, with the hemimethylated EcoRV sequence, was 10.9 mM.
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PMID:Sequence-specific binding of DNA by the EcoRV restriction and modification enzymes with nucleic acid and cofactor analogues. 766 56

The ability of heat shock to induce functional protection against ultraviolet B (UVB) light was examined in keratinocytes cultured from human skin. Cell death, measured with fluorescent vital dyes, increased in a UVB dose-dependent manner (LD50 approximately 20-60 mJ/cm2). However, a 60-min heat shock at 40 degrees C or 42 degrees C, administered several hours before UVB irradiation, reduced cell death by 2.0-2.5 times. Inducible protection took time to develop, with an optimal interval of approximately 6 h between heat and UVB exposures. Heat-inducible protection was completely blocked if either cordycepin (3'-deoxyadenosine), to inhibit mRNA synthesis, or cycloheximide, to inhibit protein synthesis, were present during the heating period. To determine whether apoptosis might be involved in UVB-induced keratinocyte death in this system, evidence for endonuclease activity was sought via in situ enzymatic labeling with terminal deoxynucleotidyl transferase and biotinylated-dUTP. Labeled nuclei were detected in UVB-irradiated cultures, and heat pretreatment at 6 h prior to UVB exposure (< 60 mJ/cm2) resulted in a 50% reduction in labeled nuclei. Overall, the data show that UVB-induced cell death in human keratinocyte cultures is attenuated by a heat-inducible mechanism that requires ongoing synthesis of mRNA and protein.
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PMID:Heat shock modulates UVB-induced cell death in human epidermal keratinocytes: evidence for a hyperthermia-inducible protective response. 793 Jun 80

A one- and two-dimensional NMR study has been performed on seven A(2'-5')A(2'-5')A fragments containing 9-(3'-fluoro-3'-deoxy-beta-D-xylofuranosyl)-adenine (AF) or 3'-fluoro-3'-deoxyadenosine (AF) residues at different positions, and on the corresponding monomers. A(2'-5')A(2'-5')A served as a reference compound. The fluoro substituent governs the conformation of the sugar ring: an AF residue displays mainly N-type sugar and the ring is considerably flattened (phi N approximately 30 degrees) compared to AF residues (phi S approximately 40 degrees), which exhibit almost pure S-type conformation. Moreover, in AF moieties the rotamer distribution around torsion angle gamma (O5'-C5'-C4'-C3') and the base orientation are influenced to a large extent by the presence of the fluorine substituent. The sugar rings of nonfluorinated residues in the trimers appear rather flexible. A possible correlation between the conformational characteristics of the fluorinated fragments and their biological activity has been found: the fragments that meet the prerequisites for binding to RNase L indeed show enhanced binding to this endonuclease. Furthermore, substitution of the 3'-OH group of the second residue by hydrogen or of the 3'-OH group of the 2'-terminal residue by fluorine or hydrogen results in increased resistance towards 2'-5'-phosphodiesterase.
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PMID:Conformation analysis of 3'-fluorinated A(2'-5')A(2'-5')A fragments. Relation between conformation and biological activity. 817 55


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