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)

Addition of extract of interferon (IFN)-treated HeLa cells to transcription reactions containing activated reovirion cores decreases the yield of viral mRNA (C. Baglioni, A. De Benedetti, and G. J. Williams, 1984, J. Virol. 52, 865-871). The 2'5'-oligo(A) (2-5A)-dependent endonuclease (RNase L) cleaves specifically viral mRNA, but little 5'-triphosphate 2-5A is recovered from these reactions by DEAE-cellulose chromatography. However, in the present study we detected microM concentrations of 2-5A derivatives by binding to RNase L. Similar results were obtained when the synthetic double-stranded RNA poly(I) X poly(C) was incubated with extract from IFN-treated cells: microM concentrations of 2-5A were detected by the binding assay, but little rRNA was degraded by RNase L. 2-5A derivatives which inhibited the activation of RNase L by authentic 2-5A were eluted from DEAE-cellulose with 90 mM KCl. These inhibitors were also formed by incubating purified 2-5A with HeLa cell extract. These results indicated that 2-5A was synthesized in the incubations with reovirion cores or poly(I) X poly(C), but that it was in large part degraded to compounds inhibitory for RNase L. IFN-treated HeLa cells were incubated with poly(I X C), but little rRNA degradation was detected in spite of the presence of high concentrations of 2-5A in these cells. Most of this 2-5A was eluted with 90 mM KCl from DEAE-cellulose and was inhibitory for RNase L. This indicated that 2-5A was degraded to inhibitory derivatives also in intact cells. The structure of the degradation products of 2-5A has not been established, but they contain free terminal phosphate(s), since their binding to RNase L and the inhibition of this enzyme is abolished by digestion with phosphatase.
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PMID:Inhibition of 2',5'-oligo(A)-dependent endoribonuclease by 2',5'-oligo(A) degradation products. 242 11

The reverse transcriptase polymerase of the human T-cell lymphotropic virus/lymphadenopathy-associated virus has been cloned into an expression vector and expressed in Escherichia coli. Two polypeptides of 66 and 51 kDa molecular mass are detectable in polymerase-expressing bacterial lysates with human patient sera. They are processed from a short-lived 120-kDa polyprotein precursor equivalent to a region consisting of polymerase, protease, and endonuclease. The 51 kDa protein appears to originate from the 66-kDa molecule; additional processing products are 32- and 15-kDa proteins. The bacterially expressed polymerase is enzymatically active and exhibits the template specificities, ion requirements, and response to inhibitors of the authentic enzyme. It was purified by DEAE-cellulose-, phosphocellulose-, and poly(rC)-agarose column chromatography followed by glycerol density gradient centrifugation. It copurifies with an RNase H activity, suggesting the existence of a virus-coded DNA polymerase-RNase H complex. The purified bacterial enzyme allows a safe large-scale screening for inhibitors of both activities.
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PMID:RNase H activity associated with bacterially expressed reverse transcriptase of human T-cell lymphotropic virus III/lymphadenopathy-associated virus. 244 62

Addition of thioglycolate and DEAE-Sephadex chromatography were used to analyze the cleavage of the C(3')-O-P bond 3' to AP (apurinic/apyrimidinic) sites in DNA and to distinguish between a mechanism of hydrolysis (which would allow the nicking enzyme to be called 3' AP endonuclease) or beta-elimination (so that the nicking enzyme should be called AP lyase). For this purpose, DNA labelled in the AP sites was first cleaved by rat-liver AP endonuclease, then with the 3' nicking catalyst in the presence of thioglycolate and the reaction products were analyzed on DEAE-Sephadex: deoxyribose-5-phosphate (indicating a 3' cleavage by hydrolysis) and the thioglycolate:unsaturated sugar-5-phosphate adduct (indicating a cleavage by beta-elimination) are well separated allowing to eventually easily discard the hypothesis of a hydrolytic process and the appellation of 3' AP endonuclease. We have shown that addition of thioglycolate to the unsaturated sugar resulting from nicking the C(3')-O-P bond 3' to AP sites by beta-elimination is an irreversible reaction. We have also shown that the thioglycolate must be present from the beginning of the reaction with the nicking catalyst to prevent the primary 5' product of the beta-elimination reaction from undergoing other modifications that complicate the interpretation of the results.
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PMID:The use of thioglycolate to distinguish between 3' AP (apurinic/apyrimidinic) endonucleases and AP lyases. 247 55

The mechanism of intracellular deoxyribonucleoside-triphosphate (dNTP) imbalance death of mouse mammary tumor FM3A cells was studied. When the cells were exposed to 5-fluorodeoxyuridine, deoxyadenosine, or 2-chlorodeoxyadenosine, dNTP pool imbalance resulted. The imbalance was followed by DNA double-strand breaks and subsequent cell death. The DNA double strand breaks were directly examined by means of orthogonal-field-alternation gel electrophoresis (OFAGE). Fragmented DNA band appeared to be approximately 100-200 kbp in size. The bases of 5'-termini in the DNA were cytosine and thymine. The imbalance induced endonuclease has been isolated by DEAE-agarose column chromatography.
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PMID:dNTP imbalance and DNA double strand breaks in mouse FM3A cells and the mechanism of cell death. 257 44

Two apurinic/apyrimidinic- (AP-) specific endonuclease activities have been isolated from the cells of Dictyostelium discoideum by fractionation on DEAE-cellulose, CM-cellulose and Sephadex G-75. These activities, designated A and B, have apparent molecular weights of 49000 and 40000, respectively. Although their precise reaction optima differ somewhat, both A and B quantitatively nick AP DNA best at pH 8.0-8.5 in low salt (less than 100 mM NaCl); both require Mg2+. These activities are apparently specific only for AP sites in DNA. The low activities observed on heavily ultraviolet-irradiated DNA, gamma-irradiated DNA and osmium tetroxide-treated DNA are consistent with the small numbers of secondary AP sites expected in these DNAs. Both A and B produce single-strand nicks in AP DNA that result in termini that serve as good primers for Escherichia coli polymerase I. Hence, A and B appear to be Class II AP endonucleases which yield 3'-OH termini at nicks on the 5' side of baseless sugars. It is unclear whether A and B are independently coded proteins, different post-translational modifications of the same gene product, or whether one is an artifact arising from the isolation. Many of the properties of these D. discoideum AP endonuclease activities are similar to those of the predominant AP endonucleases observed in bacterial, plant and animal cells. They will be of use in the characterization of excision repair in this organism.
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PMID:Apurinic/apyrimidinic-specific endonuclease activities from Dictyostelium discoideum. 258 May 57

A single-strand-specific endonuclease which converted negatively supercoiled DNA to open-circular and linear DNA was purified to homogeneity with Hb-Sepharose 4B, DEAE Trisacryl M, HA-Ultrogel and PBE-94 chromatofocusing from extracts of Streptomyces tendae ATCC 31160. Bio-Gel P-200 chromatography and electrophoresis in SDS-PAGE indicated the native protein was a monomer with a molecular weight of approximately 40-kDa. This enzyme did not hydrolyze double-stranded linear DNA but digested RNA and circular single-strand DNA. Sequence specificity for nicking of negatively supercoiled DNA was not detected.
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PMID:Purification and characterization of an endonuclease (E.C. 3.1.30.1) from Streptomyces tendae. 283 46

Three kinds of deoxyribonucleases (peaks A, B, and C) were separated from purified rat liver nuclei on DEAE-cellulose column chromatography, and their characteristics were partially studied. Peaks A and B had endonuclease activities under acidic conditions with low substrate specificity and did not require divalent metal ions. Peak C had an exonuclease activity under alkaline conditions with substrate specificity for denatured DNA or single stranded homopolymer and required divalent cations. Peak C degraded 3'-terminally mismatched substrate much faster than 3'-terminally matched substrate.
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PMID:Studies on nuclear acid deoxyribonucleases and proofreading exonuclease isolated from rat liver nuclei. 285 6

A new restriction endonuclease, EcoO109, has been isolated from Escherichia coli H709c by polyethyleneimine (PEI) precipitation, DEAE-cellulose chromatography and heparin agarose chromatography. The yield was high, more than 3000 units/g of wet cells. The EcoO109 endonuclease recognizes and cleaves a nucleotide sequence of (formula: see text), in the presence of 10 mM Mg2+. The enzyme will be useful for structural analysis and molecular cloning of DNA because of the stability, high yield and easy handling of the producer strain.
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PMID:Restriction endonuclease EcoO109 from Escherichia coli H709c with heptanucleotide recognition site 5'-PuG/GNCCPy. 300 Aug 88

1-beta-D-Arabinofuranosylcytosine (ara-C) inhibits nuclear DNA replication in Chinese hamster ovary cells by an efficient chain termination mechanism without affecting the rate at which cells traverse G1 and enter S [Heintz, N. H., & Hamlin, J. L. (1983) Biochemistry 22, 3557-3562]. Here we have employed ara-C to enrich for replication intermediates formed during initiation of DNA synthesis in synchronized CHOC 400 cells, a methotrexate-resistant derivative of Chinese hamster ovary cells that contains approximately 1000 copies of an early replicating 150-kb chromosomal domain. This highly amplified domain includes the gene for dihydrofolate reductase (DHFR). CHOC 400 cells were collected at the G1/S boundary of the cell cycle with aphidicolin prior to release into S in the presence of both [methyl-3H] thymidine and various concentrations of ara-C. Chromatographic fractionation of restriction endonuclease digests over benzoylated naphthoylated DEAE-cellulose (BND-cellulose) showed that high concentrations of ara-C inhibited the maturation of chromosomal replication intermediates containing ssDNA (replication forks) into dsDNA for up to 60 min. The effect of ara-C on the sequence complexity of replication intermediates formed during early S phase was determined by hybridizing purified intermediates labeled with 32P in vitro to Southern blots of genomic DNA derived from both methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells. In the absence of ara-C, 32P-labeled ssDNA BND-cellulose fractions from cultures released into S for 30-60 min hybridized to a spectrum of restriction fragments encompassing 40-50 kb of the amplified DHFR domain.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Replication intermediates formed during initiation of DNA synthesis in methotrexate-resistant CHOC 400 cells are enriched for sequences derived from a specific, amplified restriction fragment. 300 61

The 5'- and 3'-tRNA processing nucleases have been isolated from rat liver mitochondria. The two activities co-purified through heparin-agarose and phenyl-Sepharose columns and then efficiently separated on a DEAE-cellulose column. The 5' processing nuclease was found in the flow-through fraction, and the 3' processing activity eluted with 0.5 M KCl. Both enzymes were greater than 500-fold purified over the high speed supernatant of a mitoplast extract. The 159-base pre-tRNATyr used as a substrate in this study was synthesized in vitro and contained the Escherichia coli suppressor III tRNATyr plus a 49-base leader sequence and a 25-base trailing sequence. The 5' processing nuclease converted the pre-tRNATyr into two discrete RNA species, identified as the 5'-processed intermediate and the 5' flanking fragment, by endonucleolytic cleavage at the 5' end of the mature tRNATyr sequence. The 3' processing nuclease was inactive with the intact pre-tRNATyr as substrate but efficiently converted the 5'-processed intermediate to the mature tRNATyr, indicating an obligatory order of processing in which 5' maturation was necessary before cleavage by the 3' processing nuclease could occur. The mitochondrial enzymes exhibited optimal activity in the presence of about 2 mM Mg2+, but both enzymes were nearly fully active without addition of exogenous Mg2+ to the reaction mixtures. In contrast, a partially purified 5' processing endonuclease present in the postmitochondrial cytosolic fraction required higher [Mg2+] for activity, thus providing a means for differentiating between these similar enzyme activities obtained from the cytosolic and mitochondrial fractions.
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PMID:Separation and characterization of 5'- and 3'-tRNA processing nucleases from rat liver mitochondria. 330 31


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