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)

Single-strand DNA binding protein (SSB) from Escherichia coli abolishes transfection of E.coli by viral M13mp2 DNA at levels that inhibit transfection by M13mp2 replicative form (RF) DNA by approx. 25%. Synthesis of M13mp2 RF DNA (SS leads to DS) has been carried out using DNA polymerase I (Klenow fragment) and a unique 15-nucleotide primer. A time course for in vitro synthesis showed that the increase in transfection in the presence of SSB paralleled DNA synthesis after an initial lag period for transfection. Digestion of replication products with restriction endonucleases and S1 endonuclease indicates that only those molecules that are fully or almost fully duplex transfect competent cells in the presence of SSB.
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PMID:Preferential transfection with M13mp2 RF DNA synthesized in vitro. 630 39

Chemically synthesized leu-enkephalin gene was fused to a large Eco RI-Bam HI fragment of pBR322 along with a Eco RI fragment of Ch4A phage DNA carrying the promoter and most of the E.coli beta-galactosidase gene. The resulting recombinant DNA was used to transform E. coli cells. Transformants were screened for Tc-sensitivity, Am-resistance, and beta-galactosidase constitutional synthesis. Restriction endonuclease analysis combined with DNA sequencing of the plasmid DNAs revealed a complete nucleotide leu-enkephalin sequence and Eco RI lac-operon fragment in two possible orientations. Radioimmunoassay for leu-enkephalin activity in BrCN-treated bacterial extracts showed that in vivo leu-enkephalin is synthesized only in strains carrying plasmids with the proper lac-fragment orientation. About 5.10(4) molecules of the former are synthesized per single E. coli cell. One of the clones was used for leu-enkephalin purification. Using 100 g of cells it is possible to obtain about 2 mg of practically pure leu-enkephalin.Images
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PMID:Leu-enkephalin purification from E. coli cells carrying the plasmid with fused synthetic leu-enkephalin gene. 700 33

Many eukaryotic organisms, including humans, remove ultraviolet (UV) damage from their genomes by the nucleotide excision repair pathway, which requires more than 10 separate protein factors. However, no nucleotide excision repair pathway has been found in the filamentous fungus Neurospora crassa. We have isolated a new eukaryotic DNA repair gene from N.crassa by its ability to complement UV-sensitive Escherichia coli cells. The gene is altered in a N.crassa mus-18 mutant and responsible for the exclusive sensitivity to UV of the mutant. Introduction of the wild-type mus-18 gene complements not only the mus-18 DNA repair defect of N.crassa, but also confers UV-resistance on various DNA repair-deficient mutants of Saccharomyces cerevisiae and a human xeroderma pigmentosum cell line. The cDNA encodes a protein of 74 kDa with no sequence similarity to other known repair enzymes. Recombinant mus-18 protein was purified from E.coli and found to be an endonuclease for UV-irradiated DNA. Both cyclobutane pyrimidine dimers and (6-4)photoproducts are cleaved at the sites immediately 5' to the damaged dipyrimidines in a magnesium-dependent, ATP-independent reaction. This mechanism, requiring a single polypeptide designated UV-induced dimer endonuclease for incision, is a substitute for the role of nucleotide excision repair of UV damage in N.crassa.
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PMID:A eukaryotic gene encoding an endonuclease that specifically repairs DNA damaged by ultraviolet light. 777 97

The hsdR, hsdM and hsdS genes coding for R.EcoK restriction endonuclease, both with and without a temperature sensitive mutation (ts-1) in the hsdS gene, were cloned in pBR322 plasmid and introduced into E.coli C3-6. The presence of the hsdSts-1 mutation has no effect on the R-M phenotype of this construct in bacteria grown at 42 degrees C. However, DNA sequencing indicates that the mutation is still present on the pBR322-hsdts-1 operon. The putative temperature-sensitive endonuclease was purified from bacteria carrying this plasmid and the ability to cleave and methylate plasmid DNA was investigated. The mutant endonuclease was found to show temperature-sensitivity for restriction. Modification was dramatically reduced at both the permissive and non-permissive temperatures. The wild type enzyme was found to cleave circular DNA in a manner which strongly suggests that only one endonuclease molecule is required per cleavage event. Circular and linear DNA appear to be cleaved using different mechanisms, and cleavage of linear DNA may require a second endonuclease molecule. The subunit composition of the purified endonucleases was investigated and compared to the level of subunit production in minicells. There is no evidence that HsdR is prevented from assembling with HsdM and HsdSts-1 to produce the mutant endonuclease. The data also suggests that the level of HsdR subunit may be limiting within the cell. We suggest that an excess of HsdM and HsdS may produce the methylase in vivo and that assembly of the endonuclease may be dependent upon the prior production of this methylase.
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PMID:Cloning, production and characterisation of wild type and mutant forms of the R.EcoK endonucleases. 844 49

The genes from Moraxella bovis encoding the MboI restriction--modification system were cloned and expressed in Escherichia coli. Three open reading frames were found in the sequence containing the genes. These genes, which we named mboA, mboB, and mboC, had the same orientation in the genome. Genes mboA and mboC encoded MboI methyltransferases (named M.MboA and M.MboC) with 294 and 273 amino acid residues, respectively. The mboB gene coded for MboI restriction endonuclease (R.MboI) with 280 amino acid residues. Recombinant E.coli-MBOI, which contained the whole MboI system, overproduced R.MboI. R.MboI activity from E.coli-MBOI was 480-fold that of M.bovis. The amino acid sequences deduced from these genes were compared with those of other restriction--modification systems. The protein sequences of the MboI system had 38-49% homology with those of the DpnII system.
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PMID:Gene structure and expression of the MboI restriction--modification system. 850 28

Endonuclease IV of Escherichia coli has been implicated by genetic studies in the repair of DNA damage caused by the antitumor drug bleomycin, but the lesion(s) recognized by this enzyme in vivo have not been identified. We used the sensitive primer activation assay, which monitors the formation of 3'-OH groups that support in vitro synthesis by E.coli DNA polymerase I, to determine whether endonuclease IV-specific damage could be detected in the chromosomal DNA of cells lacking the enzyme after in vivo treatment with bleomycin. Chromosomal DNA isolated after a 1 h bleomycin treatment from wild-type, endonuclease IV-deficient (nfo-) and endonuclease IV-overproducing (p-nfo; approximately 10-fold) strains all supported modest polymerase activity. However, in vitro treatment with purified endonuclease IV activated subsequent DNA synthesis with samples from the nfo- strain (an average of 2.6-fold), to a lesser extent for samples from wild-type cells (2.1-fold), and still less for the p-nfo samples (1.5-fold). This pattern is consistent with the presence of unrepaired damage that correlates inversely with the in vivo activity of endonuclease IV. Incubation of the DNA from bleomycin-treated nfo- cells with polymerase and dideoxynucleoside triphosphates lowered the endonuclease IV-independent priming activity, but did not affect the amount of activation seen after endonuclease IV treatment. Primer activation with DNA from the nfo- strain could also be obtained with purified E.coli exonuclease III in vitro, but a quantitative comparison demonstrated that endonuclease IV was > or = 5-fold more active in this assay. Thus, endonuclease IV-specific damage can be detected after in vivo exposure to bleomycin. These may be 2-deoxy-pentos-4-ulose residues, but other possibilities are discussed.
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PMID:In vitro detection of endonuclease IV-specific DNA damage formed by bleomycin in vivo. 860 Apr 56

Ionizing radiation and normal cellular respiration form reactive oxygen species that damage DNA and contribute to a variety of human disorders including tumor promotion and carcinogenesis. A major product of free radical DNA damage is the formation of 8-oxoguanine, which is a highly mutagenic base modification produced by oxidative stress. Here, Drosophila ribosomal protein S3 is shown to cleave DNA containing 8-oxoguanine residues efficiently, The ribosomal protein also contains an associated apurinic/apyrimidinic (AP) lyase activity, cleaving phosphodiester bonds via a beta,delta elimination reaction. The significance of this DNA repair activity acting on 8-oxoguanine is shown by the ability of S3 to rescue the H2O2 sensitivity of an Escherichia coli mutM strain (defective for the repair of 8-oxoguanine) and to abolish completely the mutator phenotype of mutM caused by 8-oxoguanine-mediated G-->T transversions. The ribosomal protein is also able to rescue the alkylation sensitivity of an E.coli mutant deficient for the AP endonuclease activities associated with exonuclease III (xth) and endonuclease IV (nfo), indicating for the first time that an AP lyase can represent a significant source of DNA repair activity for the repair of AP sites. These results raise the possibility that DNA repair may be associated with protein translation.
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PMID:A Drosophila ribosomal protein contains 8-oxoguanine and abasic site DNA repair activities. 864 Dec 96

Drosophila ribosomal protein PO was overexpressed in Escherichia coli to allow for its purification, biochemical characterization and to generate polyclonal antibodies for Western analysis. Biochemical tests were originally performed to see if overexpressed PO contained DNase activity similar to that recently reported for the apurinic/apyrimidinic (AP) lyase activity associated with Drosophila ribosomal protein S3. The overexpressed ribosomal protein was subsequently found to act on AP DNA, producing scissions that were in this case 5' of a baseless site instead of 3', as has been observed for S3. As a means of confirming that the source of AP endonuclease activity was in fact due to PO, glutathione S-transferase (GST) fusions containing a Factor Xa cleavage site between GST and PO were constructed, overexpressed in an E.coli strain defective for the major 5'-acting AP endonucleases and the fusions purified using glutathione-agarose affinity column chromatography. Isolated fractions containing purified GST-PO fusion proteins were subsequently found to have authentic AP endonuclease activity. Moreover, glutathione-agarose was able to deplete AP endonuclease activity from GST-PO fusion protein preparations, whereas the resin was ineffective in lowering DNA repair activity for PO that had been liberated from the fusion construct by Factor Xa cleavage. These results suggested that PO was a multifunctional protein with possible roles in DNA repair beyond its known participation in protein translation. In support of this notion, tests were performed that show that GST-PO, but not GST, was able to rescue an E.coli mutant lacking the major 5'-acting AP endonucleases from sensitivity to an alkylating agent. We furthermore show that GST-PO can be located in both the nucleus and ribosomes. Its nuclear location can be further traced to the nuclear matrix, thus placing PO in a subcellular location where it could act as a DNA repair protein. Other roles beyond DNA repair seem possible, however, since GST-PO also exhibited significant nuclease activity for both single- and double-stranded DNA.
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PMID:Drosophila ribosomal protein PO contains apurinic/apyrimidinic endonuclease activity. 893 86

Based on parameters governing promoter activity and using regulatory elements of the lac, ara and tet operon transcription control sequences were composed which permit the regulation in Escherichia coli of several gene activities independently and quantitatively. The novel promoter PLtetO-1 allows the regulation of gene expression over an up to 5000-fold range with anhydrotetracycline (aTc) whereas with IPTG and arabinose the activity of Plac/ara-1 may be controlled 1800-fold. Escherichia coli host strains which produce defined amounts of the regulatory proteins, Lac and Tet repressor as well as AraC from chromosomally located expression units provide highly reproducible in vivo conditions. Controlling the expression of the genes encoding luciferase, the low abundance E.coli protein DnaJ and restriction endonuclease Cfr9I not only demonstrates that high levels of expression can be achieved but also suggests that under conditions of optimal repression only around one mRNA every 3rd generation is produced. This potential of quantitative control will open up new approaches in the study of gene function in vivo, in particular with low abundance regulatory gene products. The system will also provide new opportunities for the controlled expression of heterologous genes.
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PMID:Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. 909 30

An enzyme that plays an important role in the repair of oxidative DNA damage is the 3'-phosphodiesterase. This activity, which repairs damaged DNA 3'-termini,can be detected using several available biochemical assays. We present a method to detect 3'-phosphodiesterase activity of renatured proteins immobilized in polyacrylamide gels. The model substrate, labeled with [alpha-32P]dCTP, contains 3'-phosphoglycolate termini produced by bleomycin-catalyzed cleavage of the self-complementary alternating copolymer poly(dGdC). The DNA substrate is incorporated into the gel matrix during standard SDS-PAGE. Active 3'-phosphodiesterase enzymes are detected visibly by the loss of radioactivity at a position corresponding to the mobility of the enzyme during SDS-PAGE. Using this procedure, two Escherichia coli 3'-phosphodiesterases, exonuclease III and endonuclease IV, are readily detected in crude cell extracts or as homogeneous purified proteins. Extracts of mutant cells lack activity at the positions of exonuclease III and endonuclease IV but retain activity in the position of a much larger protein (Mr approximately 100 kDa). The identification of this novel 100 kDa E.coli 3'-phosphodiesterase demonstrates the potential value of the activity gel method described here.
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PMID:In situ activity gel for DNA repair 3'-phosphodiesterase. 910 75


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