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)

Escherichia coli contains multiple enzymes that hydrolyze deoxyribose fragments (phosphoglycolaldehyde, PGA) from the 3' termini of a synthetic DNA substrate. The major such activities are the main bacterial apurinic endonucleases, exonuclease III and endonuclease IV. In a double mutant deficient in both of these oxidation repair enzymes, Mg++-dependent 3'-PGA diesterase was detected at 3% the level found in wild-type bacteria. Gel filtration fractionated this residual diesterase activity into two peaks of Mr 40,000-52,000 (Pool A) and Mr 22,000-30,000 (Pool B) with differing abilities to remove 3'-phosphates from DNA. These multiple repair activities were resolved in 3'-PGA diesterase activity gels. The exonuclease III and endonuclease IV bands were identified using the purified proteins and by their specific absence from strains defective for the respective structural genes. Gel filtration Pool B yielded two activity bands of apparent Mr 25,000 and 28,000, but Pool A did not form a new band in the activity gels. Incubation of activity gels in different transition metals or boiling of the samples before electrophoresis also served to distinguish the various activities. The possible identities of the novel E. coli 3'-PGA diesterases and the importance of multiple repair enzymes for 3' damages are discussed.
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PMID:Multiple DNA repair activities for 3'-deoxyribose fragments in Escherichia coli. 246 96

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

Gel shift assays have been employed to examine the association of the thyroid hormone receptor with specific DNA sequences in the 5'-flanking DNA of the rat growth hormone (rGH) gene. This DNA is known to have structure(s) that mediate thyroid hormone effects on the rGH promoter. The receptors used were obtained from preparations purified 300-500-fold from rat liver nuclear extracts and contained about 1% pure receptors. Thyroid hormone receptor binding to DNA was assessed by monitoring protein-bound 32P-labeled restriction endonuclease fragments in parallel with L-tri[125I]iodothyronine-labeled protein-DNA complexes. The receptors were found to bind specifically to four different regions of the rGH 5'-flanking DNA (nucleotides -1730 to -1230, -530 to -230, -181 to -149, and -149 to +12) numbered with respect to the transcriptional start site. The specificity of the binding was documented by the finding that the receptor did not bind to other rGH 5'-flanking DNA sequences or to several other DNAs and by the fact that only the DNAs exhibiting specific binding could block the binding of radiolabeled DNA. The binding was also detected in NaCl concentrations up to 140 mM, reduced by Mg2+ concentrations up to 5 mM, and inhibited by 1 mM zinc. The DNA sequence-specific binding of the receptor was found to require occupancy of the receptor by the hormone (L-triiodothyronine) and could also be observed when the receptor was occupied by the thyroid hormone antagonist amiodarone. These results indicate that thyroid hormone receptors interact specifically with several sites on the 5'-flanking DNA of the rGH gene and that hormone occupancy is not required for the binding. Thus, thyroid hormone may act by stimulating a transcriptional activation function of the receptor rather than by stimulating DNA binding per se.
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PMID:The thyroid hormone receptor binds to multiple domains of the rat growth hormone 5'-flanking sequence. 283 88

The synthesis of chelating sorbents for ligand-exchange chromatography of enzymes is described. An inorganic support "Silochrom" and organic "Spheron", TSK-Gel HW 55 and cellulose were used as initial supports. The chelating sorbents contained iminodiacetic acid and iminodimethylphosphonic acid as stationary ligands. In order to obtain monofunctional sorbents, iminodiacetic acid was added in the form of its dimethyl ester. The concentration of stationary ligands on the sorbents varied from 10 to 100 mumol per ml sorbent. A chelating sorbent (in nickel form) was shown to be effective in the purification of exonuclease A5 from actinomyces. Electrophoretically homogeneous exonuclease A5 was obtained with a 25% yield. A chelating sorbent with iminodiacetic groups (in copper form) was applied to the isolation of endonuclease from Serratia marcescens directly from the culture medium. The capacity of the chelating sorbents for the endonuclease was studied as a function of the stationary ligand concentration. After one stage of purification, more than 70% pure enzyme was obtained with a yield exceeding 80%.
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PMID:Ligand-exchange chromatography of nucleases. 302 93

The 25-kDa peptidoglycan-associated outer-membrane protein and most likely porin of Vibrio cholerae is a major immunogenic species. It has been purified by ion-exchange elution on hydroxyapatite followed by gel filtration on Bio-Gel P150 both in the presence of sodium dodecyl sulfate. This protein, of greater than 90% purity as judged by Western blotting, has been used to raise antibodies in rabbits. The antisera were then used to screen V. cholerae gene banks, constructed in Escherichia coli K12, and this has enabled us to isolate several colonies harbouring the cloned gene. The plasmids in these colonies have been designated pPM451, pPM455 and pPM472. These plasmids have a 5.3 X 10(3)-base BamHI fragment of V. cholerae DNA in common. Restriction endonuclease mapping of these plasmids has been performed and the protein identified both by Western blot analysis and in E. coli K12 minicells. The protein is not efficiently expressed in E. coli K12. It is proposed to use the name ompV to describe the structural gene, present in the cloned DNA, for this V. cholerae outer membrane protein.
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PMID:Purification of the 25-kDa Vibrio cholerae major outer-membrane protein and the molecular cloning of its gene: ompV. 398 95

Purified simian virus 40 has associated with it an endonuclease activity which converts form I (double-stranded, circular) simian virus 40 deoxyribonucleic acid to a nicked form that sediments as a homogeneous peak in alkaline sucrose gradients. The enzyme is dependent on magnesium ions for activity and is completely inhibited by ethylenediaminetetraacetic acid (0.02 m) or heat (80 C for 10 min). In tris(hydroxymethyl)aminomethane-hydrochloride buffer it exhibits optimal activity between pH 6.7 and 7.1 at 37 C. Gel electrophoretic analysis of purified, disrupted virus indicates the absence of detectable host cell protein contamination.
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PMID:Endonuclease activity associated with purified simian virus 40 virions. 433 65

varphiX174 RF (replicative form) II DNA, labeled in vivo with [methyl-(3)H]thymidine, was isolated from Escherichia coli polA (DNA polymerase I-deficient) and polA(+) cells during RF replication. [(32)P]dCMP was incorporated into the gaps present in the RF II DNA with [alpha-(32)P]dCTP and T4 DNA polymerase. Sedimentation in alkaline sucrose gradients revealed that much of the incorporated (32)P was present in a heterogeneous collection of fragments shorter than unit length. Inclusion of polynucleotide ligase in the gap-filling reaction increased the average size of the (32)P-labeled fragments. Gel electrophoresis of the products formed by digestion of the (32)P-labeled RF II molecules with the restriction nuclease, endonuclease R, indicated that in the population of RF II molecules gaps could occur anywhere in the genome. Competition-annealing experiments provided evidence that the majority of the label incorporated into gaps was present in the minus strand. RF II molecules isolated from polA(+) cells were enriched for gaps in a unique region of the genome in comparison with RF II molecules isolated from polA cells. The presence of multiple gaps in the minus strand implies that it is synthesized by a discontinuous mechanism during varphiX RF replication.
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PMID:Structure of nascent phiX174 replicative form: evidence for discontinuous DNA replication. 452 6

A full-length cDNA copy of the mRNA encoding calf chymosin (also known as rennin), a proteolytic enzyme with commercial importance in the manufacture of cheese, has been cloned in an f1 bacteriophage vector. The nucleotide sequence of the cDNA was determined, and translation of that sequence into amino acids predicts that the zymogen prochymosin is actually synthesized in vivo as preprochymosin with a 16 amino acid signal peptide. In vitro translation of total poly(A)-enriched RNA from the calf fourth stomach (abomasum) and immunoprecipitation with antichymosin antiserum revealed that a form of chymosin (probably preprochymosin judging from the Mr-value) is the major in vitro translation product of RNA from that tissue. Gel-transfer hybridization of restriction endonuclease-cleaved bovine chromosomal DNA with labeled cDNA probes indicated that the two known forms of chymosin, A and B, must be products of two different alleles of a single chymosin gene.
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PMID:Molecular cloning and characterization of double-stranded cDNA coding for bovine chymosin. 618 68

A heat- and acid-stable protein which bound both native and denatured DNA but not RNA was extensively purified from extracts of Haemophilus influenzae Rd strain com-58-A. The active species had an apparent subunit molecular weight of 15,000. The interaction of the protein with denatured DNA appeared to be cooperative, as judged by the sigmoid shapes of binding curves. This cooperativity increased with increasing ionic strength and was more pronounced with sodium ions than with potassium ions. Gel filtration suggested that the native protein formed aggregates in solution. The presence of the binding protein protected single-stranded DNA from the action of S1 endonuclease; approximately 30 nucleotide residues were protected per subunit equivalent of protein. The number of subunit equivalents per cell of this protein has been estimated at 10,000. The protein, which we designate DNA-binding protein II, is most probably a major histone-line protein of H. influenzae.
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PMID:DNA-binding proteins of Haemophilus influenzae: purification and characterization of a major intracellular binding protein. 630 11

Apurinic/apyrimidinic (AP) sites are mutagenic and block DNA synthesis in vitro. Repair of AP sites is initiated by AP endonucleases that cleave just 5' to the damage. We linked a 4.1-kilobase pair HindIII DNA fragment from the region upstream of the human AP endonuclease gene (APE) to the chloramphenicol acetyltransferase (CAT) gene. Deletions generated constructs containing 1.9 kilobase pairs to 50 base pairs (bp) of the APE upstream region. Transient transfection studies in HeLa cells established that the basal APE promoter is contained within a 500-bp fragment. The major transcriptional start site in HeLa, hepatoma (HepG2), and myeloid leukemic (K562) cells was mapped to a cluster of sites approximately 130 bp downstream of a putative "CCAAT box," approximately 130 bp 5' of the first splice junction in APE. Deletion of 5' sequences to within 10 bp of the CCAAT box reduced the CAT activity by only about half, and removal of the CCAAT box region left a residual promotor activity approximately 9%. Deletion to 31 bp upstream of the transcriptional start site abolished APE promoter activity. DNA sequence analysis revealed potential transcription factor recognition sites in the APE promoter. Gel mobility-shift assays showed that both human upstream factor and Sp1 can bind their respective sites in the APE promoter. However, DNase I footprinting using HeLa nuclear extract showed that the binding of Sp1 and upstream factor is blocked by the binding of other proteins to the nearby CCAAT box region.
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PMID:Characterization of the promoter region of the human apurinic endonuclease gene (APE). 753 97


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