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)

The interaction of bacteriophage T7 specific RNA polymerase with its cognate promoter sites has been probed by selectively replacing bases in one T7 promoter site with base analogs. Base analogs such as 2,6-diaminopurine or hypoxanthine, which alter residues appearing in the minor groove of the DNA helix, prevent utilization of the promoter by T7 RNA polymerase. These analogs do not affect transcription which starts outside of the modified region. In contrast, base analogs that have alterations that appear in the major groove of the DNA helix, such as uracil, 5-bromouracil, 5-methylcytosine, 5-hydroxymethylcytosine, and [5-HgSR]pyrimidines, do not prevent utilization of the promoter. The deoxyribonucleoside analog 5'-imino-5'-deoxythymidine, an alteration appearing in the deoxyribose-phosphodiester backbone of the DNA helix, does not prevent promoter recognition. Haemophilus aegyptius restriction endonuclease III, which cleaves DNA at the sequence 5'GGCC3', does not act at sites in which the guanine residues in one of the two DNA strands have been substituted with hypoxanthine. This implicates the guanine amino group in the minor groove of the DNA helix as a possible recognition point for this restriction endonuclease.
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PMID:Transcription of T7 DNA containing modified nucleotides by bacteriophage T7 specific RNA polymerase. 35 45

The nucleotide sequence of a double stranded DNA fragment from the gene AB region of bacteriophage S13 DNA has been determined. The fragment was isolated as two adjacent shorter fragments by cleavage of S13 replicative form (RF) DNA with restriction endonuclease III from Hemophilus aegyptius. The strands of the fragments were separated electrophoretically and hydrolyzed with T4 endonuclease IV to yield short oligonucleotides which were then sequenced by partial exonuclease digestion. The complete nucleotide sequence of the restriction fragments was obtained by ordering the inter- and intrastrand overlapping oligonucleotide sequences. The adjacent fragments were 190 nucleotides in length. The sequences included a HindII site, an AluI site and two sequences which may be possible transcription initiation sequences, one with an adjacent sequence homologous to the canonical promoter site sequence T-A-T-Pu-A-T-Pu. Examination of the three possible reading frames for translation of the sequence revealed only one possible complete translation product. The postulated partial sequence of gene A protein has a highly positively charged arginine-rich area which may have importance in DNA binding.
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PMID:The nucleotide sequence of two restriction fragments located in the gene AB region of bacteriophage S13. 90 72

DNA deoxyribophosphodiesterase (dRpase) of E. coli catalyzes the release of deoxyribose-phosphate moieties following the cleavage of DNA at an apurinic/apyrimidinic (AP) site by either an AP endonuclease or AP lyase. Exonuclease I is a single-strand specific DNA nuclease which affects the expression of recombination and repair pathways in E. coli. We show here that a major dRpase activity in E. coli is associated with the exonuclease I protein. Highly purified exonuclease I isolated from an over-producing stain contains high levels of dRpase activity; it catalyzes the release of deoxyribose-5-phosphate from an AP site incised with endonuclease IV of E. coli and the release of 4-hydroxy-2-pentenal-5-phosphate from an AP site incised by the AP lyase activity of endonuclease III of E. coli. A strain containing a deletion of the sbcB gene showed little dRpase activity; the activity could be restored by transformation of the strain with a plasmid containing the sbcB gene. The dRpase activity isolated from an overproducing stain was increased 70-fold as compared to a normal sbcB+ strain (AB3027). These results suggest that the dRpase activity may be important in pathways for both DNA repair and recombination.
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PMID:DNA deoxyribophosphodiesterase of Escherichia coli is associated with exonuclease I. 132 27

It has been shown previously that the DNA deoxyribophosphodiesterase (dRpase) activity of Escherichia coli excises 2-deoxyribose 5-phosphate moieties at apurinic/apyrimidinic (AP) sites in DNA following cleavage of the DNA at the AP site by an AP endonuclease such as endonuclease IV of E coli. A second class of enzymes that cleave DNA at AP sites by a beta-elimination mechanism, AP lyases, leave a different sugar-phosphate product remaining at the AP site, which has been identified as the compound trans-4-hydroxy-2-pentenal 5-phosphate. It is shown that dRpase removes this unsaturated sugar-phosphate group following cleavage of a poly(dA-dT) substrate containing AP sites by the action of the AP lyase endonuclease III of E. coli. The Km for the removal of trans-4-hydroxy-2-pentenal 5-phosphate is 0.06 microM; the Km for the removal of 2-deoxyribose 5-phosphate is 0.17 microM. It was verified that the sugar-phosphate product removed by dRpase from the endonuclease III-cleaved substrate was trans-4-hydroxy-2-pentenal 5-phosphate by conversion of the product to the compound cyclopentane-1,2-dione. The dRpase activity is unique in its ability to remove sugar-phosphate products after cleavage by both AP endonucleases and AP lyases.
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PMID:Excision of sugar-phosphate products at apurinic/apyrimidinic sites by DNA deoxyribophosphodiesterase of Escherichia coli. 133 11

1,2-Dioxetanes are efficient sources of triplet excited carbonyl compounds, into which they decompose on thermal or photochemical activation. In the presence of DNA, the decomposition of dioxetanes gives rise to DNA modifications, which have been studied by means of specific repair endonucleases. Cyclobutane pyrimidine dimers, which are generated by triplet-triplet energy transfer, were detected by a UV endonuclease; they made up between 2% and 30% of the total modifications recognized by a crude repair endonuclease preparation from Micrococcus luteus. For various 1,2-dioxetanes, the yield of pyrimidine dimers was proportional to their triplet excitation flux. DNA strand breaks, sites of base loss (AP sites; recognized by exonuclease III and endonuclease IV) and dihydropyrimidines (recognized by endonuclease III) were found to represent only a small fraction of the modifications. The majority of the modifications detected were recognized by formamidopyrimidine-DNA glycosylase (FPG protein) and represent 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) residues or other yet not defined base modifications which are recognized by this enzyme. The modifications were generated in similar relative yields by thermal and photo-induced decomposition of the 1,2-dioxetanes and therefore emanate under both conditions from the excited carbonyl compounds. The formation of the FPG protein-sensitive modifications was efficiently quenched by azide anions; the Stern-Volmer quenching of these modifications was 150-fold more effective than that of the pyrimidine dimers. The relative amounts of the two types of modifications were strongly dependent on the structure of the 1,2-dioxetanes and on the concentration of molecular oxygen. Singlet oxygen appears to be involved only to some extent in the generation of the FPG protein-sensitive base modifications as their yield was only moderately (approximately 2-fold) increased in D2O as solvent. A mechanism is suggested in which oxidized guanine is predominantly formed by a single-electron-transfer reaction of the triplet excited carbonyl product derived from the 1,2-dioxetane, followed by unknown secondary oxidations, which involve molecular oxygen and/or undecomposed 1,2-dioxetane.
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PMID:Photochemical DNA modifications induced by 1,2-dioxetanes. 133 14

In Escherichia coli the mutY (or micA)-dependent DNA mismatch repair pathway can convert A degrees G and A degrees C mismatches to C.G and G.C base pairs, respectively, through a short repair-tract mechanism. The MutY protein has been purified to near homogeneity from an E. coli overproducer strain. Purified MutY has been shown to contain both N-glycosylase and 3' apurinic/apyrimidinic (AP) endonuclease activities. The N-glycosylase removes the mispaired adenines of A degrees G and A degrees C mismatches, and the AP endonuclease acts on the first phosphodiester bond 3' to the AP sites. The N-glycosylase and the nicking (combined N-glycosylase and AP endonuclease) activities copurified through multiple chromatographic steps without a change in relative specific activities. Furthermore, both N-glycosylase and AP endonuclease activities can be recovered by renaturation of a single polypeptide band from an SDS/polyacrylamide gel. Renaturation required the presence of iron and sulfide. These findings suggest that the MutY protein, like endonuclease III, is an iron-sulfur protein. DNA fragments with A degrees C mismatches were 20-fold less active than DNA with A degrees G mispairs as a substrate for purified MutY.
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PMID:Escherichia coli MutY protein has both N-glycosylase and apurinic/apyrimidinic endonuclease activities on A.C and A.G mispairs. 138 98

We have isolated an endonuclease from E. coli active on bleomycin-treated DNA. Purification on DEAE-cellulose separated this activity in strains lacking endonuclease I, endonuclease III or exonuclease III. After DEAE chromatography, the enzyme was active in the absence of divalent cations and was not inhibited by tRNA or harmane. In addition, this enzyme was stable at 45 degrees C for 20 min. These properties are consistent with this activity being endonuclease IV. This was supported by our finding no activity in a strain lacking endonuclease IV.
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PMID:Bleomycin-treated DNA is specifically cleaved only by endonuclease IV in E. coli. 168 11

DNA modifications induced either by photosensitization (illumination in the presence of methylene blue) or by chemically generated singlet oxygen (thermal decomposition of an 1,4-etheno-2,3-benzodioxin) are recognized and incised by repair endonucleases present in crude bacterial cell extracts. Only a small fraction of the incised modifications are sites of base loss (AP-sites) sensitive to exonuclease III, endonuclease IV from E. coli or to the UV-endonuclease from M. luteus. Cell extracts from E. coli strains overproducing or defective in endonuclease III recognize the modifications induced by illumination in the presence of methylene blue just as well as do those from wild-type E. coli strains. This indicates that dihydropyrimidine derivatives, which are characteristic of hydroxyl radical-induced DNA modifications, are absent. In contrast, most of the modifications induced are not recognized by a cell extract from a fpg strain defective in formamidopyrimidine-DNA glycosylase FPG protein). Furthermore, incision by a cell extract from an E. coli strain overproducing FPG protein takes place at much lower protein concentration than with the wild-type strain. Experiments with purified FPG protein confirm that this enzyme is responsible for the recognition of singlet oxygen-induced DNA base modifications.
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PMID:Enzymatic recognition of DNA modifications induced by singlet oxygen and photosensitizers. 170 Mar 66

In this study we demonstrate that the different substrate recognition properties of bacterial and human AP endonucleases might be used to quantify and localize apurinic (AP) sites formed in DNA in vivo. By using a model oligonucleotide containing a single AP site modified with methoxyamine (MX), we show that endonuclease III and IV of E. coli are able to cleave the alkoxyamine-adducted site whereas a partially purified HeLa AP endonuclease and crude cell-free extracts from HeLa cells are inhibited by this modification. In addition MX-modified AP sites in a DNA template retain their ability to block DNA synthesis in vitro. Since MX can efficiently react with AP sites formed in mammalian cells in vivo we propose that the MX modified abasic sites thus formed can be quantitated and localized at the level of the individual gene by subsequent site specific cleavage by either E. coli endonuclease III or IV in vitro.
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PMID:Processing in vitro of an abasic site reacted with methoxyamine: a new assay for the detection of abasic sites formed in vivo. 171 78

The inactivation efficiency and repair of single-strand breaks was investigated using model strand breaks created by endonucleolytic incision of damaged DNA. Phi X-174 duplex transfecting DNA containing either thymine glycols, urea residues, or abasic (AP) sites was incubated with AP endonucleases that produce breaks on the 3' side, the 5' side, or both sides of the lesion. For each lesion, incubation with Escherichia coli endonuclease III results in a single-strand break containing a 3' alpha, beta-unsaturated aldehyde (4-hydroxy-2-pentenal), while treatment of AP- or urea-containing DNA with E. coli endonuclease IV results in a single-strand break containing a 5' deoxyribose or a 5' deoxyribosylurea moiety, respectively. Incubation of lesion-containing DNA with both enzymes results in a base gap. Ligatable nicks containing 3' hydroxyl and 5' phosphate moieties were produced by subjecting undamaged DNA to DNase I. When the biological activity of these DNAs was assessed in wild-type cells, ligatable nicks were not lethal, but each of the other strand breaks tested was lethal, having inactivation efficiencies between 0.12 and 0.14. These inactivation efficiencies are similar to those of the base lesions from which the strand breaks were derived. In keeping with the current model of base excision repair, when phi X duplex DNA containing strand breaks with a blocked 3' terminus was transfected into an E. coli double mutant lacking the major 5' cellular AP endonucleases, a greater than twofold decrease in survival was observed. Moreover, when this DNA was treated with a 5' AP endonuclease prior to transfection, the survival returned to that of wild type. As expected, when DNA containing strand breaks with a 5' blocked terminus or DNA containing base gaps was transfected into the double mutant lacking 5' AP endonucleases, the survival was the same as in wild-type cells. The decreased survival of transfecting DNA containing thymine glycols, urea, or AP sites observed in appropriate base excision repair-defective mutants was also obviated if the DNA was incubated with the homologous enzyme prior to transfection. Thus, in every case, with both base lesions and single-strand breaks, the lesion was repaired in the cell by the enzyme that recognizes it in vitro. Furthermore, the repair step in the cell could be eliminated if the appropriate enzyme was added in vitro prior to transfection.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Processing of model single-strand breaks in phi X-174 RF transfecting DNA by Escherichia coli. 185 23


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