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)

Contradictory data have recently been published from two different laboratories on the presence vs absence of an intrinsic endonucliolytic activity of E. coli exonuclease III at apurinic sites in double-stranded DNA. It is shown here that an endonuclease activity of this specificity co-chromatographs exactly with exonuclease III on phosphocellulose and Sephadex G-75 columns, indicating that the endonuclease and exonuclease activities are due to the same enzyme. In addition, another E. coli endonuclease specific for apurinic sites exists, which can be separated from exonuclease III by the same chromatographic procedures.
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PMID:Relation between Escherichia coli endonucleases specific for apurinic sites in DNA and exonuclease III. 33 97

We present a method, utilizing a combination of restriction endonuclease cleavage and digestion with Escherichia coli exonuclease III and Aspergillus orizae nuclease S1, that allows us to position a restriction fragment bearing the promoter of the lacZ gene of E. coli at virtually any distance in front of any cloned gene. In particular, we have used this method to examine the effect on protein production of gene-promoter separation for the cro gene of phage lambda and to produce plasmids that, upon transformation into appropriate E. coli hosts, direct the synthesis of up to 190,000 cro protein monomers per cell.
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PMID:A general method for maximizing the expression of a cloned gene. 37 Aug 36

Restriction endonuclease Bgl II cleaves T7 DNA at a unique site (28.76% on the standard T7 map), yielding two fragments of molecular weights 18.9 x 10(6) (A) and 7.6 x 10(6) (B). Fragment B, representing the leftmost portion of the genome, has been purified by zone sedimentation. Transcription of fragment B by T7-specific RNA polymerase gives only r-strand-specific RNA. Analysis of the products by polyacrylamide gel electrophoresis reveals four major RNA species which have apparent molecular weights of 2.1 x 10(6), 1.36 x 10(6), 0.85 x 10(6) and 0.125 x 10(6), respectively. Each of these RNAs is reduced in size when transcription is carried out with fragment B, which has been shortened by treatment with Escherichia coli exonuclease III. Therefore, each of the transcripts must be terminated at the right end of fragment B. Analysis of the molecular weights of the four transcripts produced from whole and from exonucleolytically shortened fragment B suggests that these transcripts are read from promoters located at 13.5, 18.9, 22.6, and 27.9%, respectively, on the standard T7 map. Hence, there are at least four promoters governing the transcription of the class II region. Transcripts initiated at these promoters on intact T7 DNA appear to read through the class II and part of the class III genetic region and terminate at the strong terminator for T7-specific RNA polymerase near 61%. Transcription of fragment B which has been cleaved with the restriction endonuclease Hpa I seems to activate a fifth promoter for T7-specific RNA polymerase. This promoter appears to be identical to the promoter previously described by Oakley and Coleman (Proc. Natl. Acad. Sci. U.S.A. 74:4266-4270, 1977) that maps near 15% on the standard T7 map. Little or no RNA is read from T7 Bgl II fragment B, which has a mobility expected for a transcript read from this promoter. However, upon cleavage with Hpa I, this promoter is utilized approximately 10-fold more efficiently than the other class II promoters. The mechanism of this activation is not yet known.
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PMID:Mapping of class II promoter sites utilized in vitro by T7-specific RNA polymerase on bacteriophage T7 DNA. 43 May 92

The aromatic amine 9-amino-ellipticine is a synthetic DNA intercalating compound derived from the antitumor agent ellipticine, which cleaves at very low doses DNA containing apurinic sites by beta-elimination through formation of a Schiff base. This compound has been shown to potentiate the cytotoxic effect of alkylating drugs, such as dimethyl sulfate, in E. coli through a mechanism involving apurinic sites. We have studied the ability of 9-amino-ellipticine to inhibit an enzymatic repair system mimicking base-excision repair, in which E. coli exonuclease III only presents an endonuclease for apurinic/apyrimidinic site activity. 10 microM of 9-amino-ellipticine inhibits 70% of apurinic site repair. Other intercalating agents with similar affinities for DNA do not induce any inhibition. In another system designed for the direct assay of the exonuclease III-induced incisions 5' to AP sites 10 microM of 9-amino-ellipticine inhibits 65% of the endonuclease for apurinic/apyrimidinic site activity of E. coli exonuclease III. The 9-amino-ellipticine-induced formation of a 2',3'-unsaturated deoxyribose and cleavage at the 3' side of the apurinic site, and possible creation of an adduct, as suggested by Bertrand and coworkers (1989), on the 3' position of the deoxyribose seem to strongly inhibit the endonuclease for apurinic/apyrimidinic site activity. 9-Amino-ellipticine appears therefore to be the first small ligand which can inhibit, by an irreversible modification of the substrate, the repair of apurinic sites through the base excision-repair pathway at a pharmacological concentration.
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PMID:9-amino-ellipticine inhibits the apurinic site-dependent base excision-repair pathway. 169 66

Apurinic/apyrimidinic (AP) sites in cellular DNA are considered to be both cytotoxic and mutagenic, and can arise spontaneously or following exposure to DNA damaging agents. We have isolated cDNA clones which encode an endonuclease, designated HAP1 (human AP endonuclease 1), that catalyses the initial step in AP site repair in human cells. The predicted HAP1 protein has an Mr of 35,500 and shows striking sequence similarity (93% identity) to BAP 1, a bovine AP endonuclease enzyme. Significant sequence homology to two bacterial DNA repair enzymes, E. coli exonuclease III and S. pneumoniae ExoA proteins, and to Drosophila Rrp1 protein is also apparent. We have expressed the HAP1 cDNA in E. coli mutants lacking exonuclease III (xth), endonuclease IV (nfo), or both AP endonucleases. The HAP1 protein can substitute for exonuclease III, but not for endonuclease IV, in respect of some, but not all, DNA repair and mutagenesis functions. Moreover, a dut xth (ts) double mutant, which is nonviable at 42 degrees C due to an accumulation of unrepaired AP sites following excision of uracil from DNA, was rescued by expression of the HAP1 cDNA. These results indicate that AP endonucleases show remarkable conservation of both primary sequence and function. We would predict that the HAP1 protein is important in human cells for protection against the toxic and mutagenic effects of DNA damaging agents.
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PMID:Isolation of cDNA clones encoding a human apurinic/apyrimidinic endonuclease that corrects DNA repair and mutagenesis defects in E. coli xth (exonuclease III) mutants. 171 77

Escherichia coli deficient in exonuclease III (xth gene mutants) are known to be hypersensitive to hydrogen peroxide. We now show that such mutants accumulate many more DNA single-strand breaks than do wild-type bacteria upon exposure to H2O2. DNA isolated from H2O2-treated xth- cells contains strand breaks that do not efficiently support synthesis by E. coli DNA polymerase I, indicating the presence of blocking groups at the DNA 3' termini. Purified E. coli exonuclease III activates this blocked DNA to allow substantial synthesis by polymerase I in vitro. Another E. coli enzyme, endonuclease IV, also activates primers for DNA polymerase. Exonuclease III accounts for greater than 95% of the total activity in E. coli crude extracts for removal of 3'-terminal phosphoglycolaldehyde esters from model DNA substrates. Purified exonuclease III and endonuclease IV can each efficiently remove 3'-terminal phosphoglycolaldehyde in vitro. An important physiological function for exonuclease III is thus the activation of blocked 3' ends for DNA repair synthesis. Endonuclease IV can also initiate the repair of ruptured 3'-deoxyribose in DNA.
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PMID:Exonuclease III and endonuclease IV remove 3' blocks from DNA synthesis primers in H2O2-damaged Escherichia coli. 242 16

Saccharomyces cerevisiae apurinic endonucleases E cochromatographed with activity against a DNA substrate containing urea residues. The urea-recognizing activity of endonuclease E was competitively inhibited by apurinic DNA, and the heat labilities of both activities were the same. The apparent VmaxS of endonuclease E for both substrates were about the same, while the apparent Km for urea-containing DNA was about threefold greater than that for apurinic DNA. These results were similar to those obtained previously with Escherichia coli exonuclease III (Y. Kow and S. Wallace, Proc. Natl. Acad. Sci. USA 82:8354-8358, 1985) and suggest that the ability to recognize urea residues may be a general property of apurinic endonucleases.
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PMID:Apurinic endonucleases from Saccharomyces cerevisiae also recognize urea residues in oxidized DNA. 243 56

Micrococcus luteus extracts contain gamma-endonuclease, a Mg2+-independent endonuclease that cleaves gamma-irradiated DNA. This enzyme has been purified approximately 1000-fold, and the purified enzyme was used to study its substrate specificity and mechanism of action. gamma-Endonuclease cleaves DNA containing either thymine glycols, urea residues, or apurinic sites but not undamaged DNA or DNA containing reduced apurinic sites. The enzyme has both N-glycosylase activity that releases thymine glycol residues from OsO4-treated DNA and an associated apurinic endonuclease activity. The location and nature of the cleavage site produced has been determined with DNA sequencing techniques. gamma-Endonuclease cleaves DNA containing thymine glycols or apurinic sites immediately 3' to the damaged or missing base. Cleavage results in a 5'-phosphate terminus and a 3' baseless sugar residue. Cleavage sites can be converted to primers for DNA polymerase I by subsequent treatment with Escherichia coli exonuclease III. The mechanism of action of gamma-endonuclease and its substrate specificity are very similar to those identified for E. coli endonuclease III.
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PMID:Mechanism of action of Micrococcus luteus gamma-endonuclease. 342 18

Plasmid pLC1437a contains DNA from Escherichia coli K12 including fol, the structural gene for dihydrofolate reductase. The fol gene was mapped on this plasmid relative to several restriction endonuclease cleavage sites. fol was also cloned from strain RSO and the nucleotide sequence for the entire fol gene and its flanking regions from this strain was determined. The amino acid sequence predicted from the nucleotide sequence differs in only a few respects from the reported amino acid sequence of dihydrofolate reductase from E. coli B. The major RNA transcripts initiated at the fol promotor in vivo are approximately 550 and 590 nucleotides long. In addition to these, several longer transcripts (up to 1400 nucleotides) are present in lesser amounts. A new procedure is described for 3' end labeling of DNA fragments having blunt ends using E. coli exonuclease III and avian myeloblastoma virus reverse transcriptase.
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PMID:Nucleotide sequence of the E coli gene coding for dihydrofolate reductase. 615 75

Replicating simian virus 40 (SV40) chromosomes were found to be similar to other eukaryotic chromosomes in that the rate and extent of micrococcal nuclease (MNase) digestion were greater with replicating than with nonreplicating mature SV40 chromatin. MNase digestion of replicating SV40 chromosomes, pulse labeled in either intact cells or nuclear extracts, resulted in the rapid release of nascent DNA as essentially bare fragments of duplex DNA (3-7S) that had an average length of 120 base pairs and were degraded during the course of the reaction. In addition, nucleosomal monomers, equivalent in size to those from mature chromosomes, were released. On the other hand, MNase digestion of uniformly labeled mature SV40 chromosomes resulted in the release of only nucleosomal monomers and oligomers. The small nascent DNA fragments released from replicating chromosomes represented prenucleosomal DNA (PN-DNA) from the region of replication forks that encompasses the actual sites of DNA synthesis and includes Okazaki fragments. Predigestion of replicating SV40 chromosomes with both Escherichia coli exonuclease III (3'-5') and bacteriophage T7 gene 6 exonuclease (5'-3') resulted in complete degradation of PN-DNA. This result, together with the observation that isolated PN-DNA annealed equally well to both strands of SV40 restriction fragments, demonstrated that PN-DNA originates from both sides of replication forks. Over 90% of isolated Okazaki fragments annealed only to the retrograde DNA template. The characteristics of isolated PN-DNA were assessed by examining its sensitivity to MNase and single strand specific S1 endonuclease, sedimentation behavior before and after deproteinization, buoyant density in CsCl after formaldehyde treatment, and size on agarose gels. In addition, it was observed that MNase digestion of purified SV40 DNA also resulted in the release of a transient intermediate similar in size to PN-DNA, indicating that a DNA-protein complex is not required to account for the appearance of PN-DNA. These and other data provide a model of replicating chromosomes in which DNA synthesis occurs on a region of replication forks that is free of nucleosomes and is designated as prenucleosomal DNA.
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PMID:Structure of chromatin at deoxyribonucleic acid replication forks: prenucleosomal deoxyribonucleic acid is rapidly excised from replicating simian virus 40 chromosomes by micrococcal nuclease. 627 44


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