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)

TaqI endonuclease recognizes and cleaves its canonical sequence, TCGA, with complete fidelity under standard conditions. In the presence of some organic solvents, TaqI endonuclease introduced additional single-strand and double-strand cuts at sequences termed TaqI 'star' sites. Using 'middle-labeled' DNA, the relative rates of cleavage of each strand were simultaneously determined for several star sites. These star recognition sequences differed from the canonical sequence by a single base, and all potential star sites were either nicked or cleaved. Star sites within the middle labeled substrate represented ten of the twelve possible star sequences for each strand. For each group of identical star sites, one strand was consistently preferred for cleavage. Based on these preferences, a model for TaqI recognition of the TCGA sequence is proposed. According to this model, sequence discrimination is mediated by eight hydrogen bonds formed between TaqI and the cognate nucleotides within the major groove.
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PMID:The TaqI 'star' reaction: strand preferences reveal hydrogen-bond donor and acceptor sites in canonical sequence recognition. 284 30

It has been proposed that recognition of specific DNA sequences by proteins is accomplished by hydrogen bond formation between the protein and particular groups that are accessible in the major and minor grooves of the DNA. We have examined the DNA-protein interactions involved in the recognition of the hexameric DNA sequence, GAATTC, by the EcoRI restriction endonuclease by using derivatives of an oligodeoxyribonucleotide that contain a variety of base analogues. The base analogues hypoxanthine, 2-aminopurine, 2,6-diaminopurine, N6-methyladenine, 5-bromouracil, uracil, 5-bromocytosine, and 5-methylcytosine were incorporated as single substitutions into the octadeoxyribonucleotide d(pG-G-A-A-T-T-C-C). The effects of the substitutions on the interactions between the EcoRI endonuclease and its recognition sequence were monitored by determining the steady state kinetic values of the hydrolysis reaction. The substitutions resulted in effects that varied from complete inactivity to enhanced reactivity. The enzyme exhibited Michaelis-Menten kinetics with those substrates that were reactive, whereas octanucleotide analogues containing N6-methyladenine at either adenine position, uracil at the second thymine position, or 5-bromocytosine or 5-methylcytosine at the cytosine position were unreactive. The results are discussed in terms of possible effects on interactions between the enzyme and its recognition site during the reaction. An accompanying paper presents the results of a similar study using these oligonucleotides with the EcoRI modification methylase.
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PMID:The effects of base analogue substitutions on the cleavage by the EcoRI restriction endonuclease of octadeoxyribonucleotides containing modified EcoRI recognition sequences. 301 80

The crystal structure of the complex between Eco RI endonuclease and the cognate oligonucleotide TCGCGAATTCGCG provides a detailed example of the structural basis of sequence-specific DNA-protein interactions. The structure was determined, to 3 A resolution, by the ISIR (iterative single isomorphous replacement) method with a platinum isomorphous derivative. The complex has twofold symmetry. Each subunit of the endonuclease is organized into an alpha/beta domain consisting a five-stranded beta sheet, alpha helices, and an extension, called the "arm," which wraps around the DNA. The large beta sheet consists of antiparallel and parallel motifs that form the foundations for the loops and alpha helices responsible for DNA strand scission and sequence-specific recognition, respectively. The DNA cleavage site is located in a cleft that binds the DNA backbone in the vicinity of the scissile bond. Sequence specificity is mediated by 12 hydrogen bonds originating from alpha helical recognition modules. Arg200 forms two hydrogen bonds with guanine while Glu144 and Arg145 form four hydrogen bonds to adjacent adenine residues. These interactions discriminate the Eco RI hexanucleotide GAATTC from all other hexanucleotides because any base substitution would require rupture of at least one of these hydrogen bonds.
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PMID:Structure of the DNA-Eco RI endonuclease recognition complex at 3 A resolution. 302 21

The self-complementing dodecamer 5'-CGCGAATTCGCG-3' and its complexes with the antibiotic netropsin and the restriction endonuclease EcoRI provide substrates of known three-dimensional structure to study the stereochemistry and mechanism of the artificial nuclease of 1,10-phenanthroline-copper ion [(OP)2Cu+]. Analysis of the reaction products with the 5'-32P dodecamer on 20% sequencing gels has demonstrated the presence of 3'-phosphoglycolate ends in addition to 3'-phosphomonoester ends expected from previous studies. A reaction intermediate, which is a precursor to 3'-phosphomonoester termini, has been trapped; in contrast, no comparable species for the 5'-phosphomonoester termini can be detected when 3'-labeled DNAs are utilized as substrates. The reactive oxidative species formed by the coreactants (OP)2Cu+ and hydrogen peroxide is distinguishable in its chemistry from the hydroxyl radicals produced by cobalt-60 gamma-irradiation. The freely diffusible hydroxyl radicals generated by cobalt-60 irradiation produce equivalent amounts of 3'-phosphomonoester and 3'-phosphoglycolate termini whereas the 3'-phosphomonoesters are the preferred product of (OP)2Cu+ and H2O2. On the basis of the structures of the products obtained, the principal site of attack of the coordination complex is on the C-1 of the deoxyribose within the minor groove. This conclusion is supported by the footprinting of netropsin binding to the dodecamer. Crystallographic results have demonstrated that netropsin binds to the minor groove at the central AATT residue. A clear protection of attack by the coordination complex at the deoxyriboses associated with A-5, T-6, T-7, and C-9 is fully consistent with attack from the minor groove without intercalation during the course of the cleavage reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nuclease activity of 1,10-phenanthroline-copper ion: reaction with CGCGAATTCGCG and its complexes with netropsin and EcoRI. 302 54

We have prepared a series of undecadeoxynucleotides that contain changes in the functional group pattern present within the EcoRV recognition site - GATATC-. Oligonucleotides were synthesized on solid phase using normal and modified beta-cyanoethylphosphoramidites and analyzed in steady state cleavage experiments with the EcoRV restriction endonuclease. The following groups appear to interact strongly with the enzyme, since their modification or substitution renders the oligonucleotides refractory to cleavage: the exocyclic NH2-groups of both A residues, the N7 of the first A residue, the exocyclic NH2-group of the C residue and the CH3-groups of both T residues. The exocyclic NH-group of the G residue supports effective recognition, since its absence lowers the kcat of the cleavage reaction. The N7 of the second A residue and the C5 position of the C residue apparently are not recognized by EcoRV; their substitution by -CH- or modification with -Br or -CH3, resp., does not considerably change the rate of cleavage. All oligonucleotides investigated compete with the unmodified substrate for binding to the enzyme. We conclude that EcoRV recognizes its substrate presumably through hydrogen bonds to the exocyclic NH2-group and the N7 of the first A residue, the exocyclic NH2-groups of the second A and the C residue, as well as through hydrophobic interactions with both T residues.
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PMID:Analysis of the recognition mechanism involved in the EcoRV catalyzed cleavage of DNA using modified oligodeoxynucleotides. 306 81

To investigate the relative importance of each of the ribose 3'-hydroxyl groups of 2-5A (ppp5' A2'p5'A2'-p5' A) in determining binding to and activation of the 2-5A-dependent endonuclease (RNase L), the 3'-hydroxyl functionality of each adenosine moiety of 2-5A trimer triphosphate was sequentially replaced by hydrogen. The analog in which the 5'-terminal adenosine was replaced by 3'-deoxyadenosine (viz. ppp5'(3'dA)-2'p5' A2'p5' A) was bound to RNase L as well as 2-5A itself and was only 3 times less potent than 2-5A as an activator of RNase L. On the other hand, when the second adenosine unit was replaced by 3'-deoxyadenosine (viz. ppp5' A2'p5'(3'dA)2'p5' A), binding to RNase L was decreased by a factor of eight relative to 2-5A trimer and, even more dramatically, there was a 500-1000-fold drop in ability to activate the 2-5A-dependent endonuclease. Finally, when the 3'-hydroxyl substituent was converted to hydrogen in the 2'-terminal residue of 2-5A, a significant increase in both binding and activation ability occurred. We conclude that only the 3'-hydroxyl group of the second (from the terminus) nucleotide residue of 2-5A is needed for effective activation of RNase L.
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PMID:Only one 3'-hydroxyl group of ppp5' A2'p5'A2'p5' A (2-5A) is required for activation of the 2-5A-dependent endonuclease. 333 33

Genomic DNAs from twelve Japanese patients with steroid 21-hydroxylase [21-OHase; steroid 21-monooxygenase; steroid, hydrogen-donor:oxygen oxidoreductase (21-hydroxylating); EC 1.14.99.10] deficiency were analyzed by Southern blot hybridization. A 3.7-kilobase (kb) Taq I and a 1.7-kb Pvu II restriction endonuclease fragment that correspond to a 21-OHase B gene were absent from the DNA of two unrelated patients with the salt-wasting form of the disease. However, a 10.5-kb Bgl II fragment corresponding to the region encompassing the 21-OHase B gene was still present in these two patients. The genes encoding 21-OHase were cloned from one of these two patients, who was homozygous by descent for HLA-A26;B39;C4A3;C4B1;DR4. Restriction endonuclease mapping as well as partial nucleotide sequencing analysis revealed that the 21-OHase B gene of the patient has been converted to the pseudogene, 21-OHase A, as far as the critical 0.5-kb sequence was concerned. Thus, the defect was due to both chromosomes each carrying two copies of 21-OHase A pseudogene and lacking functional 21-OHase B gene.
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PMID:Gene conversion-like events cause steroid 21-hydroxylase deficiency in congenital adrenal hyperplasia. 350 Apr 73

The substrate specificity of a calf thymus endonuclease on DNA damaged by UV ligh, ionizing radiation, and oxidizing agents was investigated. End-labeled DNA fragments of defined sequence were used as substrates, and the enzyme-generated scission products were analyzed by using DNA sequencing methodologies. The enzyme was shown to incise damaged DNA at pyrimidine sites. The enzyme incised DNA damaged with UV light, ionizing radiation, osmium tetroxide, potassium permanganate, and hydrogen peroxide at cytosine and thymine sites. The substrate specificity of the calf thymus endonuclease was compared to that of Escherichia coli endonuclease III. Similar pyrimidine base damage specificities were found for both enzymes. These results define a highly conserved class of enzymes present in both procaryotes and eucaryotes that may mediate an important role in the repair of oxidative DNA damage.
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PMID:Substrate specificity of a mammalian DNA repair endonuclease that recognizes oxidative base damage. 353 12

S(1) nuclease, the single-strand specific nuclease from Aspergillus oryzae can cleave both strands of circular covalently closed, superhelical simian virus 40 (SV40) DNA to generate unit length linear duplex molecules with intact single strands. But circular, covalently closed, nonsuperhelical DNA, as well as linear duplex molecules, are relatively resistant to attack by the enzyme. These findings indicate that unpaired or weakly hydrogen-bonded regions, sensitive to the single strand-specific nuclease, occur or can be induced in superhelical DNA. Nicked, circular SV40 DNA can be cleaved on the opposite strand at or near the nick to yield linear molecules. S(1) nuclease may be a useful reagent for cleaving DNAs at regions containing single-strand nicks. Unlike the restriction endonucleases, S(1) nuclease probably does not cleave SV40 DNA at a specific nucleotide sequence. Rather, the sites of cleavage occur within regions that are readily denaturable in a topologically constrained superhelical molecule. At moderate salt concentrations (75 mM) SV40 DNA is cleaved once, most often within either one of the two following regions: the segments defined as 0.15 to 0.25 and 0.45 to 0.55 SV40 fractional length, clockwise, from the EcoR(I) restriction endonuclease cleavage site (defined as the zero position on the SV40 DNA map). In higher salt (250 mM) cleavage occurs preferentially within the 0.45 to 0.55 segment of the map.
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PMID:Cleavage of circular, superhelical simian virus 40 DNA to a linear duplex by S1 nuclease. 435 9

The induction of sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) with bleomycin (BLM), hydrogen peroxide (H2O2), short-wave ultraviolet (UV)-irradiation, and long-wave UV-irradiation was investigated in V79 cells with BrdUrd-substituted DNA. The application of a Neurospora endonuclease (NE) which specifically cleaves single-stranded DNA after these treatments showed that DNA single-strand breaks (SSBs) are induced by these agents. The SSBs are converted to double-strand breaks (DSBs) by NE and become visible as CAs on metaphase chromosomes. H2O2 and both types of UV-irradiation also led to an induction of CAs and SCEs, whereas BLM only induced aberrations. Cysteine (Cys) reduced the frequency of the induced SSB-dependent CAs in all treatments, but had no influence on the SCE frequencies after BLM and H2O2 treatment and had only a slight effect on the UV-induced SCEs. The results confirm the opinion that directly induced SSBs can contribute to the induction of CAs in cells with BrdUrd-substituted DNA, but that these SSBs are not efficiently converted to SCEs. The more recent conceptions regarding the mechanism of SCE are in accordance with these findings and the conclusions derived therefrom.
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PMID:The contribution of DNA single-strand breaks to the formation of chromosome aberrations and SCEs. 608 60


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