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 catalytic properties of the HhaII restriction endonuclease were studied using plasmid pSK11 DNA containing a single 5'-G-A-N-T-C HhaII cleavage site as substrate. Reactions were followed by two methods: 1) gel electrophoretic analysis of nicked circular and linear DNA products, or 2) release of 32P-labeled inorganic phosphate from specifically labeled HhaII sites in a reaction coupled with bacterial alkaline phosphatase. The enzyme is optimally active at 37 degrees C in 10 mM Tris-HCl (pH 9.1) and 4-10 mM MgCl2 without added NaCl. Activity is stabilized by the presence of 2-mercaptoethanol and 0.2% Triton X-100 or 50 microgram/ml bovine serum albumin. At enzyme concentrations below 10 nM and using pSK11 as substrate, initial kinetic rates were dependent on the order of mixing of reactants. A lag of 3-4 min was observed if enzyme or substrate was added last. Preincubation of substrate and enzyme followed by initiation of the reaction with MgCl2 or preincubation of the enzyme with nonspecific DNA followed by initiation with substrate eliminated or reduced the lag, respectively, and speeded up the reactions. Under a wide range of reaction conditions, nicked pSK11 DNA accumulated early, while linear molecules appeared later, suggesting that HhaII cleaves one strand at a time in separate binding events. The apparent Km for covalently closed pSK11 DNA molecules was approximately 17 nM, and the turnover number for the conversion of covalent to nicked sites was 1.1 single strand scissions/min. Pre-steady state kinetic analysis indicated that cleavage of the first phosphodiester bond in a site is first order with a rate constant of about 0.8 min-1, while cleavage of the second phosphodiester bond is first order with a rate constant of about 0.2 min-1.
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PMID:Catalytic properties of the HhaII restriction endonuclease. 299 12

A sensitive enzyme-linked immunosorbent capture assay using biotin and streptavidin (capture B/SA ELISA) was developed using type-specific monoclonal antibodies for typing of herpes simplex virus. Rabbit anti-herpes simplex virus immunoglobulin G was used as the capturing antibody, and biotin-linked type-1-specific mouse monoclonal antibody or rabbit type-1- or type-2-specific polyclonal antibody served as the detecting antibody. The captured antigen was detected by an ELISA with alkaline phosphatase-conjugated streptavidin, which reacted with biotin molecules on the detector antibody. The capture B/SA ELISA was compared with other methods for efficiency and reliability in typing. Results obtained by restriction endonuclease digestion of the radiolabeled viral genome were used to determine the type (1 or 2) of clinical isolates. These results were then used as a reference for determining the accuracy of the capture B/SA ELISA, as well as that of the immunofluorescence method, both of which are easily adaptable for use in the clinical laboratory. The three methods were in perfect agreement. It was determined that both the capture B/SA ELISA and the immunofluorescence method using monoclonal antibodies provided typing results with 100% specificity and 100% sensitivity and thus were accurate and reliable. However, the ELISA was the method of choice because of its simplicity, rapidity, and use of nonradioisotopic reagents.
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PMID:Typing of herpes simplex virus by capture biotin-streptavidin enzyme-linked immunosorbent assay and comparison with restriction endonuclease analysis and immunofluorescence method using monoclonal antibodies. 302 48

The potentiation of radiation damage, which can be accomplished by the inhibition of repair, is estimated from published studies of repair deficient mutants. Sensitization factors as high as 10 have been achieved. Because it has previously been suggested that the most probable lethal lesion is a DNA double strand break (DSB), it is not surprising that cells deficient in repairing this type of damage are the most radiosensitive. The structures of DNA DSBs and other Locally Multiply Damaged Sites (LMDS) (involving both single strand breaks (SSB) and base damage sites) are reviewed, together with the processes by which cells may attempt to repair these lesions. Repair processes occur in competition with damage fixation, again, mechanisms of damage fixation are predicted from studies in model systems. A strategy for inhibiting the repair processes is devised that consists of holding the first SSB constituent of the LMDS open by repairing in the presence of deoxynucleoside analogues, such as ara-C, so that there is a higher probability of the formation of a DSB upon cleavage of the second site (on the other strand) by hydrolysis of a labile bond or by endonuclease cleavage of a base damaged site. To achieve preferential sensitization of tumor vs. normal tissue it may be possible to take advantage of the deficiency in alkaline phosphatase in tumor vs. normal vasculature, that is, in analogy with treatment with WR-2721. The deoxynucleoside analogue would be delivered together with the phosphate ester (deoxynucleotide) of the correct deoxynucleoside, for example, ara-C, in the presence of deoxycytidine monophosphate (dCMP). Higher alkaline phosphatase levels in normal tissue capillaries would hydrolyse the dCMP to deoxycytidine, which competes effectively with ara-C in repair replication.
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PMID:Mechanisms of DNA repair and their potential modification for radiotherapy. 352 85

We describe a system to generate cDNA or genomic libraries from DNA segments that have blunt termini. Background and rearrangement levels are low, but efficiencies are high and the procedural times very short. T4 ligase in the presence of polyethylene glycol produces high Mr oligomers of vector and insert. These concatemers are reduced to vector-insert monomers at a high frequency by subsequent cleavage with a restriction endonuclease, which recognises the insert rarely, if at all, and the vector once. The monomers are recircularised under standard ligation conditions prior to transformation. Thus insertion conditions are optimised independently of those for recircularisation. All reading frames for expression libraries are generated by short BAL 31 cleavage followed by the blunt-end cloning procedure. Similarly, genomic expression libraries can be made by BAL 31 or mung-bean nuclease treatment after cleavage with DNase I is the presence of Mn2+. The technique is suitable for any DNA segment that is blunt-ended or can be made so. When the vector is treated with alkaline phosphatase, recombinants are generated at a frequency greater than 90% and have single inserts. Yields are 3-5 X 10(6) colony-forming units per micrograms of insert.
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PMID:Rapid and efficient method for cloning of blunt-ended DNA fragments. 359 40

phi X174 RFI DNA treated with bleomycin (BLM) under conditions permitting nicking does not serve as a template-primer for Escherichia coli DNA polymerase I. Purified exonuclease III from E. coli and extracts from wild-type E. coli strains are able to convert the BLM-treated DNA to suitable template-primer, but extracts from exonuclease III deficient strains are not. Brief digestion by exonuclease III is enough to create the template-primer, suggesting that the exonuclease III is converting the BLM-treated DNA by a modification of 3' termini. The exonucleolytic rather than the phosphatase activity of exonuclease III appears to be involved in the conversion. Comparative studies with micrococcal nuclease indicate that BLM-created nicks do not have a simple 3'-P structure. Bacterial alkaline phosphatase does not convert BLM-treated DNA to template-primer. The endonuclease VI activity associated with exonuclease III does not incise DNA treated with BLM under conditions not allowing nicking, in contrast to DNA with apurinic sites made by acid treatment, arguing that conversion does not require the endonuclease VI action on uncleaved sites.
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PMID:Synthesis by DNA polymerase I on bleomycin-treated deoxyribonucleic acid: a requirement for exonuclease III. 616 81

Neocarzinostatin (NCS) induces repair in a xeroderma pigmentosum lymphoblastoid line deficient in the ability to repair DNA damage induced with (acetoxyacetyl-amino)fluorene. Repair was demonstrated by the induction of repair synthesis and by the disappearance of NCS-induced single-strand breaks and/or alkaline-labile sites in DNA. Estimation of NCS-induced repair patch size, based on the density shift induced in DNA by extensive shear after incubation of treated cells in medium with bromodeoxyuridine or by calculation from the extent of restoration of DNA sedimentation profiles in alkaline sucrose gradients and the amount of repair synthesis measured by the BND cellulose method, indicated that only a few nucleotides were inserted per repaired region. NCS-treated bacteriophage T7 DNA requires incubation with alkaline phosphatase to make it a substrate for DNA polymerase I. NCS-reacted T7 DNA, even after phosphatase treatment, is not a substrate for a DNA polymerase alpha obtained from human lymphoma cells. NCS-treated T7 DNA did serve as a substrate for the DNA polymerase alpha when incubated with an apurinic/apyrimidinic (AP) endonuclease with associated 5'-3'-exonuclease activity. The results suggest that NCS-induced AP sites could be intermediates for the in vivo repair synthesis.
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PMID:Repair of neocarzinostatin-induced deoxyribonucleic acid damage in human lymphoblastoid cells: possible involvement of apurinic/apyrimidinic sites as intermediates. 625 59

Endonuclease V of bacteriophage T4 has been purified to physical homogeneity from T4D-infected Escherichia coli 1100. The enzyme, whose molecular weight is 16,000, possessed two distinct catalytic activities, a pyrimidine dimer-DNA glycosylase and an apurinic/apyrimidinic endonuclease. They acted on UV-irradiated poly(dA) . poly(dT) in a sequential manner; the glycosylase cleaved the N-glycosyl bond between the 5'-pyrimidine of a dimer and the corresponding sugar and then the endonuclease hydrolyzed a phosphodiester bond on the 3'-side of the apyrimidinic site. The 5'-termini thus generated were phosphorylated by T4 polynucleotide kinase only after they had been subjected to direct photoreversal and then treated with alkaline phosphatase. By using two phage mutants, uvs-5 and uvs-13, it was shown that occurrence of an amber mutation in the denV gene caused a simultaneous loss of the two activities. Suppression of the mutation of uvs-5 rendered both activities partially active. When the mutation of uvs-13 was suppressed, a mutant form of enzyme that possessed only a glycosylase activity was produced. This suggests that there are two distinct domains in a single enzyme, each of which corresponds to one of the activities.
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PMID:Purification and characterization of normal and mutant forms of T4 endonuclease V. 627 6

(2'-5')Oligoadenylate synthetase [(2'-5')A synthetase], which synthesizes a series of oligoadenylates ppp-(A2'p)n5'A [collectively referred to as (2'-5')A], has been described previously in rat liver cells, where its concentration varied with the growth status of this organ--i.e., it decreased during the early phase of rat liver regeneration after partial hepatectomy. Because double-stranded RNA, the only known activator of this enzyme, has been detected in rat liver nuclei, (2'-5')A synthesis could occur in this tissue in vivo. Analysis of rat liver cell extract after HPLC by the endonuclease-based radiobinding assay revealed several components with retention times similar to (2'-5')A trimer- and tetramer-like material. A further characterization of these compounds by their susceptibility to alkaline phosphatase and snake venom phosphodiesterase, their resistance to micrococcal nuclease, and their ability to activate an endonuclease indicated the natural occurrence of oligonucleotides indistinguishable from authentic (2'-5')A in rat liver cells. Using the combination of the radiobinding assay and a simplified (2'-5')A extraction procedure that does not involve HPLC, we further show that the early drop of (2'-5')A synthetase activity during rat liver regeneration was accompanied by a similar decrease in intracellular (2'-5')A concentration. The three characteristic phases of the (2'-5')A synthetase kinetics during the first 40 hr of liver regeneration were mimicked by the kinetics of the synthesis of the (2'-5')A oligonucleotides themselves: between 6 and 20 hr after hepatectomy, there was a sharp decrease in (2'-5')A concentration; between 20 and 24 hr, the concentration of (2'-5')A reached a minimum; at 36 hr or after the first wave of DNA synthesis (the major event of liver regeneration), the (2'-5')A concentration returned to normal. In this characterization of the (2'-5')A oligonucleotide family in a functional tissue of an animal that had not been previously treated with interferon or infected with virus, the data are compatible with a physiological role of the (2'-5')A system acting as an intracellular component of the regulatory mechanisms leading to cell proliferation or differentiation.
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PMID:(2'-5')Oligoadenylate in rat liver: modulation after partial hepatectomy. 630 30

Plasmids containing the chromosome region of Escherichia coli encoding phoM, whose product is a positive regulator of alkaline phosphatase expression, were isolated from the Clarke and Carbon plasmid bank. A 9.9-kilobase EcoRI fragment of plasmid pLC17-39 (subcloned into pBR322) was able to complement both phoM and thrB mutations. Restriction endonuclease analysis and in vitro mutagenesis of the hybird plasmids enabled the localization of the phoM gene locus to 3 kilobases of the cloned chromosomal fragment. The phoM gene product was identified, with maxicell techniques, as a protein with an approximate molecular weight of 55,000. A phoM-lacZ protein fusion was constructed by using a plasmid carrying the phoM gene and a derivative of phage lambda, lambda plac Mu2. Restriction endonuclease analysis of the plasmid carrying the fusion indicated that phoM is transcribed in a clockwise direction on the circular E. coli chromosome. Analysis of strains bearing the fusion on a multiple-copy plasmid or integrated at the lambda attachment site of the chromosome indicated that the synthesis of the phoM gene product was unaffected by phosphate limitation of growth. The expression of the phoM gene was studied in strains with mutations in genes encoding effectors of the pho regulon. A threefold increase in phoM expression was seen in a phoU strain in comparison with the wild-type strain.
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PMID:Identification of the phoM gene product and its regulation in Escherichia coli K-12. 633 29

The present investigation explored the genotypic heterogeneity of Porphyromonas gingivalis using restriction endonuclease analysis and ribotyping of 64 P. gingivalis isolates, recovered from the periodontal pockets of 3 beagle dogs, 2 of which were reared together. The isolates originated from both healthy and periodontal disease affected sites and thereby enabled the study of bacterial genotype with respect to (i) individual host, (ii) ecological niche (site within host) and (iii) level of periodontal health. Whole genomic DNA was extracted from each isolate and digested by the restriction endonuclease KpnI. Digestion fragments were separated by electrophoresis and transferred onto nylon membranes. The blots were hybridized with a digoxigenin-labeled 16S rDNA probe, and hybridization bands were detected using an anti-digoxigenin antibody conjugated with alkaline phosphatase and enhanced chemiluminescence. Fourteen genomic fingerprints and 13 ribotypes were observed among the 64 isolates. As many as 8 distinct fingerprints were detected within a single host and up to 4 fingerprints within a single periodontal pocket. The dogs reared together shared 2 common clonal types but also exhibited clonal types unique to each dog. No clear association between clonal type and periodontal health status could be made. The results revealed an extensive intra-host genotypic heterogeneity of P. gingivalis strains in the beagle dog and indicated that ribotyping was a sensitive method for differentiating clonal types within species.
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PMID:Host-related genotypic heterogeneity of Porphyromonas gingivalis strains in the beagle dog. 747 65


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