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)

High molecular weight DNA from pleroceroid larvae of the tapeworm Spirometra mansonoides was purified from isolated nuclei by conventional techniques. The DNA so isolated has a melting temperature (Tm) of 87 degrees C and a guanine plus cytosine (G/C) content of 44%. 5-Methyl cytosine could not be detected in plerocercoid DNA by HPLC analysis of DNA hydrolysates, by radiolabeling 5'-termini of MspI digests with polynucleotide kinase, or by comparing restriction patterns generated by MspI and HpaII. Renaturation kinetics demonstrated that the genome of S. mansonoides contains repetitive as well as single copy sequences and has a genome size estimated at approx. 1.6 X 10(9) bp. Hybridization was carried out between plerocercoid DNA and cDNAs for human beta-actin, alpha-tubulin and growth hormone (hGH). Rationale for this analysis was based on known homologies among actin and tubulin genes in numerous species and on apparent similarities between hGH and a plerocercoid growth factor that may be reflected in similar DNA sequence. Scanning densitometry of dot blots demonstrated that the hGH probe annealed to the same extent at low stringency (1 M NaCl, 55 degrees C) to DNA from plerocercoids, rat liver and chicken erythrocytes; but this interaction was less than to DNA from human lymphocytes, calf thymus and mouse skin. Similar results were obtained when restriction endonuclease digests of these DNAs were analyzed by Southern transfer. Little or no hybridization of the growth hormone probe to plerocercoid DNA was evident at higher stringency (1 M NaCl, 65 degrees C). In contrast, human tubulin and actin probes showed extensive hybridization to pleroceroid restriction fragments under the high stringency conditions.
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PMID:Molecular characterization of the Spirometra mansonoides genome: renaturation kinetics, methylation, and hybridization to human cDNA probes. 236 5

We have developed a strategy by which the nature of phosphodiester bond breaks produced by various DNA-repair endonucleases and also other nucleases, can be characterized. A purified apurinic/apyrimidinic (AP) specific endonuclease from a permanently established mouse plasmacytoma cell-line (MPC-11) has been examined with respect to the exact incision site generated at the baseless site. By the aid of enzymatic treatment with calf intestinal phosphatase, the 3'-phosphatase activity of T4-polynucleotide kinase, chemical modification with piperidine in addition to the Maxam-Gilbert sequencing procedure, followed by separation on a DNA-sequencing gel, the nature of the cleaved phosphodiester bond, both 3' and 5' to the cleavage site, has been established. The AP-specific endonuclease investigated was classified as a class II AP-endonuclease according to the four possible classes of AP-endonuclease with respect to the termini produced. By use of this technique each single damaged and cleaved site can be investigated separately.
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PMID:Analysis of cleavage products of DNA repair enzymes and other nucleases. Characterization of an apurinic/apyrimidinic specific endonuclease from mouse plasmacytoma cells. 245 3

The properties of a DNA-repair endonuclease isolated from mouse plasmacytoma cells have been further studied. It acted on ultraviolet-light-irradiated supercoiled DNA, and the requirement for a supercoiled substrate was absolute at ultraviolet light doses below 1.5 kJ m-2. At higher doses relaxed DNA could also serve as a substrate, but the activity on this DNA was due mostly to hydrolysis of ultraviolet-light-induced apurinic/apyrimidinic (AP) sites by the AP-endonuclease activity associated with the enzyme. The latter enzyme activity did not require a supercoiled form of the DNA. The enzyme also introduced nicks in unirradiated d(A-T)n. The nicked ultraviolet-light-irradiated DNA served as a substrate for DNA polymerase I, showing that the nicks contained free 3'-OH ends. Treatment of the nicked ultraviolet-light-irradiated DNA with bacterial alkaline phosphatase followed by T4 polynucleotide kinase, resulted in the phosphorylation of the 5' ends of the nicks, indicating that the nicks possessed a 5'-phosphate group; 5'- and 3'-mononucleotide analyses of the labelled DNA suggested that the enzyme introduced breaks primarily between G and T residues. The enzyme did not act on any specific region on the supercoiled DNA molecule; it produced random nicks in ultraviolet-light-modified phi X 174 replicative form I DNA. Antibodies raised against ultraviolet-light-irradiated DNA inhibited the activity. DNA adducts such as N-acetoxy-2-acetylaminofluorene and psoralen were not recognized by the enzyme. It is suggested that the enzyme has a specificity directed toward helical distortions.
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PMID:Properties of a DNA repair endonuclease from mouse plasmacytoma cells. 258 76

Yeast tRNA ligase, from Saccharomyces cerevisiae, is one of the protein components that is involved in the splicing reaction of intron-containing yeast precursor tRNAs. It is an unusual protein because it has three distinct catalytic activities. It functions as a polynucleotide kinase, as a cyclic phosphodiesterase, and as an RNA ligase. We have studied the binding interactions between ligase and precursor tRNAs containing two photoreactive uridine analogues, 4-thiouridine and 5-bromouridine. When irradiated with long ultraviolet light, RNA containing these analogues can form specific covalent bonds with associated proteins. In this paper, we show that 4-thiouridine triphosphate and 5-bromouridine triphosphate were readily incorporated into a precursor tRNA(Phe) that was synthesized, in vitro, with bacteriophage T7 RNA polymerase. The analogue-containing precursor tRNAs were authentic substrates for the two splicing enzymes that were tested (endonuclease and ligase), and they formed specific covalent bonds with ligase when they were irradiated with long-wavelength ultraviolet light. We have determined the position of three major cross-links and one minor cross-link on precursor tRNA(Phe) that were located within the intron and near the 3' splice site. On the basis of these data, we present a model for the in vivo splicing reaction of yeast precursor tRNAs.
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PMID:Binding interactions between yeast tRNA ligase and a precursor transfer ribonucleic acid containing two photoreactive uridine analogues. 285 71

DNA-dependent ATPase IV has been purified to near homogeneity from the Novikoff rat hepatoma. The enzyme is devoid of DNA polymerase, RNA polymerase, exonuclease, endonuclease, phosphomonoesterase, 3'- or 5'-phosphodiesterase, polynucleotide kinase, protein kinase, topoisomerase, helicase or DNA reannealing activities at a detection level of 10(-5) to 10(-7) relative to the ATPase activity. The enzyme is a monomer of Mr 110,000, has a sedimentation coefficient of 5.9 S, a Stokes radius of 40 A and a frictional coefficient of 1.32. In the presence of Mg2+ ion and a polynucleotide effector, ATPase IV hydrolyzes either ATP or dATP to the nucleoside diphosphate plus Pi. Other ribo- or deoxyribonucleoside triphosphates are not substrates. ATPase IV utilizes double-stranded DNA and single-stranded DNA as effector; however, it does not utilize poly(dT). The Km for dsDNA or ssDNA is 2.2 microM (nucleotide). A variety of ATP analogues were found to be competitive inhibitors of ATPase IV.
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PMID:Purification and enzymological characterization of DNA-dependent ATPase IV from the Novikoff hepatoma. 296 5

The mutagenic and carcinogenic substance benzo[a]pyrene reacts with DNA following activation to its corresponding 7,8-diol 9,10-epoxide (BPDE), and the major DNA adduct (BP-N2-Gua) is formed when the C(10)-position of BPDE reacts with the N2-position of guanine. It is unknown if this adduct is a premutagenic lesion in vivo. Herein, the construction and characterization of an M13mp19-based, E. coli vector that contains BP-N2-Gua located in the unique PstI restriction endonuclease recognition site at nucleotide position 6249 in the (-)-strand is described (designated, BP-N2-Gua-M13mp19). First, the oligonucleotide 5'-TGCA-3' was reacted with BPDE and a product (5'-T(BP-N2)GCA-3') was isolated by HPLC that, when enzymatically digested to deoxynucleosides, yielded an adduct that comigrated on HPLC with an authentic BP-N2-Gua deoxynucleoside standard. Second, the 5'-hydroxyl group of 5'-T-(BP-N2)GCA-3' was phosphorylated with ATP and T4 polynucleotide kinase, and the product (5'-pT(BP-N2)GCA-3') was purified by HPLC. This product is stable when heated at 80 degrees C at both neutral and alkaline pH. Third, M13mp19 was manipulated such that the sequence 5'-pTGCA-3' was selectively removed from the (-)-strand in its unique PstI recognition site, and 5'-pT(BP-N2)GCA-3' was ligated into this gap with T4 DNA ligase and ATP. The product of this reaction (BP-N2-Gua-M13mp19) was shown to be insensitive to cleavage by PstI, which suggests that a modification is located in the PstI recognition site. The most likely modification is the adduct BP-N2-Gua.
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PMID:Construction of an Escherichia coli vector containing the major DNA adduct of activated benzo[a]pyrene at a defined site. 297 26

The DNA intermediates and final products formed by the Type I restriction endonuclease, EcoB, were further characterized. DNA cleaved on only one strand (hemi-restricted DNA) contains gaps of approximately 70-100 nucleotides, while the fully restricted products contain 3'-single-stranded tails averaging approximately 70-100 nucleotides for each strand cleaved. The gaps and tails are formed with the release of an equal number of nucleotides as small oligonucleotides that are soluble in acid. After purification, neither the hemi-restricted nor the fully restricted DNAs are cleaved again by EcoB. There is no apparent specificity for which strand of a duplex is initially cleaved by EcoB, nor is there specificity with respect to the composition of the 3'-terminal nucleotide formed on the DNA or the 3'- or 5'-terminal nucleotides of the acid-soluble oligonucleotides released during DNA cleavage. The structure formed at the 5' terminus of the DNA product which blocks phosphorylation by T4 polynucleotide kinase remains unknown, but its removal with phage lambda exonuclease allows at least some reutilization of recognition sites by EcoB as well as phosphorylation of the newly formed 5' termini. To explain the complex mechanism of this enzyme, it is suggested that the unidentified 5'-tails prevent wasteful rerestriction from occurring, whereas the 3'-single-stranded tails create DNA which, when nonhomologous to chromosomal DNA, cannot be rescued because such tails are not substrate for DNA polymerases. However, when homologous chromosomal DNA exists, the randomly cleaved large fragments with these tails can easily be assimilated by recA-mediated genetic recombination, thus stimulating DNA exchange between related organisms.
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PMID:The DNA restriction endonuclease of Escherichia coli B. II. Further studies of the structure of DNA intermediates and products. 298 10

Three single-stranded oligonucleotide probes, 22 bases long, homologous to unique regions of herpes simplex virus (HSV) types 1 (HSV-1) and 2 (HSV-2) and a region common to both were chemically synthesized with use of a modified phosphochloridite protocol. For hybridization experiments each probe was labeled with use of polynucleotide kinase and [gamma-32P] ATP to a specific activity of approximately 2 X 10(9) cpm/micrograms. Two hundred one clinical isolates of HSV (96 HSV-1 and 105 HSV-2) collected from vesicles in the mucocutaneous junction of the mouth or from the genital area were analyzed. There was a 99% (199 of 201) agreement between hybridization and monoclonal antibody typing; the two discrepant isolates of HSV-2 that were negative by monoclonal antibody typing were confirmed as HSV-2 by restriction endonuclease analysis. The probes detected between 10(4) and 10(5) HSV infectious units and from 150 to 600 HSV-infected Vero cells. No binding was detected between any of the three probes and isolates of cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus.
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PMID:Typing of herpes simplex virus with synthetic DNA probes. 300 36

The duplex genome of Escherichia coli virus M13mp10 was modified at a unique site to contain N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG8-ABP), the major carcinogen-DNA adduct of the human bladder carcinogen 4-aminobiphenyl. A tetradeoxynucleotide containing a single dG8-ABP residue was synthesized by reacting 5'-d(TpGpCpA)-3' with N-acetoxy-N-(trifluoracetyl)-4-aminobiphenyl, followed by high-performance liquid chromatography purification of the principal reaction product 5'-d(TpG8-ABPpCpA)-3' (yield 15-30%). Characterization by fast atom bombardment mass spectrometry confirmed the structure as an intact 4-aminobiphenyl-modified tetranucleotide, while 1H nuclear magnetic resonance spectroscopy established the site of substitution and the existence of ring stacking between the carcinogen residue and DNA bases. Both 5'-d(TpG8-ABPpCpA)-3' and 5'-d(TpGpCpA)-3' were 5'-phosphorylated by use of bacteriophage T4 polynucleotide kinase and were incorporated into a four-base gap uniquely positioned in the center of the recognition site for the restriction endonuclease PstI, in an otherwise duplex genome of M13mp10. In the case of the adducted tetranucleotide, dG8-ABP was located in the minus strand at genome position 6270. Experiments in which the tetranucleotides were 5' end labeled with [32P]phosphate revealed the following: the adducted oligomer, when incubated in a 1000-fold molar excess in the presence of T4 DNA ligase and ATP, was found to be incorporated into the gapped DNA molecules with an efficiency of approximately 30%, as compared to the unadducted d(pTpGpCpA), which was incorporated with 60% ligation efficiency; radioactivity from the 5' end of each tetranucleotide was physically mapped to a restriction fragment that contained the PstI site and represented 0.2% of the genome; the presence of the lesion within the PstI recognition site inhibited the ability of PstI to cleave the genome at this site; in genomes in which ligation occurred, T4 DNA ligase was capable of covalently joining both modified and unmodified tetranucleotides to the gapped structures on both the 5' and the 3' ends with at least 90% efficiency. Evidence also is presented showing that the dG8-ABP-modified tetranucleotide was stable to the conditions of the recombinant DNA techniques used to insert it into the viral genome.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A probe for the mutagenic activity of the carcinogen 4-aminobiphenyl: synthesis and characterization of an M13mp10 genome containing the major carcinogen-DNA adduct at a unique site. 330 Jul 70

We have determined the levels of DNA polymerase, DNA ligase, a DNase acting on single-stranded DNA, an endonuclease making single-strand breaks in double - stranded DNA and polynucleotide kinase in fibroblasts obtained from nine normal persons and from nine patients with Xeroderma Pigmentosum; the pathological lines belong to the different described clinical forms and to the three different complementation groups described so far. All the enzymes are present in the normal lines and in the Xeroderma lines. The levels are quite variable, but the values obtained in the pathological lines lie within the ones observed in the normal population.
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PMID:Levels of some enzymes acting on DNA in xeroderma pigmentosum. 441 76


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