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)

A modification of Hong's systematic DNA sequencing strategy is described. The original procedure has been simplified and transfectant yield increased. After DNase I limited cleavage in the presence of Mn2+, the single-cut linear DNA does not have to be separated from supercoiled or open circular DNA on an agarose gel. After ligation, the DNA is digested with a second restriction endonuclease for which a unique cleavage site resides between the insert and the first restriction endonuclease cutting site. The original intact DNA is linearized whereas the deleted subclone is not. The background is decreased to an undetectable level. This DNA sequencing strategy was tested on a 1.4-kb DNA fragment containing the araC regulatory gene from Erwinia carotovora. A set of subclones sufficient to sequence the fragment on both strands was produced in 2 days and the yield was at least 60-fold higher than in the original protocol.
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PMID:An improved DNA sequencing strategy. 299 13

The endogenous endonuclease activity of chromatin in isolated rat liver nuclei in the presence of Mn2+, Mg2+ and Ca2+ + Mg2+ was studied. The existence of a Mn2+-dependent endonuclease activity not coupled with the Ca2+, Mg2+-dependent endonuclease was demonstrated, which was weaker than the former one in isolated cell nuclei but higher than in the preparation of Ca2+, Mg2+-dependent nuclease obtained by gel filtration through Toyopearl HW 60F. The Mn2+-dependent splitting of chromatin predominantly occurs at linker DNA of distal parts of chromatin loops. A split-off of purified DNA was more universal than in the presence of Ca2+, Mg2+-dependent endonuclease; the hydrolysis rate of native and denaturated DNA appeared to be the same.
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PMID:[Mn2+-dependent endonuclease activity of chromatin]. 301 67

To study Moloney murine leukemia virus (M-MulV) proteins associated with the integration of proviral DNA into the host chromosome, we isolated endonuclease activities from purified virion preparations of the wild type and two of its replication mutants. A major endonuclease activity was identified in virions of M-MuLV; the enzyme catalyzed nicks in double-stranded DNA in the presence of either Mn2+ or Mg2+ and was stimulated by ATP. The endonuclease nicked DNA adjacent to all four nucleotides with some preference for G and C. The same enzyme, and in comparable amounts, was isolated from two virus replication mutants: dl2905, deficient in the processing of Pr65gag and Pr200gag-pol, and dl50401, deficient for the virus integration function. In the process of these experiments, the residual reverse transcriptase in mutant dl2905 was shown to be the mature size, implying that the uncleaved precursor lacks enzymatic activity. It appears that the major endonuclease activity found in virions of M-MuLV is not encoded by either the gag or pol genes.
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PMID:Characterization of endonuclease activities in Moloney murine leukemia virus and its replication-defective mutants. 303 6

The distribution of different types of DNase in rat liver nuclei was determined after a purification procedure involving ion exchange chromatography and gel filtration. As major enzymes Ca2+, Mg2+-dependent endonuclease, Mn2+-dependent endonuclease and an acid endonuclease were identified, sharing 60, 20 and 10% of the total activity, respectively. Mn2+-dependent endonuclease is a novel enzyme with a molecular mass of 30 +/- 5 kilodaltons. The synergistic effect of Ca2+ and Mg2+ ions for the Mn2+-dependent enzyme was lower by an order of magnitude than that of the Ca2+ and Mg2+-dependent endonuclease. The Ca2+ and Mg2+-dependent nuclease activity represents a heterogeneous population of enzymes. One of the cation dependent enzymes (Mr 25 +/- 5 kD) is stimulated by ATP the ATP optimum being 0.1 mM and the Mg2+ requirement 1 mM. The ATP-dependent endonuclease belongs to the minor endonucleases separable from the major ones.
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PMID:Bivalent cation and ATP requirements of endonucleases from rat liver nuclei. 313 3

A scheme for the isolation of Ca,Mg-dependent endonuclease from human spleen lymphocyte nuclei has been developed. The isolation procedure resulted in protein preparations (Mr = 57 kD) possessing an enzymatic activity and stable upon storage for over a period of one year. The enzyme is an endonuclease which predominantly cleaves double-stranded DNA by a mixed single- and double-hit mechanism with the formation of 5'-phosphate and 3'-OH terminal groups. Its maximal activation is induced by Ca2+ plus Mg2+. The enzyme is also active in the presence of Mn2+, Ca2+, Mg2+ and Zn2+ and is inhibited by Co2+. NaCl and KCl (0.15-0.2 M) and p-chloromercuribenzoate (1 mM) also inhibit the enzyme. ATP has no activating effect.
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PMID:[Isolation and analysis of Ca/Mg-dependent endonuclease from cell nuclei of human splenocytes]. 317 64

An endonuclease specific for cruciform junctions has been purified from yeast cells treated with a DNA-damaging agent. The activity was followed through five chromatographic steps by assaying for the linearization of supercoiled plasmid DNA, which extrudes cruciform structures in vitro. The sites of cleavage on pColIR215 were sequenced, and nicks were located to positions symmetrically opposed across the cruciform junction. The products of cleavage were unit length linear duplexes that contained terminal hairpin loops. In contrast to pColIR215, the cleavage patterns of pXG540 plasmid DNA were found to be complex, and cuts were found up to 40 bases from an (A-T)34 sequence that extrudes into a cruciform. Little or no activity could be detected on single-stranded DNA, linear duplex DNA, or nicked circular duplex DNA. The nuclease was insensitive to RNase but was inactivated by treatment with proteinase K. Mg2+ was required as cofactor and could not be replaced by Mn2+, Ca2+, Co2+, or Cu2+. The native molecular weight of the activity was approximately 200,000 as estimated by gel filtration.
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PMID:Purification and properties of a nuclease from Saccharomyces cerevisiae that cleaves DNA at cruciform junctions. 330 13

An endonuclease activity (termed endonuclease G) that selectively cleaves DNA at (dG)n X (dC)n tracts has been partially purified from immature chicken erythrocyte nuclei. Sites where n greater than or equal to 9 are cleaved in a manner that resembles types II and III restriction nucleases. The nicking rate of the G-strand is 4- to 10-fold higher than that of the C-strand depending on the length of the (dG)n X (dC)n tract and/or nucleotide composition of the flanking sequences. Endonuclease G hydrolyzes (dG)24 X (dC)24 of supercoiled DNA in a bimodal way every 9-11 nucleotides, the maxima in one strand corresponding to minima in the opposite, suggesting that it binds preferentially to one side of the double helix. The nuclease produces 5' phosphomonoester ends and its activity is dependent on Mg2+ or Mn2+. The wide distribution and high relative activity of endonuclease G in a variety of tissues and species argues for a general role of the enzyme. The striking correlation between genetic instability and poly(dG) X poly(dC) tracts in DNA suggests that these sequences and endonuclease G are involved in recombination processes.
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PMID:Endonuclease G: a (dG)n X (dC)n-specific DNase from higher eukaryotes. 358 64

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

The ability of Escherichia coli DNA polymerase I and T7 DNA polymerase to bypass bulky C-8 guanyl-2-aminofluorene adducts in DNA was studied by in vitro DNA synthesis reactions on a site-specific aminofluorene-modified M13mp9 template. This site-specifically modified DNA was prepared by ligating an oligonucleotide containing a single aminofluorene adduct into a gapped heteroduplex of M13mp9 DNA (Johnson, D. L., Reid, T. M., Lee, M.-S., King, C. M., and Romano, L. J. (1986) Biochemistry 25, 449-456). The resulting covalently closed duplex DNA molecule was then cleaved with a restriction endonuclease, denatured, and annealed to a primer on the 3' side of the adduct to form a template specifically designed to study bypass. In this system, any synthesis that was not blocked by the bulky aminofluorene adduct would proceed to the 5' terminus of the single-stranded template, while synthesis interrupted by the adduct would terminate at or near the adduct location. We have measured DNA synthesis on this template and find that the amount of radiolabeled nucleotide incorporated by either E. coli DNA polymerase I (large fragment) or T7 DNA polymerase was much greater than would be predicted if the aminofluorene adduct were an absolute block to DNA synthesis. Furthermore, the products of similar reactions electrophoresed on polyacrylamide gels showed conclusively that the majority of the DNA synthesized by either the T7 DNA polymerase or E. coli DNA polymerase I bypassed the aminofluorene lesion. Substitution of Mn2+ for Mg2+ as the divalent cation resulted in even higher levels of translesion synthesis.
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PMID:Evidence for in vitro translesion DNA synthesis past a site-specific aminofluorene adduct. 366 96

A potent endonuclease identified in a crude fraction of soluble proteins from bovine heart mitochondria has been purified 2500-fold and partially characterized. Physical studies of the enzyme indicate a Stokes radius of 30.3 A and a sedimentation coefficient, S20 degrees, w, of 4.1 yielding a native molecule weight of 59,000 and a frictional coefficient of 1.2. Analysis of extensively purified fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a major band at 29,000 Da accounting for 50% of the total protein and suggesting a dimeric subunit structure. The endonuclease maintains two distinct pH optima: pH 5.1-5.5 and 7-8. Both acid and neutral activities nick supercoiled M13 circular double-stranded replicative form I DNA and fragment single-stranded DNA templates to generate 5'-phosphoryl-3'-hydroxyl breaks. The endonuclease requires a divalent cation (preferring Mn2+ over Mg2+) and is sensitive to N-ethylmaleimide and moderate levels of salt. Analysis of the digestion products of double-stranded DNA after prolonged nuclease treatment yields a mixture of oligonucleotides, 13% of which are di- and trinucleotides. Despite the enzyme's ability to degrade DNA to oligonucleotides under some conditions, a strikingly nonrandom pattern of cleavage is observed when a restriction fragment composed of bovine D-loop DNA is used as a template. In this case, a strong preference for guanine tracts is seen.
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PMID:Purification and characterization of the potent endonuclease in extracts of bovine heart mitochondria. 381 85


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