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)

In this paper, finite-difference solutions to the nonlinear Poisson-Boltzmann (NLPB) equation are used to calculate the salt dependent contribution to the electrostatic DNA binding free energy for both the lambda cI repressor and the EcoRI endonuclease. For the protein-DNA systems studied, the NLPB method describes nonspecific univalent salt dependent effects on the binding free energy which are in excellent agreement with experimental results. In these systems, the contribution of the ion atmosphere to the binding free energy substantially destabilizes the protein-DNA complexes. The magnitude of this effect involves a macromolecular structure dependent redistribution of both cations and anions around the protein and the DNA which is dominated by long range electrostatic interactions. We find that the free energy associated with global ion redistribution upon binding is more important than changes associated with local protein-DNA interactions (ion-pairs) in determining salt effects. The NLPB model reveals how long range salt effects can play a significant role in the relative stability of protein-DNA complexes with different structures.
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PMID:Salt effects on protein-DNA interactions. The lambda cI repressor and EcoRI endonuclease. 815 53

The reaction requirements and kinetic properties of the in vitro endonuclease activity of the bacteriophage lambda terminase and its large subunit, gene product (gp) A, have been analyzed. Optimal cleavage reaction activity for both proteins requires Mg2+, a pH between 8.5 and 9.0, and is enhanced by ATP or ATP analogs. Under these conditions both terminase and gpA generate aberrant nicks in and around cosN. Optimal nicking specificity of terminase is observed under conditions of 50-100 mM salt, 5 mM spermidine, 1.5 mM ATP, and a pH between 7.0 and 7.5. Specific activity of terminase is greatly reduced under these conditions, and gpA is completely inactive at all protein concentrations tested. Under optimal reaction conditions, gpA endonuclease activity differs from that of the holoenzyme in that it can only be detected at high concentrations, is strongly protein concentration-dependent, and can not be stimulated by the Escherichia coli protein integration host factor. Addition of purified gpNu1 partially, but not completely, minimized these differences, suggesting that the role of gpNu1 in the holoenzyme is to modulate the basal endonuclease of gpA.
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PMID:The in vitro endonuclease activity of gene product A, the large subunit of the bacteriophage lambda terminase, and its relationship to the endonuclease activity of the holoenzyme. 817 93

A restriction endonuclease, MgoI, an isoschizomer of Sau3AI, was purified from Mycobacterium gordonae TRC1318. As compared to Sau3AI, the yield of MgoI was seven-eightfold higher, the enzyme was four times more stable at 37 degrees C, and in addition, had optimal activity over a much broader range of salt concentrations.
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PMID:Purification and characterization of restriction endonuclease MgoI from Mycobacterium gordonae. 837 May 36

The effect of formalin fixation on DNA and on polymerase chain reaction (PCR) amplification was investigated. Lambda phage DNA fixed in buffered formalin showed incomplete digestion on restriction endonuclease treatment. The resistance to restriction digestion was dependent on the temperature of fixation, but not affected by salt concentration of the fixative. Lambda phage DNA fixed in unbuffered formalin showed poor PCR amplification due to degradation of DNA during fixation. Lambda phage DNA fixed in buffered formalin evaded degradation and suited for template of amplification. Feasibility of formalin-fixed tissues as sources for PCR amplification was also investigated with primers producing 128 bp fragment of c-Ki-ras exon 2. Although DNA from tissues fixed for 3 months showed amplification, there was no amplification from tissues kept in unbuffered formalin for longer than 6 months.
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PMID:The effect of formalin fixation on restriction endonuclease digestion of DNA and PCR amplification. 837 78

DNA of yeast artificial chromosomes (YACs) was prepared for microinjection by separation from most of the natural yeast chromosomes on a pulsed-field gel, treatment with agarase, and centrifugation. A salt concentration of 100 mM NaCl was necessary to protect the DNA from shear during these procedures. Injection of a 590-kb YAC, yGART2, into Chinese hamster ovary cells gave rise to cells expressing the 40-kb human GART gene carried on the YAC. Nine of 12 cell lines analyzed contained an intact stretch of at least 110 kb of YAC DNA surrounding the GART gene, and one cell line contained at least 480 kb, but not the entire 590 kb, intact. Mouse L A-9 cells were similarly injected with DNA of a 230-kb YAC containing the human beta-globin gene cluster and a mammalian selectable marker. Seven of 10 of the resulting cell lines contained both YAC vector arms plus the intact 140-kb SfiI fragment spanning the beta-globin gene. Three cell lines were analyzed by RecA-assisted restriction endonuclease (RARE) cleavage and found to contain the entire intact 210-kb YAC insert. Introduction of similarly prepared DNA into mammalian cells by lipofection gave rise to cell lines with multiple YAC fragments that were generally shorter than the YAC fragments found in microinjected cell lines. The results show that microinjection of gel-purified YAC DNA into mammalian cells is an efficient method of transferring DNA fragments several hundred kilobase pairs in size into mammalian cells.
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PMID:Microinjection of intact 200- to 500-kb fragments of YAC DNA into mammalian cells. 838 48

The effect of formalin fixation on DNA and extraction of DNA from fixed tissues was investigated to retrieve archival tissue samples stored in pathology departments for molecular biological studies. Aldehyde fixatives resulted in degradation of DNA at room temperature but not at 4 degrees C. The degradation also occurred in formalin when the pH or the salt concentration was low, or the formic acid level was high. Restriction endonuclease digestion of fixed DNA was incomplete after formalin fixation and this was also temperature-dependent. Thus, relatively intact DNA was obtained from the tissues fixed in buffered formalin at 4 degrees C or fixed with microwave irradiation. The use of modified tissue-lysing buffer containing 4M urea allowed extraction of high-molecular-weight DNA suitable for Southern blot analysis from fixed and embedded tissues. In conclusion, fixation with buffered formalin at 4 degrees C permitted extraction of DNA of sufficient quality for Southern blot analysis.
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PMID:The effect of formalin fixation on DNA and the extraction of high-molecular-weight DNA from fixed and embedded tissues. 839 Jun 45

Actinobacillus actinomycetemcomitans, a periodontopathic gram-negative bacterium, produces a leukotoxin that is a member of the RTX cytotoxin family. Although genes may function in toxin secretion, the leukotoxin is not secreted extracellularly but remains associated with the bacterial cell surface. We report here that this toxin-cell surface association is mediated by nucleic acids and directly demonstrate that the extracellular secretion of toxin occurs in growing cultures with increased ionic strength of medium. All examinations were performed with freshly harvested A. actinomycetemcomitans 301-b from anaerobic fructose-limited chemostat cultures. The occurrence of cell surface-localized DNA was shown by directly digesting whole cells with the restriction endonuclease EcoRI or HindIII, which yielded many DNA fragments. The cell surface DNA constituted about 20% of the total cellular DNA. The leukotoxin was released from the whole cells by digestion with DNase I as well as restriction endonucleases. Because the leukotoxin binds ionically to DNA, it is dependent on the ionic strength of buffers or media. Accordingly, the toxin was released from cells suspended in saline at pH 7.5 in the presence of increasing amounts of MgCl2 (0 to 10 mM) or NaCl (0 to 50 mM). Moreover, a considerable quantity of leukotoxin was detected in the culture supernatant of fructose-limited chemostat cultures when sodium succinate solution was pumped into the steady state as an additional salt (30 and then 50 mM). This toxin-DNA association was also found in well-characterized strains including not only the leukotoxin-producing ATCC 29522 but also the toxin production-variable ATCC 29523 and the non-leukotoxin-producing ATCC 33384 when these strains were grown in the chemostat culture.
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PMID:Association of Actinobacillus actinomycetemcomitans leukotoxin with nucleic acids on the bacterial cell surface. 840 88

Intron-encoded endonucleases are distinguished by their ability to catalyze the cleavage of double-stranded DNA with high specificity. I-PpoI endonuclease, an intron-encoded endonuclease from the slime mold Physarum polycephalum, is a small enzyme (2 x 20 kDa) that catalyzes the cleavage of a large asymmetric DNA sequence (15 base pairs). Here, the interactions of I-PpoI with its substrate were examined during both binding (in the absence of Mg2+) and catalysis (in the presence of Mg2+). Using circular permutation assays, I-PpoI was shown to bend its substrate by 38 +/- 4 degrees upon binding. Two independent methods, gel mobility shift assays and fluorescence polarization assays, revealed that I-PpoI binds tightly to its substrate. Values of Kd range from 3.3 to 112 nM, increasing with increasing NaCl concentration. Similar salt effects on the values of Km were observed during steady-state catalysis. At low salt concentrations, the value of kcat/Km for the cleavage of an oligonucleotide duplex approaches 10(8) M-1 s-1. Although other divalent cations can replace Mg2+, catalysis by I-PpoI is most efficient in the presence of an oxophilic metal ion that prefers an octahedral geometry: Mg2+ > Mn2+ > Ca2+ = Co2+ > Ni2+ > Zn2+. Together, these results provide the first chemical insight into substrate binding and turnover by an intron-encoded endonuclease.
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PMID:Substrate binding and turnover by the highly specific I-PpoI endonuclease. 854 43

A novel method was used to characterize the long range susceptibility of Schizosaccharomyces pombe chromosomal DNA to endogenous endonuclease cleavage. Analyses of pulsed field gel experiments revealed two periodicities in the distribution of endogenous endonuclease hypersensitive sites. Endonuclease cleavage sites occurred, roughly at 30-509 kilobase pairs (kb) intervals under physiological conditions (25 mM KCl). At higher salt concentrations (250 mM KCl or 0.2 M and 0.9 M NaCl), endonuclease hypersensitive sites occurred at 200-300 kb intervals. Endonuclease hypersensitive sites in different chromosomal regions were monitored during different stages of the cell cycle. DNA sequencing around the endonuclease hypersensitive sites revealed the presence of clusters of A+T-rich motifs, autonomously replicating sequences (ARSs) in sequences (a characteristic of the scaffold-associated regions (SARs) and the presence of a CTG trinucleotide at most sites.
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PMID:Endogenous endonuclease hypersensitive sites in Schizosaccharomyces pombe chromosomes. 857 99

To evaluate the relative importance of alternating d(CG) sequence length, DNA supercoiling, and salt in left-handed Z-DNA formation, plasmids containing short d(CG)n sequences (n = 3-17) with the capability of replicating in either Escherichia coli or the halophilic archaeum Halobacterium halobium were constructed. Z-DNA conformation in the d(CG)n sequences was assayed by (i) a band shift assay using the Z-DNA-specific Z22 monoclonal antibody (ZIBS assay); (ii) an S1 nuclease cleavage-primer extension assay to map B-Z junctions; and (iii) a BssHII restriction inhibition assay. Using the ZIBS assay on plasmids purified from E. coli, the transition from B-DNA to Z-DNA occurred from d(CG)4, to d(CG)5, with 20% of d(CG)4, and 90% of d(CG)5 in Z-DNA conformation. These findings were consistent with the results of S1 nuclease cleavage observed at B-Z junctions flanking d(CG)4 and d(CG)5 sequences. Resistance to BssHII restriction endonuclease digestion was observed only in supercoiled plasmids containing d(CG)8 or longer sequences, indicating that shorter d(CG)n sequences are in dynamic equilibrium between B- and Z-DNA conformations. When a plasmid containing d(CG)4, was isolated from a topA mutant of E. coli, it contained 25% greater linking deficiency and 40% greater Z-DNA conformation in the alternating d(CG) region. In plasmids purified from H. halobium, which showed 30% greater linking deficiency than from E. coli, 20-40% greater Z-DNA formation was found in d(CG)4-6 sequences. Surprisingly, no significant difference in Z-DNA formation could be detected in d(CG)3-17 sequences in plasmids from either E. coli or H. halobium in the NaCl concentration range of 0.1-4 M.
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PMID:Analysis of left-handed Z-DNA formation in short d(CG)n sequences in Escherichia coli and Halobacterium halobium plasmids. Stabilization by increasing repeat length and DNA supercoiling but not salinity. 862 98


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