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At least 793 records · Page 44Linked to original sources
PubMed1986

Sequence-dependent S1 nuclease hypersensitivity of a heteronomous DNA duplex.

Using cloned (dG-dA)n X (dC-dT)n DNA duplexes [GA)n) as models of homopurine-homopyrimidine S1-hypersensitive sites, we show that cleavage of the alternate (non-B, non-Z) DNA structure by S1 nuclease is length-dependent, in both supercoiled and linear forms, which are similar because of the identity of their nicking profiles. However, the length of flanking sequences, the presence of borders, and the DNA topology affect the equilibrium between the alternate structure and B-DNA. The B form of (GA)38 has a 10.4-base pair helical repeat, but the two phosphodiester backbones have different conformations (heteronomous DNA with a dinucleotide repeat unit). Extension experiments reveal that the alternate structure is also heteronomous, in agreement with the nicking patterns generated by S1 and mung bean nucleases and by venom phosphodiesterase. Sensitivity to the latter enzyme at pH 9.0 indicates that the alternate DNA does not appear only in the low pH of the S1 nuclease reaction. Moreover, Hoogsteen G-CH+ base-pairing does not seem to be a prerequisite for the appearance of sensitivity because S1 still recognizes the structure even when all Gs are methylated at N-7. This is consistent with the results of chemical probing of the structure using dimethyl sulfate and diethyl pyrocarbonate at various pH values, which show absence of protection at guanine N-7. However, diethyl pyrocarbonate treatment at low pH results in hyper-reactivity of A residues.

Base Sequence↗
PubMed1988

Immunogenic duplex nucleic acids are nuclease resistant.

Several derivatives of the synthetic duplex DNA poly(d(GC] were prepared. These analogs contained ribo- and phosphorothioate substitutions, namely, poly(r(GC], poly(rGdC), poly(d(sGC], poly(d(GsC], and poly(d(sGsC]. The nucleic acids were complexed to methylated BSA and injected into C57BL/6 mice. Poly(d(GC] was not immunogenic, whereas the analogs gave a strong response. Analysis of the sera revealed that antibody populations were present, which recognized A, B, and Z form nucleic acids. In particular, immunization with poly(d(sGC] and poly(d(GsC] produced antibodies that bound poly(d(GC] and poly(d(IC]. The nuclease sensitivity of these polymers also was investigated. Poly(d(GC] was rapidly degraded by pancreatic DNase I, whereas the phosphorothioate derivatives were resistant, and the ribo-substituted polymers were resistant to both RNases and DNase I. Similarly, the nucleases present in mouse serum digested calf thymus DNA and ribosomal rRNA while both types of analog persisted indefinitely. These results suggest the hypothesis that duplex DNAs are generally not immunogenic because they are rapidly degraded by serum nucleases. Therefore, they escape immune detection.

Animals↗
PubMed1987

Identification of a glucocorticoid-induced nuclease in thymocytes. A potential "lysis gene" product.

Glucocorticoids initiate a cytolytic process in lymphoid cells that is characteristic of programmed cell death. In vivo treatment of adrenalectomized rats with glucocorticoids results in the rapid degradation of the thymocyte genome at internucleosomal sites. This DNA degradation occurs prior to cell death, and considerable evidence indicates that this nucleolytic event is central to the initiation of lymphocytolysis. To further characterize this process, we have searched for the gene products in thymocytes which may be responsible for steroid-induced DNA degradation. Adrenalectomized rats were treated in vivo with dexamethasone or a vehicle control; nuclear thymocyte proteins were extracted with 0.6 M NaCl and analyzed for protein content or nuclease activity on sodium dodecyl sulfatepolyacrylamide gels containing calf thymus DNA. Glucocorticoid treatment resulted in the induction of two major protein families, a 30-32-kDa protein doublet and a series of 3-4 proteins of 12-19 kDa, both of which express prominent DNase activity. Induction of the lower molecular weight nucleases increased with time after steroid treatment and paralleled the time course of glucocorticoid-mediated DNA degradation. Nuclease induction was blocked by the glucocorticoid antagonist RU 486, indicating a steroid receptor-mediated process. When nuclei from glucocorticoid-resistant cells were incubated with nuclear extracts from glucocorticoid-treated rats, the DNA was cleaved at internucleosomal sites, whereas extracts from vehicle-treated animals were virtually inactive. Based on these findings we propose that glucocorticoids, acting via a receptor-mediated pathway, induce a nucleolytic "lysis gene" product(s) responsible for lymphocytolysis.

DNA↗
PubMed1979

[Nuclease from Aspergillus oryzae, specific for single-stranded regions of nucleic acids].

A single-stranded specific nuclease has been purified from amyloryzine obtained from the mould fungi Aspergillus cryzae. The nuclease under study resembles the enzymes described in the literature in its ability to hydrolyze single-stranded nucleic acids. However, the enzyme essentially differs from previously known nucleases in some catalytic properties, particularly in its ability for degradation of poly A. It has been shown that the enzyme also hydrolyzes the synthetic dinucleotide pTpT to mononucleoside phosphates.

Aspergillus↗
PubMedDate not supplied

[Isolation and some properties of homogenous nuclease S1 from alpha-amyloryzin].

A purification method for isolating homogeneous single-strand specific nuclease S1 from alpha-amyloryzin has been developed. The yield was about 16% and purification factor--9000. Nuclease S1 thus obtained was proved to be free of contaminations of any others nucleolytic enzymes. It is shown for the first time that ribo- and deoxy-dinucleosidemonophosphates are hydrolyzed by nuclease S1 to form 5'-nucleotides with pH optimum for ApA equal to 4.6.

Aspergillus oryzae↗
PubMed1981

The effect of histone hyperacetylation on the nuclease sensitivity and the solubility of chromatin.

We have examined the effects of histone hyperacetylation upon nuclease digestion of nuclei and subsequent fractionation of chromosomal material in the presence of MgCl2. DNase I shows a maximum sensitivity towards hyperacetylated nuclei at somewhat elevated ionic strengths (150-200 mM NaCl), whereas micrococcal nuclease exhibits no specificity for acetylated nuclei over a broad range of ionic strengths. Fractionation in the presence of MgCl2 of hyperacetylated nuclei digested with micrococcal nuclease results in a substantial increase in the amount of soluble chromatin relative to that obtained with control nuclei. This increased yield of Mg2+-soluble chromatin results from the recruitment into this fraction of oligonucleosomes containing extremely hyperacetylated histones. These results suggest that contiguous nucleosomes containing highly acetylated histones may be altered in their ability to interact with themselves and with other nucleosomes.

Acetylation↗
PubMedDate not supplied

[Effect of nuclease S1 on DNA of the highly oncogenic simian adenovirus SA7(C8)].

The effect at specific nuclease S1 on DNA and the complex viral DNA-terminal protein of the highly oncogenic simian adenovirus SA7(C8) was studied. It was shown that nuclease S1 did not digest the bound between DNA and terminal protein in the complex but residual amino acid(s) was cleaved out after digestion with pronase. The DNA obtained after nuclease S1 action could be ligated and its 5'-ends were phosphorylated by polynucleotide kinase.

Adenoviridae↗
PubMed1984

Terminally directed hydrolysis of duplex ribonucleic acid catalyzed by a species of the BAL 31 nuclease from Alteromonas espejiana.

The extracellular nuclease activities of Alteromonas espejiana sp. BAL 31 are mediated by at least two distinct protein species that differ in molecular weights and catalytic properties. The two species that have been purified to homogeneity and characterized, the "fast" (F) and "slow" (S) enzymes, both possess an exonuclease activity that shortens both strands of duplex DNA, with the F nuclease displaying a much greater (approximately 19-fold) turnover number for this degradation than the S species. In the present article, it is shown that the F species also mediates the terminally directed hydrolysis of a linear duplex RNA, gradually shortening molecules of this substrate through a mechanism that results in the removal of nucleotides from both the 3' and the 5' ends. This degradation proceeds with very infrequent introduction of scissions away from the termini as demonstrated by gel electrophoretic examination of the products of partial degradation, both in duplex form and after denaturation by reaction with CH3HgOH, and by electron microscopic characterization of duplex partially degraded molecules. The apparent Michaelis constant and turnover number have been determined. At equimolar enzyme concentrations in the limit of high substrate concentration, the F nuclease will degrade duplex RNA at a rate 0.021 +/- 0.010 (S.D.) times that for a duplex DNA of comparable guanine + cytosine content. The S species, by contrast, shows very little activity against the duplex RNA substrate relative to that of the F enzyme.

Endodeoxyribonucleases↗
PubMed1978

[Cleavage of bacteriophage PM2 DNA by S1 nuclease].

The kinetics of cleavage of superhelical PM2 DNA by the single strand-specific S1 nuclease is studied at various salt concentrations (0.01--1 MNaCl) by electrophoresis in neutral and alkaline agarose gels. Cleavage of different DNA forms (superhelical DNA I, relaxed circular DNA II and linear DNA III) is described by the kinetic equations of the first order, and respective rate constants k1, k2, and k3 are determined at all salt concentrations used. It is shown that a high salt concentration (not lower than 0.2 MNaCl) is necessary for high specificity of S1 nuclease action on DNA regions with impaired base pairing. Examination of the S1 nuclease preparation action on superhelical PM2 DNA may be used as a convenient and reliable assay of its specificity.

DNA, Bacterial↗
PubMedDate not supplied

Use of DEAE-cellulose filters in the S1 nuclease method for bacterial deoxyribonucleic acid hybridization.

Polynucleotide sequence relatedness can be studied with the S1 nuclease method by a fast and accurate procedure using DEAE-cellulose filters (DE81 Whatman). After S1 treatment of DNA-DNA hybrid molecules formed in 0.42 M saline solution, free nucleotides (digestion products) but not DNA are eluted from DE81 filters in appropriate salt concentration such as 5% Na2HPO4.12 H2O. The relative binding ratio and the thermal stability of heteroduplexes are determined for 7 strains of Enterobacteriaceae. The conclusions resulting from S1 nuclease and DE81 filters assay are similar to those obtained with S1 nuclease and trichloracetic acid precipitation.

Base Sequence↗
PubMed1980

DNA damage in rat 9L cells treated with nitrogen mustard and 1,3-bis(2-chloroethyl)-1-nitrosourea assayed by viscoelastometry and S1 nuclease.

The techniques of viscoelastometry and S1 nuclease digestion were applied to the analysis of DNA damage in rat 9L cells treated with nitrogen mustard and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Results of the S1 nuclease assay permitted quantitation of the amount of single-strand (or alkali-labile) break formation as well as DNA interstrand cross-link formation. In the presence of 2% detergent, only cells treated with nitrogen mustard showed evidence of DNA cross-link formation as determined by this assay. Viscoelastic analysis of cell lysates under denaturing conditions (pH 12.15) showed that cells treated with nitrogen mustard led to substantial increases in both the viscoelastic retardation time and recoil, consistent with the presence of DNA cross-links, while treatment with BCNU led to decreases in these two properties, consistent with the induction of single-strand breaks. Viscoelastic analysis of cell lysates under nondenaturing conditions (pH 11.15) showed that nitrogen mustard produced an increase in retardation time, consistent with single-strand break induction, along with a fast recoiling component that eventually led to gel-like behavior, suggesting the possibility of drug-induced intermolecular DNA-DNA cross-links. BCNU treatment resulted in a decrease in retardation time. This decrease in consistent with induction of DNA interstrand cross-links by BCNU and shows that the single-strand breaks observed at denaturing conditions were due to the presence of alkali-labile sites rather than true strand breaks. While other methods using denaturing conditions have resulted in evidence for DNA cross-links following BCNU treatment, both viscoelastic and S1 nuclease experiments showed negative results in this regard. Further work is needed to clarify this point.

Animals↗
PubMedDate not supplied

[Nucleases. Biological role].

In the first part of the review (R. I. Tatarskaya, 1976, Mol. Biol. 10, 235--259) the role of nucleases in the transcription, DNA replication, recombination and reparation was briefly described and discussed. The second part comprises some data on the role of nucleases in tRNA, rRNA, mRNA and DNA processing and mRNA degradation. Brief chapters "Restriction" and "Nucleases and viruses" are included as well.

Base Sequence↗
PubMed1993

Preparation, properties and application of Aspergillus oryzae S1 nuclease covalently bound to aminobutyl-Bio-Gel P-2 through its carbohydrate moiety.

Purified Aspergillus oryzae S1 nuclease, when covalently coupled to aminobutyl-(AB)-Bio-Gel P-2, via its carbohydrate moiety, retained 40-50% activity of the soluble enzyme. Optimization of coupling conditions showed that the most active immobilized preparations are obtained when 50-60 units of 1 mM periodate-oxidized enzyme are allowed to react with 1 ml (packed volume) of AB-Bio-Gel P-2 at 4 degrees C, in the presence of 20% (v/v) ethylene glycol, for 15 h. Immobilization did not change the pH and temperature optima of the enzyme, but it increased the temperature-stability. Immobilization brought about an approx. 2-fold increase in the Km and a slight decrease in the Vmax. On repeated use, the bound enzyme retained 60-65% of its initial activity after six cycles. Immobilized S1 nuclease could be stored, in a wet state, for more than 45 days without any significant loss in its initial activity. The application of AB-Bio-Gel- and concanavalin A-Sepharose-bound S1 nuclease in removing restriction-endonuclease-generated single-stranded tails in plasmid DNA is demonstrated.

Aspergillus oryzae↗
PubMed1998

Recognition of single-stranded DNA by nuclease P1: high resolution crystal structures of complexes with substrate analogs.

The reaction mechanism of nuclease P1 from Penicillium citrinum has been investigated using single-stranded dithiophosphorylated di-, tetra-, and hexanucleotides as substrate analogs. The complexes crystallize in tetragonal and orthorhombic space groups and have been solved by molecular replacement. The high resolution structures give a clear picture of base recognition by P1 nuclease at its two nucleotide-binding sites, especially the 1.8 A structure of a P1-tetranucleotide complex which can be considered a P1-product complex. The observed binding modes are in agreement with a catalytic mechanism where the two closely spaced zinc ions activate the attacking water while the third, more exposed zinc ion stabilizes the leaving 03' oxyanion. Stacking as well as hydrogen bonding interactions with the base 5' to the cleaved phosphodiester bond are important elements of substrate binding and recognition. Modelling of a productive P1-substrate complex based on the solved structures suggests steric hindrance as the likely reason for the resistance of Rp-phosphorothioates and phosphorodithioates. Differences with the highly homologous nuclease S1 from Aspergillus oryzae are discussed.

Catalysis↗
PubMed2000

Correlation between changes in nuclear magnetic resonance order parameters and conformational entropy: molecular dynamics simulations of native and denatured staphylococcal nuclease.

Recent work has suggested that changes in NMR order parameters may quantitatively reflect changes in the conformational entropy of a protein ensemble. The extent of the mathematical relationship between local entropy changes as seen by NMR order parameters and the full protein entropy change is a complex issue. As a step towards a fuller understanding of this problem, molecular dynamics calculations of both native and denatured staphylococcal nuclease were performed. The N-H bond vector motion, in both explicit and implicit solvent, was analyzed to estimate local and global entropy changes. The calculated N-H bond vector order parameters from simulation agreed on average with experimental values for both native and denatured structures. However, the inverted-U profile of order parameters versus residue number observed experimentally for denatured nuclease was only partially reproduced by simulation of compact denatured structures. Comparisons made across the full set of simulations revealed a correlation between the N-H order parameter-based conformational entropy change and the total quasiharmonic-based conformational entropy change between the native and denatured structures. The calculations showed that about 25% of the total entropy change was reflected by changes in simulated S2 values. This result suggests that NMR-derived order parameters may be used to provide a reasonable estimate of the total conformational entropy change on protein folding.

Magnetic Resonance Spectroscopy↗
PubMed1981

Electron microscopic studies of rat sperm heads treated with urea, dithiothreitol, and micrococcal nuclease.

Urea and dithiothreitol can decondense the chromatin in some of rat sperm heads. By this treatment, we have observed that in the nuclei of rat sperm the chromatin is organized into two morphologically distinct portions, namely: the compact chromatin rods of about 450 to 1,000 A thick, and the interlacing fibers about 250-290 A in thickness. When these treated sperm are further digested with micrococcal nuclease, the small fibers disappear, whereas the chromatin rods are still present in the "urea-nuclease pellet." From the available evidence, we suggest that the chromatin rods represent the highly packed nucleoprotamine, whereas the small fibers represent the more loosely organized nucleohistone.

Animals↗
PubMed1995

Charge and size effects in the capillary zone electrophoresis of nuclease A and its variants.

The migration behavior of nuclease A from Staphylococcus aureus and 11 of its variants in capillary zone electrophoresis (CZE) was investigated in the light of their three-dimensional structure known from X-ray crystallography and nuclear magnetic resonance (NMR) measurements. Nuclease A (molecular mass 16.8 kDa, pKa 10.3) and the variants differ only in a single amino acid residue and have a very similar crystal structure. With the use of coated quartz capillaries and suitable buffers, the protein migration was investigated at pH from 2.8 to 9.5 without interference by wall adsorption. Although the selectivity of the electrophoretic system for the proteins was mainly determined by their charge differences, certain variants having the same net charge could also be readily separated under nondenaturing conditions. For instance, the mobility of variant K116A was sufficiently higher than that of K116G so that they could be separated by CZE. The structures of both variants are the same except for the solvent-exposed loop containing residue 116. For this reason, the difference in electrophoretic mobilities can be attributed to the fact that in K116G the backbone of the 112 to 117 amino acids protrudes slightly from the protein, with a concomitant increase in the hydrodynamic radius with respect to that of K116A. Consequently, K116G shows a smaller mobility than K116A due to its larger hydrodynamic radius despite its smaller molecular mass. The interpretation of the experimentally measured mobilities of such closely related proteins therefore requires not only consideration of their electrostatic charge but also the fine details of their molecular structures.

Amino Acid Sequence↗
PubMed1986

Digestion of the chicken beta-globin gene chromatin with micrococcal nuclease reveals the presence of an altered nucleosomal array characterized by an atypical ladder of DNA fragments.

The structure of the chicken adult beta-globin gene chromatin in immature and mature erythrocyte nuclei has been analysed using micrococcal nuclease digestion. The resulting DNA fragments were blotted onto DBM-papers and probed with labelled DNA fragments spanning the adult beta-globin gene and its 5'- and 3'-flanking regions. The structure of the nucleosomes within and in the regions flanking the adult beta-globin gene appears to be altered in at least two ways in erythrocyte chromatin, when compared with either bulk or inactive ovalbumin gene chromatin. First, oligomeric DNA fragments containing the beta-globin gene are released faster than those of either bulk or ovalbumin gene chromatin. Second, although the difference in size of the liberated oligomeric DNA fragments is similar to the nucleosomal repeat length of bulk and ovalbumin gene chromatin, the individual oligomers are approximately 100 bp shorter than their bulk or ovalbumin gene counterparts, most noticeably when the nuclease digestion is performed at 37 degrees C. This results in an atypical ladder of approximately 300, 500, 700, 900 bp instead of the canonical chicken erythrocyte ladder which is an integral multiple of 207 bp. The same ladder was obtained from immature erythrocytes, in which the beta-globin gene is actively transcribed, and from mature erythrocytes, in which it is considered to be inactive with RNA polymerase molecules clustered in the 5' moiety of the gene. This indicates that the alteration of the nucleosomal structure is not due to transcription per se.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗