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Query: EC:3.1.31.1 (micrococcal nuclease)
 2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Electron microscopic studies of rat sperm heads treated with urea, dithiothreitol, and micrococcal nuclease. 731 26

Core particles prepared by micrococcal nuclease digestion of pig liver chromatin have been adsorbed on hydroxyapatite and dissociated by gradual increase in ionic strength and finally by urea and guanidine. By this method non-histone proteins have been found to be associated with the core particles. Proteins tightly bound to the core particle DNA (i.e. dissociated only by urea and guanidine) have also been found: these are proteins with a limited heterogeneity, with respect to their molecular weights, since only six components are present with molecular weights ranging from 71000 to 20000. They show, furthermore, a peculiar amino acid composition. Other tightly bound proteins have been shown to be present only in the spacer regions. The existence of two different classes of tightly bound proteins probably reflects different modes of binding to the DNA, which are compatible or incompatible, respectively, with the simultaneous binding of the histone octamer.
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PMID:Tightly bound non-histone proteins in nucleosomes from pig-liver chromatin. 732 67

DNA isolated from mammalian cell nuclear reveals discrete size patterns when partially digested with micrococcal nuclease. The DNA repeat lengths from different tissues within a species or from different species may vary. These differences have been attributed to the presence of different species of histone H1. To examine the nature of regulation of DNA repeat lengths and their possible relationship to histone H1, we have selected several mouse and human cell lines that differ in their DNA repeat lengths and examined them and their cell hybrids. 24 mouse X human and five mouse X mouse hybrid cell lines were analyzed. All the interspecific hybrids exhibited the repeat pattern characteristic of the murine parent. The mouse intraspecific hybrids had a repeat pattern of only one of the parents. We conclude that the partial human chromosome complements retained in the hybrids assume the repeat lengths exhibited by the mouse cells. Because H1 histones have been implicated in the determination of DNA repeat lengths, we also investigated the regulation of H1 histone expression in these cell hybrids. Purified H1 histones were radioactively labeled in vitro, and individual subfractions were subjected to proteolysis followed by gel electrophoresis. The resulting partial peptide maps off H1 histone subfractions A and B were distinguishable from one another and from different cell lines. In the mouse X human hybrids analyzed, only the mouse H1 histones were detected. These observations were extended to H2b by analysis of the hybrid cell histone by Triton-acid-urea gels. Neither the DNA repeat length nor histone expression is affected by the presence of any specific human chromosome. The fact that human genes are expressed in these hybrids suggests that the H1 histones of one species is able to interact with the chromatin of another species in a biologically funtional conformation. Analysis of the intraspecific PG19 X B82 (mouse X mouse) hybrids reveals the presence of H1 histone subfractions of the B82 mouse cells. Because these hybrids exhibit the nucleosome repeat length only of the PG19 cells, it appears that if histone H1 plays a role in determining the repeat length it does so in consort with other nonhistone chromosomal proteins.
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PMID:Histone gene expression and chromatin structure in mammalian cell hybrids. 741 91

Spectroscopic methods were used to examine the sequential build up of structure in the denatured state of staphylococcal nuclease. The 'free energy distance' between the native and denatured states was manipulated by altering conditions in solution (for example altering urea or glycerol concentration) and by changing the amino acid sequences. Initial studies employed a fragment of nuclease, referred to as delta 131 delta, which lacks six structural residues from the amino terminus and one structural residue from the carboxy-terminus. Nuclear magnetic resonance analysis of this fragment in solution revealed a modest quantity of dynamic structure which is native-like in character. With the addition of urea, 12 new HN peaks appeared in the 1H-15N correlation spectrum, presumably as a result of the breakdown of residual structure involving the first three beta strands. With the addition of glycerol, there was a rapid increase in the quantity of beta sheet structure detected by circular dichroism spectroscopy. At very high glycerol concentrations, an increase in helical structure became apparent. These data in addition to previously published results suggest that: (i) a beta-meander (strands beta 1-beta 2-beta 3) and the second alpha helix (alpha 2) are among the most stable local structures; (ii) the five-strand beta-barrel forms in a reaction which does not require the presence of several other native substructures; and (iii) the last step on the equilibrium folding pathway may be the formation and packing of the carboxy terminal alpha helix (alpha 3) to give the native state.
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PMID:Initial studies of the equilibrium folding pathway of staphylococcal nuclease. 777 Apr 83

The distributions of chromium-DNA adducts and DNA-protein crosslinks induced by treatment of intact CHO cells with carcinogenic chromium were examined in distinct chromatin subfractions: a chromatin subfraction released by digestion of isolated nuclei with micrococcal nuclease (1SF, 14% of total nuclear DNA), bulk chromatin (74% of total DNA) and a nuclear matrix fraction (12% of total DNA). The identity of the matrix fraction was confirmed by hybridization of DNA from each subfraction with a cDNA probe prepared from total mRNA isolated from CHO cells, which showed that the 1SF and nuclear matrix fractions were 2.3- and 3.8-fold enriched in actively transcribed genes respectively, compared to total unfractionated DNA. Immediately following treatment of cells with 150 microM sodium chromate for 2 h the binding of chromium to each chromatin fraction was found to be non-uniform. Compared with total unfractionated nuclei, the nuclear matrix fractions were enriched in chromatin-bound chromium (3.4-fold), whereas the bulk chromatin fraction was relatively depleted (0.5-fold). Approximately 13% of nuclear chromium was associated with the detergent-soluble lipid component of nuclei. A similar distribution of chromatin-bound chromium was also apparent 24 h after the chromate treatment. Immediately after the 2 h chromate treatment, chromium-DNA adducts were detected in all the chromatin subfractions. Total nuclear and bulk chromatin DNA contained similar levels of this type of damage. The 1SF fraction was depleted approximately 3-fold in this type of damage compared with total nuclear DNA. In contrast, the nuclear matrix was markedly enriched in chromium-DNA adducts (approximately 4-fold compared with total nuclear DNA) at this time. As previously demonstrated, chromium-DNA adducts in total nuclear DNA decreased within the first 24 h, but thereafter persisted at a similar level. Chromium-DNA adducts in nuclear matrix DNA also reached maximum levels at the end of the 2 h treatment and decreased to 68% and 39% of this level by 24 and 48 h after treatment respectively. In contrast, the adduct levels in the 1SF and bulk chromatin fractions did not change up to 48 h after treatment. Chromium-induced DNA-protein crosslinks, which were stable to 8 M urea and 2% SDS, occurred almost exclusively in the nuclear matrix fraction. The crosslinks in this fraction reached a maximum level at the end of the 2 h treatment, but returned to control levels 24 h later.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Preferential formation and repair of chromium-induced DNA adducts and DNA--protein crosslinks in nuclear matrix DNA. 803 23

The exchange kinetics of over 70% of the 143 backbone amide hydrogens in staphylococcal nuclease H124L (nuclease H124L), both in its unligated state and in its ternary complex with Ca2+ and thymidine 3',5'-bisphosphate, have been quantified by nitrogen-15 resolved proton nuclear magnetic resonance spectroscopy. Protection factors for the slowly exchanging hydrogens in unligated nuclease H124L at 37 degrees C and pH 5.5 were found to vary by over one order of magnitude. This range of protection factors has been interpreted in the framework of global and local structural fluctuations. The three most highly protected hydrogens (K24, L25, M26) map to strand 2 of the central five-stranded beta-barrel. The free energy change for the opening reaction which exposes these hydrogens to the solvent (delta G(degree)op) was calculated from the exchange rates in the native and denatured states, the latter values being estimated from model peptide exchange studies [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158]. Close agreement was found between delta G(degree)op and delta G(degree)u, the free energy change of unfolding as measured by urea denaturation experiments. Exchange of these hydrogens thus appears to occur via global unfolding of the protein. One region exhibited somewhat lower protection factors: it mapped to the C-terminal portions of helix 2 and helix 3 and to part of the intervening segment. This region has been identified as a minor hydrophobic domain of nuclease [Shortle, D., Stites, W. E., & Meeker, A. K. (1990) Biochemistry 29, 8033-8041].(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Hydrogen exchange in unligated and ligated staphylococcal nuclease. 821 67

The 32P-postlabelling methodology for analysis of DNA adducts derived from carcinogens containing one aromatic ring (e.g., safrole, styrene oxide, benzene metabolites, 1-nitrosoindole-3-acetonitrile) or a bulky non-aromatic moiety (e.g., mitomycin C, diaziquone) is reviewed. Six steps are involved: digestion of DNA to 3'-nucleotides, enrichment of adducts, 32P-labelling of adducts, separation of labelled adducts by TLC, detection, and quantitation. The first step, DNA digestion with micrococcal nuclease and spleen phosphodiesterase, is applicable to DNA modified with most carcinogens independent of their size and structure. Of the two commonly used procedures for enrichment of aromatic adducts in DNA digests, the nuclease P1 treatment is substantially more effective than butanol extraction for small aromatic and bulky non-aromatic adducts. For initial purification of these adducts from unadducted material after 32P-labelling, multi-directional polyethyleneimine (PEI)-cellulose TLC using 1 M sodium phosphate, pH 6.0, as the D1 solvent is not suitable, because they are not retained on PEI-cellulose under these conditions. A higher concentration of sodium phosphate (e.g., 2.3 M) or development with D1 and D3 solvents in the same direction helps to retain adducts of safrole and of benzene metabolites. Also, transfer of adducts from multiple cut-outs above the origin after D1 chromatography, as adopted for analysis of I-compounds, is potentially applicable. However, initial purification by reverse-phase TLC, followed by in situ transfer and resolution by PEI-cellulose TLC has been found to be most effective for these adducts. Reverse-phase TLC at 4 degrees C or in a stronger salt solution further improves retention of some adducts (e.g., mitomycin C and diaziquone adducts). For adduct separation by PEI-cellulose TLC, salt solutions with or without urea are used.
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PMID:32P-postlabelling analysis of small aromatic and of bulky non-aromatic DNA adducts. 822 92

The reversible unfolding of two dissimilar proteins, phosphoglycerate kinase from Bacillus stearothermophilus (PGK) and Staphylococcus aureus nuclease (SAN), was induced with two denaturants, urea and guanidinium chloride (GuHCl). For each protein, structural transitions were monitored by intrinsic fluorescence intensity changes arising from a unique tryptophan residue. In the case of SAN the single, native tryptophan residue was used, whereas for PGK two versions, one with a tryptophan at position 315 and one at 379, were constructed genetically. The resultant folding curves were analyzed by considering the change in the solvation free energy of internal amino acid residues as the denaturant concentration was varied. We derive the following simple relationship: -RT ln K = delta Gw + n delta Gs,m[D]/Kden. + [D]) where K is the equilibrium constant describing the distribution of folded and unfolded forms at a given denaturant concentration [D], delta Gw is the free energy change for the transition in the absence of denaturant, and n is the number of internal side chains becoming exposed. delta Gs,m and Kden. are constants derived empirically from the solvation energies of model compounds and represent the behavior of an average internal side chain between 0 and 6 M GuHCl and 0 and 8 M urea. For proteins of known structure these values can easily be derived, and for others, average values in guanidinium chloride (delta Gs,m = 0.775 kcal/mol and Kden. = 5.4 M) or urea (delta Gs,m = 1.198 kcal/mol and Kden. = 25.25 M) can be used in the analysis. Results show that the parameters n and delta Gw are independent of the denaturant used for all 12 transitions studied. This supports the hypothesis that the unfolding activity of urea and GuHCl can be accounted for by their effect on the solvation energy of amino acid side chains which are buried in the folded but exposed in the unfolded protein. This simple analytical treatment allows the "cooperativity" of protein folding to be interpreted in terms of the number of side chains becoming exposed to the solvent in a given step and allows accurate estimation of the free energy irrespective of the denaturant concentration needed to induce the transition.
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PMID:The energetics and cooperativity of protein folding: a simple experimental analysis based upon the solvation of internal residues. 847 98

In a previous report [Alexandrescu, A. T., Abeygunawardana, C., & Shortle, D. (1994) Biochemistry 33, 1063-1072], NMR methods were used to characterize the residual structure in delta 131 delta, a large fragment of staphylococcal nuclease that serves as a model denatured state under nondenaturing conditions. On the basis of a large number of missing amide protons for the residues that form a three-strand antiparallel beta sheet in the native state, it was concluded that this beta meander may be highly populated in delta 131 delta, with severe line broadening due to relatively slow exchange between different conformational states. In the present report, results from circular dichroism spectroscopy and NMR spectroscopy indicate strands beta 2-beta 3 form a beta hairpin at urea concentrations below 6 M. Amide proton resonances from several hydrophobic residues adjacent to this beta hairpin disappear in concert with all of the beta 2-beta 3 residues, suggesting a local, non-native hydrophobic interaction may help stabilize the beta hairpin. At concentrations below 3 M, all amide resonances from strand beta 1 in delta 131 delta also disappear, suggesting that beta 1 may combine with the beta 2-beta 3 hairpin to form a native-like beta meander. In addition, the hydrophobic helix alpha 2 decreases from approximately 30% population in 0 M urea to approximately 10%-15% at 6 M urea, whereas helix alpha 1 goes from 10%-15% populated in 0 M urea to undetectable in 6 M urea. Characterization of a second, distinctly different denatured state, WT nuclease at pH 3.0 and low salt, reveals that this low-density acid-denatured state is structurally similar to delta 131 delta at low concentrations of urea. From these and previously published data, a tenative equilibrium folding pathway can be constructed for staphylococcal nuclease which describes the relative strengths and interdependencies of the chain-chain interactions involved in forming the native state.
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PMID:The equilibrium folding pathway of staphylococcal nuclease: identification of the most stable chain-chain interactions by NMR and CD spectroscopy. 851 46

Several mixed disulfide variants of staphylococcal nuclease have been produced by disulfide bond formation between nuclease V23C and methane, ethane, 1-propane, 1-n-butane, and 1-n-pentane thiols. Although CD spectroscopy shows that the native state is largely unperturbed, the stability toward urea-induced unfolding is highly dependent on the nature of the group at this position, with the methyl disulfide protein being the most stable. The variant produced by modification with iodoacetic acid, however, gives a CD spectrum indicative of an unfolded polypeptide. Thiol-disulfide exchange equilibrium constants between nuclease V23C and 2-hydroxyethyl disulfide have been measured as a function of urea concentration. Because thiol-disulfide exchange and unfolding are thermodynamically linked, the effects of a mutation (disulfide exchange) can be partitioned between various conformational states. In the case of unmodified V23C and the 2-hydroxyethyl protein mixed disulfide, significant effects in the nonnative states of nuclease are observed. Truncated forms of staphylococcal nuclease are thought to be partially folded and may be good models for early folding intermediates. We have characterized a truncated form of nuclease comprised of residues 1-135 with a V23C mutation after chemical modification of the cysteine residue. High-resolution size-exclusion chromatography indicates that modification brings about significant changes in the Stokes radius of the protein, and CD spectroscopy indicates considerable differences in the amount of secondary structure present. Measurement of the disulfide exchange equilibrium constant between this truncated protein and 2-hydroxyethyl disulfide indicate significant interactions between position 23 and the rest of the protein when the urea concentration is lower than 1.5 M.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interactions in nonnative and truncated forms of staphylococcal nuclease as indicated by mutational free energy changes. 852 79


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