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

With the use of a reconstituted poly(ADP-ribosyl)ating enzyme system and three purified nucleases, micrococcal nuclease (MN), bull seminal RNase (BS RNase) and Ca2+, Mg2+-dependent endonuclease (BS DNase), as model acceptor proteins for ADP-ribose, the effect of ionic strength on the modification reaction was examined in detail. When these three nucleases were extensively poly(ADP-ribosyl)ated in this system at a low ionic strength (5 mM Tris), they were all inhibited by about 80% and the chain length of the polymer covalently bound to the nucleases was 13 to 23 ADP-ribose units. The observed inhibition was markedly prevented by increasing the ionic strength in the reaction mixture with a concomitant decrease in the polymer size bound to the nucleases. The NaCl concentrations required for decreasing the extent of the inhibition to half of the maximum were calculated to be 20, 50, and 100 mM for MN, BS RNase, and BS DNase, respectively. These values are similar to the NaCl concentrations required for decreasing the average chain lengths of the polymer to half, suggesting that the length of polymer is closely correlated to the extent of inhibition of these nucleases. DNA-binding affinities of these nucleases, expressed in terms of the NaCl concentrations required for eluting the enzymes from DNA-cellulose, were 140, 280, and 340 mM for MN, BS RNase, and BS DNase, respectively. Considering that maintainance of a ternary complex of poly(ADP-ribose) synthetase, acceptor and DNA may be essential for the modification reaction, the relatively strong salt effect observed in the modification of MN may be explained by its low DNA-binding affinity.
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PMID:Effect of ionic strength on chain elongation in ADP-ribosylation of various nucleases. 371 Oct 53

The distribution of methylated purines in different regions of liver chromatin DNA has been examined after treating rats with [14C]dimethylnitrosamine (2 mg/kg). At different times after administration of the carcinogen, liver nuclei were isolated and fractionated by micrococcal nuclease digestion and low and high salt extractions into an active chromatin fraction, two fractions comprising the bulk of the genome, and a nuclear matrix fraction. Regions of active chromatin and nuclear matrix tended to be methylated more readily than bulk chromatin, with respect to formation of both O6-methylguanine and N-methyl purines. Removal of both 7-methylguanine and 3-methyladenine (by repair and depurination reactions) occurred at a relatively uniform rate in all chromatin fractions. In contrast, repair of O6-methylguanine proceeded more rapidly from active chromatin than from bulk chromatin, whereas repair of this lesion from nuclear matrix DNA was much slower than for bulk DNA. Pretreatment of rats for 4 weeks with non-radioactive dimethylnitrosamine before the administration of [14C]dimethylnitrosamine enhanced the rate of repair of radioactive O6-methylguanine from all chromatin fractions. Nevertheless the rate of loss of the adduct was still faster from active chromatin and slower from matrix DNA than from the bulk of the genome. Since pretreatment also elevated the rate of liver DNA synthesis especially in the nuclear matrix fraction, there is an increased probability of the fixation of mutations due to the presence of O6-methylguanine in this selected region of the genome. The implications of this persistent O-alkylation of matrix DNA, and rapid repair of O6-alkylguanine in active chromatin for the toxicity and carcinogenicity of alkylating agents are discussed.
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PMID:Selective repair of methylated purines in regions of chromatin DNA. 374 23

In order to investigate the functional organization of the progesterone receptor in chromatin we characterized the physical-chemical properties of the receptor bound chromatin fragments released by micrococcal nuclease and DNase I digestion. The crude nuclear fraction was isolated from T 47 D cells, previously exposed to 0.1 microM [3H]ORG 2058. The parameters determined in low and high salt concentrated buffers were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding abilities to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. Micrococcal nuclease digestion solubilized a receptor form sedimenting as a single peak at 4.4 S with a Rs = 7.78 nm and an estimated Mr = 144,000. About 53% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. 0.4 M KCl dissociated this receptor form into a smaller receptor sedimenting at 3.3 S with Rs = 5.53 nm and a calculated Mr = 76,000. A similar receptor form was extracted by 0.6 M KCl from the undigested crude nuclear fraction. DNase I digestion solubilized a receptor form sedimenting at 3.3 S with a Rs = 6.87 nm and a calculated Mr = 94,000. About 26% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. Dissociation of this receptor form by 0.4 M KCl resulted in a receptor sedimenting at 2.8 S with a Rs = 6.53 nm and an estimated Mr = 76,000. These results suggest: The progesterone receptor in chromatin is associated with several molecules probably proteins which complexed it to DNA. Some of these molecules still associated with the progesterone receptor could be released by nucleases digestion. Micrococcal nuclease releases a larger portion of these molecules than those release by DNase I.
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PMID:Characterization of the progesterone receptor solubilized by micrococcal nuclease and DNase I digestion. 374 26

Micrococcal nuclease, DNase I, and trypsin have been employed to study the kinetics of core particle self-assembly by salt jump from 2.0 to 0.2 M NaCl. A few seconds after the initiation of the reassociation reaction, the bulk of core particle DNA becomes protected from digestion by micrococcal nuclease, whereas free DNA, under the same conditions, is completely hydrolyzed. The central and C-terminal regions of core histones are also protected from trypsin digestion immediately after the 2.0-0.2 M NaCl salt jump. Moreover, the extent of degradation produced by trypsin is the same for samples digested a few seconds after the salt jump and for samples digested 20 min after the salt jump. With DNase I, minor structural differences have been detected between samples obtained at different times during the reaction. However, even in this case our results indicate that many of the characteristic histone-DNA contacts within the core particle are made a few seconds after the initiation of the self-assembly reaction. Furthermore, core particles have been labeled with the fluorescent reagent N-(1-pyrenyl)maleimide (NPM), which was previously used as a sensitive probe for nucleosome conformation. Extensive DNase I or trypsin digestion of NPM-labeled core particles in 0.2 M NaCl does not produce significant changes in excimer fluorescence. This allows us to conclude that the covalent continuity of DNA is not required for the maintenance of the folded conformation of the core particle and that the trypsin-resistant domains of core histones play a fundamental role in the stabilization of this structure.
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PMID:Nucleosome core particle self-assembly kinetics and stability at physiological ionic strength. 380 41

The proteolytic fragments of the nuclear estrogen receptor in the MCF-7 cell line were characterized following limited digestion with chymotrypsin and trypsin. Nuclei were isolated from cells previously exposed to 10 nM [3H]estradiol. The proteolytic digestion was performed either on the micrococcal nuclease hydrolysate or on intact nuclei. The molecular weights (Mr) were calculated from the sedimentation coefficients determined on a sucrose gradient and from the Stokes radii estimated by gel filtration. Digestion of the nuclei with micrococcal nuclease solubilized a receptor form of Mr = 151,000. This receptor form was degraded by chymotrypsin to a receptor of Mr = 33,000 and by trypsin to a receptor of Mr = 60,000. Digestion of intact nuclei with chymotrypsin solubilized a receptor form of Mr = 62,000 which dissociated in 0.4 M KCl to a receptor of Mr = 32,000. Digestion of intact nuclei with trypsin followed by micrococcal nuclease solubilized a receptor form of Mr = 75,000 which was further dissociated by 0.4 M KCl to a receptor form of Mr = 60,000. The ability of the receptor forms to bind DNA was tested using DNA-cellulose column chromatography. About 40% of the micrococcal nuclease solubilized receptor form, compared to about 7% of the chymotrypsin degraded receptor and to about 13% of the trypsin degraded receptor forms, all bound to the column and could be eluted by high salt concentrated buffer. We conclude that the nuclear estrogen receptor in the MCF-7 cell line can be partially degraded either in the micrococcal nuclease hydrolysate or in intact nuclei by chymotrypsin or trypsin generating protein moieties, probably receptor fragments of Mr = 33,000 and 60,000 respectively. Both fragments retain their estradiol binding domain and it may be hypothesized that the heavier fragment retains its chromatin binding domain.
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PMID:Analysis of the nuclear estrogen receptor from MCF-7 cells by limited proteolysis. 382 Nov 6

We [Rocha, E., Davie, J.R., van Holde, K.E., & Weintraub, H. (1984) J. Biol. Chem. 259, 8558-8563] have previously reported that the transcriptionally competent beta-globin gene domain is selectively enriched in chromatin fractions eluted with solutions of approximately physiological ionic strength from micrococcal nuclease digested mature chicken erythrocyte nuclei. In this report, we demonstrate that beta-globin chromatin is eluted as oligonucleosomes while vitellogenin, a transcriptionally inactive gene, is eluted as mononucleosomes as is the bulk of sequences found in this fraction. Following removal of the salt, the eluted chromatin was made 100 mM KCl and separated into aggregation-prone and aggregation-resistant fractions. Globin sequences were present in both fractions and had the greatest enrichment in the aggregation-prone fraction which contained H1 and H5, H1 being more abundant. A procedure is presented in which H1 is selectively removed from the erythrocyte nuclei. Following the selective removal of H1 and subsequent fractionation, globin but not vitellogenin oligonucleosomes were present in the aggregation-resistant chromatin fraction. The results indicate the beta-globin domain is a mosaic of aggregation-resistant and aggregation-prone regions with the latter being associated with H1 and H5. Vitellogenin sequences were associated principally with aggregation-prone regions complexed with H5.
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PMID:Selective solubilization of beta-globin oligonucleosomes at low ionic strength. 382 4

It has been suggested that nascent DNA located at the DNA replication fork may exhibit enhanced sensitivity to radiation damage. To evaluate this hypothesis, Chinese hamster ovary cells (CHO) were labeled with 125I-iododeoxyuridine (125IUdR) either in the presence or absence of aphidicolin. Aphidicolin (5 micrograms/ml) reduced cellular 125IUdR incorporation to 3-5% of the control value. The residual 125I incorporation appeared to be restricted to low molecular weight (sub-replicon sized) fragments of DNA which were more sensitive to micrococcal nuclease attack and less sensitive to high salt DNase I digestion than randomly labeled DNA. These findings suggest that DNA replicated in the presence of aphidicolin remains localized at the replication fork adjacent to the nuclear matrix. Based on these observations an attempt was made to compare the lethal consequences of 125I decays at the replication fork to that of 125I decays randomly distributed over the entire genome. Regardless of the distribution of decay events, all treatment groups exhibited identical dose-response curves (D0: 101 125I decays/cell). Since differential irradiation of the replication complex did not result in enhanced cell lethality, it can be concluded that neither the nascent DNA nor the protein components (replicative enzymes, nuclear protein matrix) associated with the DNA replication site constitute key radiosensitive targets within the cellular genome.
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PMID:Cell lethality after selective irradiation of the DNA replication fork. 392 25

When mouse brain nuclei are optimally digested with micrococcal nuclease, most of the chromatin is soluble in a 180 mM salt/1 mM EDTA buffer [1]. At this ionic concentration, chromatin maintains its native structure [2]. In an attempt to selectively extract different fractions of chromatin from digested nuclei, we have examined the differential solubility of chromatin in the 180 mM salt buffer containing concentrations of MgCl2 ranging from 2 to 0 mM. The results suggest that digested chromatin may be fractionated into specific soluble chromatin fractions which correspond to nuclease-sensitive chromatin, bulk chromatin, and heterochromatin. These soluble fractions have a high molecular weight (up to 20 kbp), and contain a full complement of histones as well as a complex assortment of non-histone proteins. The residual insoluble fraction may be equivalent to a native, nuclear matrix-bound chromatin fraction.
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PMID:Fractionation of micrococcal nuclease-digested chromatin solubilized at physiologic ionic strength. 396 93

The pattern of micrococcal nuclease digestion of chromatin from different organs of Allium cepa has been studied. The DNA from small oligonucleosomes appears to be highly degraded and heterogeneous. In solutions of intermediate ionic strength (0.15 M NaCl) histones H2A and H2B form dimers, however at high salt concentrations (2 M NaCl) they tend to form complexes of higher order, such as tetramers. It is proposed that a correlation exists between the ability of these proteins to form tetramers and the particular stability of internucleosome interactions.
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PMID:Structural studies on Allium cepa L. chromatin: enhanced stability of internucleosome interactions in plant chromatin. 401 50

Androgen receptors were attached covalently in situ to their nuclear acceptor sites with the contact site cross-linker, formaldehyde. Chromatin, prepared from sonicated nuclei of rat prostate, was labeled by isotope exchange with [3H]dihydrotestosterone and found to contain 19,000 +/- 900 (mean +/- S.E.) salt-extractable androgen receptors/nucleus which sedimented in the 3-4 S region of 7.6-76% (v/v) glycerol gradients and at a density of approximately 1.28-1.35 g/ml in CsCl gradients. After incubation of the chromatin with 0.5% (w/v) formaldehyde for 1 h at 4 degrees C, there was a 90% reduction in the concentration of free androgen receptors and an increase in the density of the androgen binding sites recovered from CsCl gradients. Extensive digestion of the cross-linked chromatin with micrococcal nuclease liberated 18% of the androgen receptors as 3-4 S entities and caused an overall decrease in the density of the receptor-acceptor complexes. Ribonuclease digestions had no effect on the androgen receptors cross-linked to chromatin. Mild digestion of the cross-linked preparations with trypsin, alone or in combination with micrococcal nuclease, resulted in the release of 74% and 97% of the androgen receptors, respectively. Together, these findings imply that two classes of receptor-acceptor complexes are present in prostatic chromatin--one, containing about 20% of the androgen receptors in which the receptors are in direct contact with DNA but not with proteins and the other, containing most of the androgen receptors in which the receptors are adjacent to acceptor proteins but not to DNA.
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PMID:In situ cross-linking of androgen receptors to nuclear acceptor sites of rat prostate with formaldehyde. 401 1


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