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)

All of the clinically available nitrosourea antitumor agents produce serious treatment-limiting bone marrow toxicity. A reduction in this toxicity can be achieved by attaching the chloroethylnitrosourea cytotoxic group to C2 (chlorozotocin) or C1 (1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea, GANU) of glucose. Both glucose analogs are less myelotoxic in mice than 1-(2-chloroethyl)-3-cyclohepyl-1-nitrosourea (CCNU) or 1-(4-amino-2-methylpyrimidin-5-yl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), while retaining comparable antitumor activity against the murine L1210 leukemia. To define the nuclear mechanisms for this reduced myelotoxicity, alkylation of L1210 and murine bone marrow DNA was quantitated. With the use of the endonuclease micrococcal nuclease and DNase I, the sites of alkylation within the chromatin substructure were determined. Experiments were performed on L1210 leukemia or bone marrow cells that had been incubated in vitro for 2 hr with 0.1 mM [14C]chloroethyl drug. The quantitative alkylation of DNA by GANU was 1.3-fold greater in L1210, as compared to bone marrow, cells. This ratio of DNA alkylation is comparable to the 1.3 ratio we previously reported for chlorozotocin [L. C. Panasci, D. Green and P. S. Schein, J. clin. Invest. 64, 1103 (1979)]. In contrast, the ratio of alkylation (L1210:bone marrow DNA) for the myelotoxic ACNU was 0.66, similar to 0.59 for CCNU. Nuclease digestion experiments demonstrated that chlorozotocin and GANU preferentially alkylated internucleosomal linker regions of bone marrow chromatin, while nucleosome core particles were the preferred targets of CCNU and ACNU. The reduced myelotoxicity of chlorozotocin and GANU may be correlated with the advantageous ratio of L1210:bone marrow DNA alkylation and preferential alkylation of internucleosomal regions of bone marrow chromatin.
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PMID:Correlation of nitrosourea murine bone marrow toxicity with deoxyribonucleic acid alkylation and chromatin binding sites. 710 31

Possible mechanisms of internucleosomal DNA fragmentation in thymocytes of irradiated rats were studied. It was shown that thymocyte nuclei contain at least two nucleases that cleave DNA between nucleosomes--a Ca2+/Mg2+-dependent nuclease and an acidic one which does not depend on bivalent ions. 2 and 3 h after irradiation at a dose of 10 Gy the initial rate of DNA cleavage by Ca2+/Mg2+-dependent nuclease in isolated nuclei increased three and seven times, respectively, but the kinetics of DNA digestion by acidic nuclease did not change. The experiments with cycloheximide indicated that Ca2+/Mg2+-dependent endonuclease turns over at a high rate. The activity of the cytoplasmic acidic and Mg2+-dependent nucleases was shown to increase (by 40 and 50%, respectively) 3 h after irradiation. The effect is caused by the de novo synthesis of the nucleases. At the same time the activity of nuclear nucleases did not essentially change. The chromatin isolated from rat thymocytes 3 h after irradiation did not differ in its sensitivity to some exogenic nucleases (DNAase I, micrococcal nuclease and nuclease from Serratia marcescens) from the control. Thus, Ca2+/Mg2+-dependent endonuclease seems to be responsible for the postirradiation internucleosomal DNA fragmentation in dying thymocytes.
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PMID:The involvement of nuclear nucleases in rat thymocyte DNA degradation after gamma-irradiation. 715 95

Electrophoresis of endonuclease-split rat liver chromatin in polyacrylamide and agarose gels of a monotonous concentration revealed different separation patterns. The first pattern exhibited a spectrum of chromatin fragments of discrete sizes, while the second one -- two fractions, i. e. DNP and DNA. The polyacrylamide gel concentration gradient allowed to achieve the best resolution of individual classes of chromation fragments with individual subfractions in sub-, mono- and trinucleosomes. A comparison of micrococcal nuclease-split chromatin fragments of vertebrates revealed nucleosomes of varying sizes. The heterogeneity of the nucleosomes was also found during isotachophoresis and free flow electrophoresis. An analysis of individual subfractions of the mononucleosomes revealed differences in the composition and number of proteins as well as in the kinetics of basic and acidic titration. The chromatin fractions obtained by discontinuous endonucleolysis differed in their composition from the individual subfractions of histones.
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PMID:[Structural heterogeneity of chromatin as revealed by electrophoretic methods]. 723 91

Mild digestion of Xenopus nuclei with micrococcal nuclease results in the cleavage of oocyte-type 5S RNA genes once every four nucleosomes, or about once per tandem repeating unit of 5S DNA. This specific cleavage pattern is observed with nuclei from somatic cells where oocyte-type 5S genes are never transcribed (blood and liver) and with cultured cell nuclei where these genes are in a DNAase I-sensitive chromatin conformation and low level transcription is observed. Cleavage of protein-free DNA with micrococcal nuclease does not result in a specific digestion pattern. The similarity of the nuclease-generated repeat length and the sequence repeat length of oocyte-type 5S genes suggested a sequence-specific arrangement of nucleosomes on these DNA sequences. Restriction endonuclease analysis indicates that micrococcal nuclease preferentially cleaves in a restricted region within the 5S repeating unit, about 200 bp from the single Hind III site. Using specific end-labeled DNA probes derived from cloned 5S DNA we can recognize at least four possible modes of organization of the nucleosomes on 5S DNA. In each of these phase arrangements, functionally significant regions of the 5S gene (start of transcription, middle control region and transcription termination site) are found in or near nucleosome linkers.
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PMID:Nonrandom alignment of nucleosomes on 5S RNA genes of X. laevis. 743 6

An RNA-containing endonuclease that catalyzes the excision and maturation of the 16S ribosomal RNA (rRNA) from the rRNA primary transcript (pre-rRNA) in the hyperthermophilic archaeon Sulfolobus acidocaldarius has been characterized. The ribonucleoprotein was inactivated by micrococcal nuclease treatment and inactivation was reversed by reconstitution with bulk RNA. A 159-nucleotide RNA with sequence and structural similarity to U3 small nucleolar RNAs of eukaryotes copurified with the endonuclease activity. Oligonucleotide-targeted ribonuclease H inactivation of the U3-like RNA component also abolished processing activity. A motif within the U3 homolog is complementary to the region around the three cleavage sites in the pre-RNA substrate. Thus, U3-mediated processing of pre-rRNA is not specific to eukaryotes; its origin predates the divergence of archaea and eukaryotes.
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PMID:Ribosomal RNA precursor processing by a eukaryotic U3 small nucleolar RNA-like molecule in an archaeon. 929 34

We have investigated protein-DNA interactions in the proximal promoter of the human amyloid precursor protein (APP) gene in temporal lobe neocortical nuclei isolated from control and Alzheimer disease (AD) affected brains. We report that the human APP 5' promoter sequence from -203 to +55 bp, which has been previously reported to contain essential regulatory elements for APP gene transcription, lies in a deoxyribonuclease I, micrococcal nuclease- and restriction endonuclease-sensitive, G+C-rich nucleosome-free gap flanked both 5' and 3' by typical nucleosome structures. As analyzed by electrophoretic mobility shift assay, this extended internucleosomal linker DNA is heavily occupied by nuclear protein factors, and interacts differentially with nuclear protein extracts obtained from HeLa and human brain neocortical nuclei. This suggests that the chromatin conformation of the APP gene promoter may vary in different cell types, and may correlate with differences in APP gene expression. Human recombinant transcription factors AP1, SP1 and TFIID (but not AP2 or brain histones H1, H2B and H4) interact with the -203 to +55 bp of the human APP promoter sequence. Only minor differences were observed in the chromatin structure of the immediate APP promoter between non-AD and AD affected neocortical nuclei, suggesting either that post-transcriptional processes, or that regulatory elements lying elsewhere in the APP gene may be important in the aberrant accumulation of the APP gene product.
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PMID:Protein-DNA interactions in the promoter region of the amyloid precursor protein (APP) gene in human neocortex. 801 72

We designed a new method for quantitative analysis of the degree of DNA fragmentation, a characteristic feature of apoptosis. A photograph of an agarose gel electrophoresis of fragmented DNA was incorporated by an optical density scanner or equivalent equipment, and the integrations of middle molecular size area (10,000 to 300 bp), single nucleosomal size area (smaller than 300 bp) and total lane area were calculated. We defined fragmentation rate or (%)FR as the amount of fragmented DNA expressed as the percentage of the total amount of DNA, assigning the coefficients of 1 and 0.5, respectively, to the fragments completely digested into single nucleosomal length and to those partially digested (between 10 k to 300 bp). A standard calibration curve was constructed from triplicate experiments of target nuclei digestion by micrococcal nuclease, which revealed that this method covered a wide range of nuclease activity. We also confirmed that our method was applicable to an autodigestion assay of isolated nuclei which represented endogenous endonuclease activities. This method may be a useful tool for quantitative analysis of endonuclease activities capable of producing nucleosomal-size DNA fragmentation.
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PMID:A new method for quantitative estimation of the degree of DNA fragmentation utilizing agarose gel electrophoresis. 808 25

Apoptosis is the predominant form of cell death observed in a variety of physiological and pathological conditions such as cancer involution, insect metamorphosis, the development of the immune and nervous systems, and embryogenesis. The typical nuclear changes taking place in apoptotic cells include extensive condensation of chromatin and internucleosomal DNA fragmentation into units of 200 base pairs. However, the mechanisms responsible for both chromatin condensation and DNA fragmentation have yet to be elucidated. In this study, micrococcal nuclease and the divalent cations, Ca2+ and Mg2+, were applied to isolated nuclei in an attempt to reconstitute in vitro the digestion of genomic DNA associated with apoptosis. Micrococcal nuclease was found to induce a typical pattern of DNA fragmentation, but did not give rise to chromatin condensation, whereas Ca2+/Mg2+ induced both chromatin condensation and DNA fragmentation in isolated mouse liver nuclei. When the endonuclease inhibitor ZnCl2 was used, the DNA fragmentation induced by Ca2+/Mg2+ in nuclei could be completely inhibited, but chromatin condensation still occurred. For comparison, intact liver cells were treated with valinomycin, a potassium ionophore, which gave rise to an atypical cell death, with chromatin condensation appearing without DNA fragmentation. Our results suggest that endonuclease activation in apoptosis is neither necessary nor sufficient to induce chromatin condensation, and that DNA fragmentation and chromatin condensation may be triggered through separate pathways during apoptosis.
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PMID:Separate metabolic pathways leading to DNA fragmentation and apoptotic chromatin condensation. 829 67

A comparative study of several parameters of the cell nuclei of hybridoma MLC-1c and its parent cells--myeloma X-63.Ag8.653 and spleen lymphocytes of Balb/c mice, has been carried out. The results of cytogenetic studies suggest that although the hybridoma and myeloma cell lines used in this study are rather stable, they contain some proportion of the altered chromosomal material. Two-dimensional electrophoresis performed according to O'Farrell revealed that the similarity between the relative presentation and reciprocal location of the nuclear proteins expressed by the myeloma and the hybridoma was greater than that between these cell lines and lymphocytes. Probing of the chromatin structure by micrococcal nuclease showed no significant differences in the degree of nuclease resistance of chromatin between myeloma, hybridoma and lymphoid cells. A comparative study of the Ca/Mg-dependent endonuclease activity of the nuclei in situ and in nuclear extracts demonstrated that whereas its content in lymphocytes was rather high, in myeloma and hybridoma it was practically absent. At the same time, cell nucleus extracts of the myeloma and the hybridoma contained high amounts of DNA-binding proteins which were undetectable in mouse spleen lymphocytes.
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PMID:[Comparative characteristics of some parameters of the cell nucleus in the series myeloma-hybridoma-lymphocyte]. 848 24

Aurintricarboxylic acid (ATA), an inhibitor of Ca(2+)-dependent endonuclease activity, is often used to implicate a role for increased intracellular calcium in mechanistic toxicology studies. We report here on the ability of ATA to inhibit the activity of several NAD(H)/NADP(H)-requiring enzymes (purified or cellular homogenates), including lactic dehydrogenase, alcohol dehydrogenase, cytochrome c reductase, ethoxycoumarin o-dealkylase, isocitric dehydrogenase, glutathione reductase and glucose-6-phosphate dehydrogenase. These results were compared with the ability of ATA to inhibit micrococcal nuclease and rat liver Ca(2+)-dependent endonuclease activity in similar incubations. With the exception of alcohol dehydrogenase, ATA was a potent inhibitor of each of the purified enzymes, with IC50s ranging from 0.5 to 82 microM. In cell homogenates, however, ATA was from 10 to 100-fold less potent at inhibiting these enzymes. When exogenous protein was added to purified enzyme incubations, the effect of ATA was similarly diminished. Our results demonstrate that ATA inhibits a wide range of NAD(H)/NADP(H)-requiring enzymes in in vitro incubations using purified enzymes, but that the inhibitory effects are markedly reduced in incubations which more closely resemble a cellular milieu.
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PMID:Inhibition of NAD(H)/NADP(H)--requiring enzymes by aurintricarboxylic acid. 855 68


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