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)

The aim of the present study was to reassume the chromatin changes occurring in lymphoid tissues of mice treated with alkylating agents of the nitrogen-mustard type in relation to recent evidence on the nucleosomal organization of chromatin and to our new data on the regular character of chromatin degradation in lymphoid tissues of irradiated mice. DNA was isolated from nuclei at various intervals (1-18 h) after treatment of mice and subjected to gel electrophoresis in polyacrylamide gels. Thymus chromatin from treated mice has been shown to degrade in a regular fashion and to yield discrete DNA fragments, resembling those that originate in lymphoid tissues of irradiated mice or in thymus nuclei digested with micrococcal nuclease in vitro. With increasing interval after treatment higher amounts of smaller DNA fragments appear. Chromatin in spleen cells responds to treatment in a similar way, whilst no degradation in vivo takes place in liver chromatin. Chromatin of LS/BL lymphosarcoma cells in mice treated with alkylating agents or with irradiation suffers from a similar regular degradation. The results stress the significance of the action of liberated or activated endogenous nuclease(s) in the development of chromatin damage in lymphoid cells after treatment with alkylating agents.
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PMID:Regular character of chromatin degradation in lymphoid tissues after treatment with biological alkylating agents in vivo. 58 77

Four different footprinting techniques have been used to probe the DNA sequence selectivity of Thia-Net, a bis-cationic analogue of the minor groove binder netropsin in which the N-methylpyrrole moieties are replaced by thiazole groups. In Thia-Net the ring nitrogen atoms are directed into the minor groove where they could accept hydrogen bonds from the exocyclic 2-amino group of guanine. Three nucleases (DNAase I, DNAase II, and micrococcal nuclease) were employed to detect binding sites on the 160bp tyr T fragment obtained from plasmid pKM delta-98, and further experiments were performed with 117mer and 253mer fragments cut out of the plasmid pBS. MPE.Fe(II) was used to footprint binding sites on an EcoRI/HindIII fragment from pBR322. Thia-Net binds to sites in the minor groove containing 4 or 5 base pairs which are predominantly composed of alternating A and T residues, but with significant acceptance of intrusive GC base pairs. Unlike the parent antibiotic netropsin, Thia-Net discriminates against homooligomeric runs of A and T. The evident preference of Thia-Net for AT-rich sites, despite its containing thiazole nitrogens capable of accepting GC sites by hydrogen bonding, supports the view that the biscationic nature of the ligand imposes a bias due to the electrostatic potential differences in the receptor which favour the ligand reading alternating AT sequences.
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PMID:DNA-sequence specific recognition by a thiazole analogue of netropsin: a comparative footprinting study. 165 46

The backbone 1H and 15N resonances of unligated staphylococcal nuclease H124L (recombinant protein produced in Escherichia coli whose sequence is identical to the nuclease produced by the V8 strain of Staphylococcus aureus) have been assigned by three-dimensional (3D) 1H-15N NOESY-HMQC NMR spectroscopy at 14.1 tesla. The protein sample used in this study was labeled uniformly with 15N to a level greater than 95% by growing the E. coli host on a medium containing [99% 15N]ammonium sulfate as the sole nitrogen source. The assignments include 82% of the backbone 1HN and 1H alpha resonances as well as the 15N resonances of non-proline residues. Secondary structural elements (alpha-helices, beta-sheets, reverse turns, and loops) were determined by analysis of patterns of NOE connectivities present in the 3D spectrum.
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PMID:Solution studies of staphylococcal nuclease H124L. 1. Backbone 1H and 15N resonances and secondary structure of the unligated enzyme as identified by three-dimensional NMR spectroscopy. 173 48

Samples of staphylococcal nuclease H124L (cloned protein overproduced in Escherichia coli whose sequence is identical with that of the nuclease isolated from the V8 strain of Staphylococcus aureus) were labeled uniformly with carbon-13 (26% ul 13C), uniformly with nitrogen-15 (95% ul 15N), and specifically by incorporating nitrogen-15-labeled leucine ([98% 15N]Leu) or carbon-13-labeled lysine ([26% ul 13C]Lys), arginine ([26% ul 13C]Arg), or methionine ([26% ul 13C]Met). Solutions of the ternary complexes of these analogues (nuclease H124L-pdTp-Ca2+) at pH 5.1 (H2O) or pH* 5.5 (2H2O) at 45 degrees C were analyzed as appropriate to the labeling pattern by multinuclear two-dimensional (2D) NMR experiments at spectrometer fields of 14.09 and 11.74 T: 1H-13C single-bond correlation (1H[13C]SBC); 1H-13C single-bond correlation with NOE relay (1H[13C]SBC-NOE); 1H-13C single-bond correlation with Hartmann-Hahn relay (1H-[13C]SBC-HH); 1H-13C multiple-bond correlation (1H[13C]MBC); 1H-15N single-bond correlation (1H-[15N]SBC); 1H-15N single-bond correlation with NOE relay (1H[15N]SBC-NOE). The results have assisted in spin system assignments and in identification of secondary structural elements. Nuclear Overhauser enhancements (NOE's) characteristic of antiparallel beta-sheet (d alpha alpha NOE's) were observed in the 1H [13C]-SBC-NOE spectrum of the nuclease ternary complex labeled uniformly with 13C. NOE's characteristic of alpha-helix (dNN NOE's) were observed in the 1H[15N]SBC-NOE spectrum of the complex prepared from protein labeled uniformly with 15N. The assignments obtained from these multinuclear NMR studies have confirmed and extended assignments based on 1H[1H] 2D NMR experiments [Wang, J., LeMaster, D. M., & Markley, J. L. (1990) Biochemistry (preceding paper in this issue)].
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PMID:Two-dimensional NMR studies of staphylococcal nuclease. 2. Sequence-specific assignments of carbon-13 and nitrogen-15 signals from the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex. 232 33

Exonucleolytic cleavage of DNA by the recBC DNase is accompained by a DNA-dependent ATP hydrolysis that ceases when the DNA that has been digested to a limit. On the other hand, DNA that has been crosslinked by 4,5',8-trimethylpsoralen in the presence of 360-nm light remains an effective cofactor in the ATPase reaction, but is resistant to digestion by the enzyme. Psoralentreated DNA is degraded by pancreatic DNase, micrococcal nuclease, and Escherichia coli B restriction enzyme, but not by Neurospora crassa nuclease, suggesting that crosslinking did not grossly distort the duplex structure of the DNA. The psoralen-DNA is not a potent inhibitor, but competes with single-stranded DNA from bacteriophage fd for the recBC DNase to roughly the same extent as does normal duplex DNA. DNA treated with psoralen in the dark, exposed to 360-nm light in the absence of psoralen, or treated with the intercalating agents ethidium bromide, 9-aminoacridine, ICR-191, or actinomycin D, responds to the enzyme no differently from untreated DNA. However, DNA crosslinked with mitomycin C or nitrogen mustard behaves similarly to psoralen-treated DNA. The relationship of these findings to models for the function and control of the recBC ATPase and nuclease, and the advantages of psoralen as a DNA crosslinking agent, are discussed.
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PMID:Uncoupling of the recBC ATPase from DNase by DNA crosslinked with psoralen. 426 6

Covalent adducts formed by the reaction of DNA with chemical carcinogens and mutagens may be detected by a 32P-labeling test. DNA preparations exposed to chemicals known to bind covalently to DNA [N-methyl-N-nitrosourea, dimethyl sulfate, formaldehyde, beta-propiolactone, propylene oxide, streptozotocin, nitrogen mustard, and 1,3-bis(2-chloroethyl)-1-nitrosourea] were digested to a mixture of deoxynucleoside 3'-monophosphates by incubation with micrococcal endonuclease (EC 3.1.31.1) and spleen exonuclease (EC 3.1.16.1). The digests were treated with [gamma-32P]ATP and T4 polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) to convert the monophosphates to 5'-32P-labeled deoxynucleoside 3',5'-bis-phosphates. These compounds were then separated on polyethyleneimine-cellulose thin layers in ammonium formate and ammonium sulfate solutions. Autoradiograms of the chromatograms obtained by this high-resolution procedure showed the presence of nucleotides derived from chemically altered, as well as normal, DNA constituents. Maps from DNA exposed to any of the chemicals used exhibited a spot pattern typical for the particular chemical. This method detected a single adduct in 10(5) DNA nucleotides without requiring that the compound under investigation be radioactive and thus provides a useful test to screen chemicals for their capacity to damage DNA by covalent binding.
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PMID:32P-labeling test for DNA damage. 703 43

The radiation chemistry of the DNA tetranucleoside triphosphate d(ApCpGpT) was investigated. Irradiations were carried out on aqueous solutions saturated with oxygen (with and without added Cu++), nitrogen or nitrous oxide. When oxygen was present, principal products were formed by hydroxylation at the 8-position of guanine and by degradation of thymine leaving a formamido remnant. Products were also formed containing both of the aforementioned lesions at adjacent deoxyguanosine and pyrimidine nucleosides. Other products resulted from rearrangement of the thymine ring generating two diastereoisomers of the 5-methyl-5-hydroxyhydantoin modification of d(ApCpGpT). Rearrangement of the cytosine ring occurred generating imidazolidine products and a hydantoin product. The product profiles are similar when either an N2O or N2 gaseous environment is maintained. However, in the latter case the dihydrothymine modifications of d(ApCpGpT) are markedly enhanced. Other products include an 8,5' cyclized product formed from the 2'-deoxyadenosine nucleoside and the 8-hydroxyguanine modification. 6-Hydroxy-5,6-dihydrothymine and 5,6-dihydroxy-5,6-dihydrothymine modifications of the thymidine nucleoside were also observed. A strand break product formed in oxygenated solution is also produced in nitrous oxide saturated solutions. Scission of the deoxyadenosine terminus was also observed. The effect of several of these lesions on d(ApCpGpT) as substrate for nuclease P1, bovine spleen phosphodiesterase and snake venom phosphodiesterase was studied.
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PMID:Radiation chemistry of d(ApCpGpT). 749 May 1

Poly(ADP-ribose) polymerase (PADPRP) catalyzes the transfer of multiple ADP-ribose units from NAD to nuclear histone and nonhistone proteins, a reaction that appears to be important in the rejoining of DNA strand breaks during DNA repair and replication. We previously established and characterized a HeLa cell line that was stably transfected with a recombinant expression plasmid containing the mouse mammary tumor virus promoter upstream of a construct encoding PADPRP antisense RNA. We now show that after depletion of PADPRP mRNA as a result of antisense RNA expression, normal PADPRP mRNA concentrations are restored between 8 and 16 h after removal of dexamethasone (which activates the mouse mammary tumor virus promoter). By depleting antisense cells of PADPRP, we demonstrated the contribution of this enzyme to various aspects of nuclear structure and function: (a) amplification of a selectable gene encoding three early enzymes in the pyrimidine biosynthetic pathway was greatly increased in cells depleted of PADPRP; (b) chromatin structure was significantly altered in PADPRP-depleted cells, as indicated by reduced initiation and elongation of poly(ADP-ribose) chains attached to various nuclear protein acceptors, lower levels of poly(ADP-ribosyl)ation of histone H1, and an increased susceptibility of DNA to micrococcal nuclease digestion; and (c) the survival of PADPRP-depleted antisense cells exposed to the DNA alkylating and carcinogenic agent methyl methanesulfonate or nitrogen mustard was significantly reduced relative to that of control cells.
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PMID:Depletion of nuclear poly(ADP-ribose) polymerase by antisense RNA expression: influences on genomic stability, chromatin organization, and carcinogen cytotoxicity. 806 55

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

We investigated the role of W140 in the folding of Staphylococcal nuclease. For this purpose, we constructed the 19 possible substitution mutations at residue 140. Only three mutants, W140F, W140H, and W140Y, adopted native-like structures under physiological conditions and showed native-like enzymatic activities. In contrast, the other 16 mutants took on compact unfolded structures under physiological conditions and the enzymatic activities of these mutants were decreased to approximately 70% of wild-type levels. These 16 mutants maintained substrate-induced foldability. These results strongly indicate that the side-chain information encoded by residue 140 is essential to maintain a stable native structure, and that this residue must be an aromatic side chain. The order of thermal stability was wild type > W140H > W140F = W140Y. Therefore, the five-membered nitrogen-containing ring of the indole is thought to bear the essential information. In the crystal structure of staphylococcal nuclease, the five-membered ring is at the local center of the C-terminal cluster through hydrophobic interactions. This cluster plays a key role in the interaction connecting the C-terminal region and the N-terminal beta-core. Mutants other than W140H, W140F, and W140Y lost the ability to form the local core, which caused the loss of the long-range interactions between the C-terminal and N-terminal regions. Inhibitor or substrate binding to these mutants compensates for the lack of long-range interactions generated by W140.
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PMID:Elucidation of information encoded in tryptophan 140 of staphylococcal nuclease. 1557 80


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