Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.25.1 (deoxyribonuclease)
 1,471 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The deoxyribonuclease induced in KB cells by herpes simplex virus (HSV) type 1 and type 2 has been purified. Both enzymes are able to completely degrade single- and double-stranded DNA yielding 5'-monophosphonucleotides as the sole products. A divalent cation, either Mg2+ or Mn2+, is an absolute requirement for catalysis and a reducing agent is necessary for enzyme stability. The maximum rate of reaction is achieved with 5 mM MgCl2 for both HSV-1 and HSV-2 DNase. The optimum concentration for Mn2+ is 0.1 to 0.2 mM and no exonuclease activity is observed when the concentration of Mn2+ is greater than 1 mM. The rate of reaction at the optimal Mg2+ concentration is 3- to 5-fold greater than that at the optimal Mn2+ concentration. In the presence of Mg2+, the enzymes are inhibited upon the addition of Mn2+, Ca2+, and Zn2+. The enzymatic reaction is also inhibited by spermine and spermidine, but not by putrescine. Crude and purified HSV-1 and HSV-2 DNase can degrade both HSV-1 and HSV-2 DNA, but native HSV-1 DNA is hydrolyzed at only 22% of the rate and HSV-2 DNA at only 32% of the rate of Escherichia coli DNA. Although HSV-1 and HSV-2 DNase were similar, minor differences were observed in most other properties such as pH optimum, inhibition by high ionic strength, activation energy, and sedimentation coefficient. However, the enzymes differ immunologically.
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PMID:The deoxyribonuclease induced after infection of KB cells by herpes simplex virus type 1 or type 2. I. Purification and characterization of the enzyme. 20 46

Endo-exonuclease (EE) has been found in both active and inactive, but trypsin-activatable, forms in Aspergillus nidulans. Active EE was present mainly in nuclei, mitochondria, and vacuoles, while trypsin-activatable EE was mainly in the cytosol. The active form accounts for over 90% of the neutral deoxyribonuclease activity extracted from mycelia. A single strand (ss) DNA-binding EE associated with a 28 kilodalton (kDa) polypeptide was partially purified and characterized. It was found to closely resemble, in size and enzymological properties, the ss-DNA-binding EE previously purified from Neurospora crassa. Aspergillus nidulans EE was also found to be immunochemically related to the N. crassa EE and, like that enzyme, was probably derived from a polypeptide of 90 kDa or larger through proteolysis during extraction and purification. It had divalent metal ion-dependent (Mg2+, Mn2+, or Zn2+) activity on both DNA and RNA, which ultimately produced small 5'-P-terminated oligonucleotides. The nuclease activity was mixed endo- and exo-nucleolytic with ss-DNA as substrate, but largely exonucleolytic with double strand (ds) DNA. Superhelical phi X-174 DNA was nicked by EE to form relaxed circular and then linear ds-DNA, which was rapidly degraded to shorter fragments. Linearized pBR322 DNA was extensively nicked internally under conditions where there was relatively low exonuclease activity, but this nicking required that 5'-P-termini be present on the linear ds-DNA. The levels of active EE found in extracts of two recombination-deficient mutants of A. nidulans, uvsC and uvsE, dit not differ significantly from those in extracts of the wild type.
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PMID:Endo-exonuclease of Aspergillus nidulans. 216 74

Extracellular rabies virus, grown in monolayer cultures of BHK21 cells in the presence of medium supplemented with bovine serum albumin, was purified by the following procedure. Virus was precipitated from infectious tissue culture fluid by zinc acetate and was resuspended in a solution of ethylenediaminetetraacetate. The suspension was filtered through a Sephadex column and was treated with ribonuclease and deoxyribonuclease. The virions were then pelleted by centrifugation at high speed and were resuspended in buffer solution. Banding of the virus by centrifugation in a sucrose density gradient was the final step in the purification procedure. Purified preparations contained bullet-shaped virus particles of variable length and little (up to 5%) contaminating host-cell material. Most of the virions were "complete", i.e., 180 nm long, but some virus particles were shorter. The length distribution of the virions was nonrandom. Shorter virions seemed to be noninfectious and showed markedly decreased hemagglutinating activity. The complement-fixing activity and the ribonucleic acid to protein ratio of the virions were not related to the length of the virus particles. Although the properties of extracellular and intracellular viruses were similar, the procedure was not suitable for purification of intracellular rabies virus.
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PMID:Purification of rabies virus grown in tissue culture. 497 71

Acid deoxyribonuclease (EC 3.1.4.6) (DNase) from young (16 days of incubation) and old (1.5 years) chick cerebral hemispheres was purified to apparent homogeneity. Throughout the purification schedule, the behavior of "young" and "old" enzymes was similar. However, the specific activity of the purified enzyme from old brain was only one-tenth that of young enzyme. Polyacrylamide gel electrophoresis of the purified acid DNase gave a single band. Antisera against both "young" and "old" enzyme were raised and double immunodiffusion experiments revealed cross-reaction of young antigen with old antiserum and vice versa, although precipitin bands with young antigen against young antiserum and old antigen against old antiserum were more sharp. Both young and old acid DNase preparations showed an apparent molecular weight of 62,000 and many other properties like heat stability, effect of various exogenous compounds like Hg2+, Zn2+, Mg2+, etc., were also similar. The old enzyme showed slightly higher Km and decreased Vmax compared with the young enzyme. Dansylation of N-terminal amino acids and their analysis following tryptic digestion of both "young" and "old" acid DNase revealed a similar pattern. Immunotitration experiments showed that the old enzyme requires more antiserum prepared against "young" enzyme to achieve 50% inactivation, thus pointing out the presence of completely or partially inactive molecules in "old" acid DNase preparation. Circular dichroism spectra of the enzyme preparations indicated that the "old" acid DNase molecules are more rigid and have more alpha-helical structure, compared with the "young" enzyme. From these data, it is suggested that the reduction in the specific activity of old acid DNase may be, apart from other possibilities, due to conformational changes in the enzyme molecules.
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PMID:Age-dependent conformational changes in acid deoxyribonuclease of chick brain. 619 64

Bacteriophage T4 endonuclease VII is one of a class of structure-selective enzymes that resolve helical branchpoints in DNA molecules. The sequence of this protein suggests a modular organisation. We have expressed a synthetic gene encoding endonuclease VII, which has been used in a directed mutagenesis exercise, with the aim of understanding the role of different sections of the protein sequence. Towards the N-terminal end of the protein lies a section of polypeptide in which four cysteine residues distributed in a CxxC--CxxC pattern co-ordinate one atom of zinc. The N-terminal section composed of amino acid residues 1 to 65 isolated from the remaining C-terminal section also binds one mole of zinc, suggesting that this region folds autonomously. Mutation shows that the outer cysteine residues are essential for zinc binding, while the inner cysteine residues are partially degenerate in that either one of the two (but not both) can be replaced while retaining some zinc. The activity as a junction-resolving enzyme correlated qualitatively with the presence of the zinc. In the C-terminal part of the protein lies a section that is 48% identical with a sequence found in the DNA repair protein T4 endonuclease V. We can replace the section of T4 endonuclease VII with the corresponding sequence from T4 endonuclease V with no change in the pattern of cleavage on four-way junctions. The evidence supports a modular construction for T4 endonuclease VII.
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PMID:The modular character of a DNA junction-resolving enzyme: a zinc-binding motif in bacteriophage T4 endonuclease VII. 756 77

IL-2-dependent CTLL2 cells, upon IL-2 deprivation, die by apoptosis, which is accompanied by the fragmentation of genomic DNA. Two major deoxyribonuclease activities were detected in the extract of IL-2-deprived CTLL2 cells in a zymographic assay. They were designated nuc-58 and nuc-40, based on their apparent molecular mass of 58 and 40 kDa. The activity of both DNases was greatly induced in CTLL2 cells deprived of IL-2 or treated with the kinase inhibitor staurosporine. Deregulated expression of bcl-2 cDNA suppressed the induction of both nuclease activities. Nuc-58 was dependent on both Ca2+ and Mg2+ ions alone. Nuc-40 showed a preferential nuclear localization over that of nuc-58, which was found primarily in the cytoplasm. Optimal activity of both DNases required neutral pH and was inhibited by zinc ions. The physicochemical characteristics of the nucleases indicate that they are novel DNases associated with apoptosis in CTLL2 cells.
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PMID:Deoxyribonuclease induction in apoptotic cytotoxic T lymphocytes. 776 59

We describe a deoxyribonuclease activity from nuclear protein extracts of cultured Aedes albopictus mosquito cells. The nuclease cleaved linear and circular double-stranded DNA, first generating 3' OH single-stranded nicks followed by second strand cleavage, but had little or no exonucleolytic activity. Detection of this activity was optimal at pH 7.1, in the presence of a divalent cation (Mg2+, Ca2+, Mn2+, Ba2+). In the presence of Mg2+, Zn2+, Hg2+ and Cu2+ inhibited activity, sulfhydryl reagents and ATP had no effect. At physiological temperatures (18-35 degrees C), linear double-stranded DNA probes were preferentially cleaved near sites containing 3-6 consecutive deoxyadenine/thymine base pairs. Results from salt dependency and drug inhibition studies, combined with inspection of DNA sequence, suggested that DNA structure is among the parameters that determine preferred cleavage sites.
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PMID:Evidence for DNA endonuclease activity in nuclear extracts from mosquito cells. 785 41

Analyses of cleavage ends of DNA fragments in apoptotic rat thymocytes induced by gamma-ray irradiation or by treatment with dexamethasone revealed that in both cases the fragments produced had 3'-hydroxyl (OH) and 5'-phosphoryl (P) ends of DNA chains. Rat thymocyte nuclei contained at least three endonuclease activities (deoxyribonucleases alpha, beta and gamma) that were able to cleave chromatin to mononucleosomal and oligonucleosomal fragments. The nuclei of apoptotic rat thymocytes induced by gamma-ray irradiation or dexamethasone retained considerable deoxyribonuclease gamma activity, but not alpha or beta deoxyribonuclease activity. During the induction of apoptosis, treatment with cycloheximide, which suppressed apoptosis, resulted in marked decreases of deoxyribonucleases alpha and beta activities. After release of cycloheximide inhibition, DNA fragmentation associated with apoptosis occurred in the cycloheximide-treated thymocyte nuclei, in which deoxyribonuclease gamma activity was only observed. The purified deoxyribonucleases alpha and beta were divalent cation-independent acidic endonucleases, which were separated on a CM5PW column by HPLC. The molecular masses of deoxyribonucleases alpha and beta were 28 kDa and 30 kDa, respectively, as determined by TSK G-2000SW gel-filtration HPLC, and both were 32 kDa in molecular mass as determined by SDS/PAGE. In contrast, deoxyribonuclease gamma, a neutral endonuclease, required both Ca2+ and Mg2+ for full activity and was inhibited by Zn2+. The molecular mass of deoxyribonuclease gamma was 31 kDa and 33 kDa when measured by gel filtration and SDS/PAGE, respectively. Under these optimal conditions, deoxyribonuclease gamma was shown to produce 3'-OH/5'-P ends of nucleosomal DNA fragments, while deoxyribonucleases alpha and beta both formed DNA fragments with 3'-P/5'-OH ends. The ends formed by cleavage with deoxyribonuclease gamma were the same as those produced in apoptotic rat thymocytes. On the basis of these results, it seems likely that deoxyribonuclease gamma is responsible for internucleosomal cleavage of chromatin during thymic apoptosis.
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PMID:Identification of an endonuclease responsible for apoptosis in rat thymocytes. 795 53

Several plant ribotoxins, including gelonin, were reported to have additional weak nuclease activities on supercoiled DNA. The potential contribution of this activity to their cytotoxicity has not been given serious consideration due to concerns about contaminating nucleases in the protein preparations. We now report the degradation of single-stranded DNA by preparations of native plant gelonin and recombinant gelonin produced in E. coli. The DNase activity of both preparations is similarly modulated by zinc. An SDS-PAGE DNase assay identifies gelonin as the polypeptide responsible for deoxyribonuclease activity.
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PMID:Direct evidence for the deoxyribonuclease activity of the plant ribosome inactivating protein gelonin. 910 9

Genetic information is frequently disturbed by introduction of modified or mismatch bases into duplex DNA, and hence all organisms contain DNA repair systems to restore normal genetic information by removing such damaged bases or nucleotides and replacing them by correct ones. The understanding of this repair mechanism is a central subject in cell biology. This review focuses on the three-dimensional structural views of damaged DNA recognition by three proteins. The first protein is T4 endonuclease V (T4 endo V), which catalyzes the first reaction step of the excision repair pathway to remove pyrimidine-dimers (PD) produced within duplex DNA by UV irradiation. The crystal structure of this enzyme complexed with DNA containing a thymidine-dimer provided the first direct view of DNA lesion recognition by a repair enzyme, indicating that the DNA kink coupled with base flipping-out is important for damaged DNA recognition. The second is very short patch repair (Vsr) endonuclease, which recognizes a TG mismatch within the five base pair consensus sequence. The crystal structure of this enzyme in complex with duplex DNA containing a TG mismatch revealed a novel mismatch base pair recognition scheme, where three aromatic residues intercalate from the major groove into the DNA to strikingly deform the base pair stacking but the base flipping-out does not occur. The third is human nucleotide excision repair (NER) factor XPA, which is a major component of a large protein complex. This protein has been shown to bind preferentially to UV- or chemical carcinogen-damaged DNA. The solution structure of the XPA central domain, essential for the interaction of damaged DNA, was determined by NMR. This domain was found to be divided mainly into a (Cys)4-type zinc-finger motif subdomain for replication protein A (RPA) recognition and the carboxyl terminal subdomain responsible for DNA binding.
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PMID:Three-dimensional structural views of damaged-DNA recognition: T4 endonuclease V, E. coli Vsr protein, and human nucleotide excision repair factor XPA. 1094 33


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