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At least 631 records · Page 35Linked to original sources
PubMed1984

Effects of Neurospora nuclease halo (nuh) mutants on secretion of two phosphate-repressible alkaline deoxyribonucleases.

Various recently isolated nuh mutants of Neurospora crassa (i.e., mutants which show reduced nuclease haloes on DNA-sorbose plates flooded with HCl) were mapped in several new genes or gene clusters and checked for effects on DNA repair and nuclease secretion. Some of them were found to be sensitive to MMS (methylmethane sulfonate) and sterile in meiosis. Release of nuclease activities into filtrates of liquid cultures was analyzed by DEAE-Sepharose chromatography. In the wild type, three alkaline deoxyribonuclease activities (A, B, and C) can be separated after growth in sorbose minimal media [Fraser, M. J. (1979). Nucleic Acids Res. 6: 231]. When strains were grown in phosphate-free DNA sucrose media, high (200-fold derepressed) DNase levels were found, and crude dialyzed filtrates could be chromatographed. Only two peaks were found, namely, those of DNase A, a Ca2+-dependent strand-nonspecific endonuclease, and DNase B, a ss-DNA-specific Mg2+-dependent exonuclease. Of the nuh mutants analyzed by one or both of these methods, many resembled the wild type. A few showed poor derepression, since their sorbose filtrates were normal, while profiles from DNA media lacked all peaks. These grew variably in liquid media with organic phosphates and probably produced suppressors, as was regularly found for nuc-2. Other mutants, which lacked specific peaks, gave the same results with both methods. One of these, nuh-7, produced no peaks at all but secreted unusually high amounts of protein.

DNA Repair↗
PubMed1990

Nuclease treatment results in high specific purification of Creutzfeldt-Jakob disease infectivity with a density characteristic of nucleic acid-protein complexes.

Representative preparations of partially purified Creutzfeldt-Jakob disease (CJD), including disaggregated density gradient fractions, were treated with a variety of nucleases. RNases as well as exhaustive digestions with micrococcal nuclease did not significantly diminish infectivity, but resulted in an approximately 7,000-fold specific purification of infectivity with respect to nucleic acid. Protected nucleic acids included species of up to 2,000 bases in length. After nuclease treatment, infectivity co-migrated with nucleic acid-protein complexes at a density of 1.27 g/cm3 in sucrose. Substantial specific protein purification were also achieved in the gradient step (approximately 11,000-fold), where 70% the host Gp34 ("prion protein") as well as other free proteins separated from infectivity. These CJD purifications are better than those previously attained in scrapie, and may be useful for further studies of non-host protein and nucleic acid species. The data are consistent with the hypothesis that CJD-like agents are composed of nucleic acid-protein complexes.

Animals↗
PubMed1985

Nuclease S1 analysis of eubacterial 5S rRNA secondary structure.

Single-strand-specific nuclease S1 was employed as a structural probe to confirm locations of unpaired nucleotide bases in 5S rRNAs purified from prokaryotic species of rRNA superfamily I. Limited nuclease S1 digests of 3'- and 5'-end-labeled [32P]5S rRNAs were electrophoresed in parallel with reference endoribonuclease digests on thin sequencing gels. Nuclease S1 primary hydrolysis patterns were comparable for 5S rRNAs prepared from all 11 species examined in this study. The locations of base-paired regions determined by enzymatic analysis corroborate the general features of the proposed universal five-helix model for prokaryotic 5S rRNA, although the results of this study suggest a significant difference between prokaryotic and eukaryotic 5S rRNAs in the evolution of helix IV. Furthermore, the extent of base-pairing predicted by helix IV needs to be reevaluated for eubacterial species. Clipping patterns in helices II and IV appear to be consistent with a secondary structural model that undergoes a conformational rearrangement between two (or more) structures. Primary clipping patterns in the helix II region, obtained by S1 analysis, may provide useful information concerning the tertiary structure of the 5S rRNA molecule.

Aeromonas↗
PubMed1989

Influence of lectin concentration on the catalytic properties of S1 nuclease bound to Concanavalin A-sepharose.

Partially purified S1 nuclease was bound through its carbohydrate moiety to Con A-Sepharose containing increasing amounts of lectin. The retention of activity was high, varying essentially from 75% on the "low lectin" matrix (1 mg Con A/mL of Sepharose), to no detectable activity on the "high lectin" matrix (8 mg Con A/mL of Sepharose). However, approximately 50% activity could be restored in "high lectin" matrix when the coupling was carried out in the presence of glucose, suggesting that the loss of activity on the "high lectin" matrix is caused by conformational changes brought about by the multiple attachment of the enzyme to the matrix. Interaction of Con A with S1 nuclease was used to predict the nature of carbohydrate moiety and its location with respect to the active site of the enzyme. Immobilization resulted in an increase in the optimum temperature, pH, and temperature stabilities, but it did not affect the pH optimum. A marginal increase in the apparent Km was observed. The bound enzyme also showed enhanced stability toward 8 M urea. On repeated use, the bound enzyme retained more than 80% of its initial activity after 6 cycles. These results are discussed taking into consideration the factors affecting immobilized enzymes. In addition, the potential use of immobilized S1 nuclease as an analytical tool is discussed.

Binding Sites↗
PubMed1998

Role of yeast Rth1 nuclease and its homologs in mutation avoidance, DNA repair, and DNA replication.

The RTH1(RAD27) gene of Saccharomyces cerevisiae encodes a structure-specific endonuclease that cleaves 5'-ended single-stranded DNA at its junction with duplex DNA. Genetic and biochemical studies have indicated a role of Rth1 nuclease in the removal of RNA primers formed during DNA replication. The rth1Delta mutation confers temperature-sensitive lethality, and increases sensitivity to alkylating agents. The instability of repetitive DNA is greatly enhanced in the rth1Delta mutant. The conditional lethality of the rth1Delta mutation indicates that another nuclease can function in DNA replication in the absence of RTH1. RAD2, a homolog of RTH1, is required for nucleotide-excision repair. Here, we examine three other homologs of RTH1/RAD2 - YEN1, EXO1, and DIN7. Deletion of any of these genes in the rth1Delta strain has no effect on cell viability, suggesting the involvement of another, and as yet unidentified, nuclease in the maturation of Okazaki fragments. Our data also indicate that only RTH1 functions in the repair of alkylation damage. Deletions of YEN1, EXO1, DIN7, or RAD2, either singly or when combined with one another and with the rth1Delta mutation, have no effect on the rate of instability of dinucleotide repeats or on the rate of formation of large duplications in the CAN1 gene. These data provide evidence of a high degree of specificity for the role of RTH1 in DNA replication and in base-excision repair, and for the requirement of RAD2 in nucleotide-excision repair. The possibility that both Rth1 and Exo1 function in DNA mismatch repair is discussed.

Alkylation↗
PubMed2001

Expression kinetics of the late UL12 gene encoding the bovine herpesvirus 1 alkaline nuclease.

We characterized the expression kinetics of the transcript and protein generated from the bovine herpesvirus 1 (BHV1) homologue of the herpes simplex virus 1 (HSV1) UL12 gene that encodes a viral alkaline nuclease. The BHV1 UL12 coding sequence, which was previously shown to express in E. coli a protein exhibiting nuclease activity, is located at positions 82776-->84239 of the viral genome. Northern blot analysis of RNA from BHV1-infected cells with a single strand RNA probe complementary to UL12 detected four specific 3' coterminal viral transcripts of 4.2, 3.7, 2.2, and 0.7 kb that accumulated simultaneously from 6 to 24 hours post-infection (p.i.). S1 nuclease mapping of the UL12 capping site at position 82384 of the genome as well as the identification of a consensus polyadenylation signal at 84490-84495 allowed us to establish that the 2.2 kb transcript corresponds to that of UL 12. A UL 12 specific antiserum generated against a T7-Tag/UL12 fusion protein expressed in E. coli detected a 53 kDa protein in cell lysates from BHV1-infected cells, whose size correlated with that predicted (51,844 Da), which accumulated from 12 to 30 h p.i. Differences observed between the transcriptional and translational expression profiles suggest that the UL12 of BHV1 is regulated at both the translational and posttranslational levels. Surprisingly, the protein expression was strictly dependent on viral DNA synthesis, unambiguously demonstrating that BHV1 UL12 belongs to viral genes of the gamma2 class. This is in contrast to the HSV1 and pseudorabies homologues that are classified as early (beta) genes. Further studies will be required to determine whether these kinetic differences have any functional implications.

Animals↗
PubMed1978

Studies on sheep kidney nuclease. I. An improved purification method and some properties.

An improved purification method of the sheep kidney nuclease (nuclease SK) specific for single-strans nucleic acid, which includes extraction with 0.85% NaCl, treatment with DEAE-cellulose, fractionation with polyethylene glycol, phospho-cellulose chromatography, CM-Sephadex chromatography and phospho-cellulose rechromatography is described. The nuclease was purified approx. 390-fold. Identity was established by comparison with known properties. Molecular weight was estimated to be 52 000-53 000 by gel filtration on Sephadex G-100. The properties of the purified enzyme agreed well those reported previously. The purified enzyme hydrolyzed heat-denatured calf thymus DNA, yeast RNA and no hydrolytic activity for native calf thymus DNA, A2'-pA, A3'-pA, ADP, ATP, 5'-AMP and cyclic AMP.

Animals↗
PubMed1978

Studies on sheep kidney nuclease. II. Limited digestion of phiX174 single-strand DNA.

Single strand DNA of bacteriophage phiX174 was digested by sheep kidney nuclease (nuclease SK) at 37 degrees C for 2-6 h and the digests were examined by 2.5% polyacrylamide gel electrophoresis. On this electrophoresis five bands were detected corresponding to the DNA fragments by scanning the gel at 260 nm. After these DNA fragments from the gel with the electrode buffer used for the electrophoresis, the molecular weights of fragments I, II, III, IV and V were determined by sucrose density gradient centrifugation. The molecular weights of these DNA fragments were 1.7 . 10(6), 1.2 . 10(6), 8.6 . 10(5), 4.5 . 10(5) and 2.8 . 10(5). From the melting curves and the base compositions of these fragments it was suggested that phiX174 single-strand DNA might have intramolecular double helical regions, which were hardly susceptible to attack by nuclease SK.

Animals↗
PubMed1979

Characterization of alkaline nuclease from rat liver mitochondria.

The alkaline nuclease (pH optimum 9.0) has been purified about 500-fold in 25% yield from the extract of rat liver mitochondria. The enzyme cleaves yeast RNA, poly(U), poly(U), poly(C) and denatured DNA to yield oligonucleotides with 5'-phosphoryl and 3'-hydroxyl ends. The enzyme has a molecular weight of about 60 000, a sedimentation coefficient of 4 S and an isoelectric point of 9.0. The behaviors of RNAase activity of the nuclease are identical with those of DNAase activity in column chromatography as well as in catalytic nature. The affinities of RNAase activity for substrate, Mg2+, spermidine and polyvinyl sulfate are lower than those of DNAase activity. The alkaline nuclease activity measured in the homogenate of regenerating rat liver is not significantly changed.

Animals↗
PubMed1991

Ca2+/Mg(2+)-dependent nuclease: tissue distribution, relationship to inter-nucleosomal DNA fragmentation and inhibition by Zn2+.

Ca2+/Mg(2+)-dependent endonuclease has been implicated in the extensive internucleosomal DNA fragmentation that accompanies apoptosis (gene-directed cell death). We present further evidence that this enzyme is involved in apoptosis. Ca2+/Mg2+ nuclease activity was increased about 6-fold during colchicine-induced apoptosis in human chronic lymphocytic leukaemia cells. The increase in activity coincided with onset of DNA fragmentation. Spleen, liver, kidney and thymus expressed high levels of this enzyme while lung, brain, heart and testis contained little activity. Cells from tissues with high Ca2+/Mg2+ nuclease activity underwent rapid DNA fragmentation in response to a Ca2+ flux. Physiological concentrations of Zn2+ known to inhibit both apoptosis and DNA fragmentation also inhibited Ca2+/Mg2+ nuclease activity.

Animals↗
PubMed1988

Sensitivity of intergenic regions of yeast mitochondrial DNA to single-strand-specific nucleases.

The reactivity of mitochondrial DNA (mtDNA) sequences from Torulopsis glabrata and Saccharomyces cerevisiae towards single-strand-specific nucleases has been examined. AT-rich stretches located in intergenic sequences from both yeasts were cleaved by nucleases when the sequences were contained in supercoiled plasmid DNA. In particular ori/rep sequences from the mtDNA of S. cerevisiae were shown to be sensitive to the single-strand-specific nucleases. The locations of the sensitive sites were related to the organisation of the sequence domains of ori/rep and the superhelicity of the DNA, as well as the presence of particular sequences. It is proposed that distortions of the DNA duplex could be generated in mtDNA molecules in vivo and that these distortions may provide a substrate for enzymes involved in transmission, recombination and/or transcription of mtDNA.

Candida↗
PubMed1993

Separation of isoforms of Serratia marcescens nuclease by capillary electrophoresis.

Three S. marcescens nuclease isoforms differing mainly in charge (native nuclease with pI 6.8 and two minor isoforms with pI 7.3 and 7.4) were separated using several different modes of high-performance capillary electrophoresis. Separation of the isoforms by free solution capillary electrophoresis was unsatisfactory. Separation by micellar electrokinetic capillary chromatography was therefore investigated in detail and the method optimized with respect to pH and sodium dodecyl sulphate concentration; in addition, the effect of adding various substances to control dispersion and avoid analyte adsorption at the capillary wall was examined. Under optimal conditions there was almost complete baseline separation of the two isoforms with basic pI whereas there was only partial separation of the native form and the isoform with pI 7.4. With capillary isoelectric focusing there was complete baseline separation of the native nuclease and the other two isoforms.

Amino Acid Sequence↗
PubMed1988

Recognition of the DNA helix stabilizing anthramycin-N2 guanine adduct by UVRABC nuclease.

The binding of the anti-tumor antibiotic anthramycin to a defined linear DNA fragment was investigated using both exonuclease III and lambda exonuclease. We show that most of the guanine residues are reactive toward anthramycin; however, several guanine residues showed preferential reactivity for the drug. Using purified UVRA, UVRB and UVRC proteins we present evidence that these three proteins in concert are able to recognize and produce specific strand cleavage flanking anthramycin-DNA adducts. The cleavage of anthramycin adducts by UVRABC nuclease is specific and results in strand breaks at five or six bases 5' and three or four bases 3'-flanking an adduct. At some guanine residues single incisions were observed only on one side of the adduct. The 5' strand breaks observed often occurred as doublet bands on sequencing gels, indicating plasticity in the site of 5' cleavage whereas the 3' cleavage did not show this effect. When DNA fragments modified with elevated levels of anthramycin were used as substrates the activity of the UVRABC nuclease toward the anthramycin adducts decreased. Possible mechanisms for the recognition and specific cleavage of the helix-stabilizing anthramycin DNA adduct and other helix destabilizing lesions by the UVRABC nuclease are discussed.

Anthramycin↗
PubMed1988

Nucleotide and deduced amino acid sequence of stp: the bacteriophage T4 anticodon nuclease gene.

Pre-existing host tRNAs are reprocessed during bacteriophage T4 infection of certain Escherichia coli strains. In this pathway, tRNALys is cleaved 5' to the wobble base by anticodon nuclease and is later restored in polynucleotide kinase and RNA ligase reactions. Anticodon nuclease depends on prr, a locus found only in host strains that restrict T4 mutants lacking polynucleotide kinase and RNA ligase; and on stp, the T4 suppressor of prr restriction. stp was cloned and the nucleotide sequences of its wild-type and mutant alleles determined. Their comparison defined an stp open reading frame of 29 codons at 162.8 to 9 kb of T4 DNA (1 kb = 10(3) base-pairs). We suggest that stp encodes a subunit of anticodon nuclease, perhaps one that harbors the catalytic site; while additional subunits, such as a putative prr gene product, impart protein folding environment and tRNA substrate recognition.

Amino Acid Sequence↗
PubMed1991

Initiation of the UvrABC nuclease cleavage reaction. Efficiency of incision is not correlated with UvrA binding affinity.

The incision steps of Escherichia coli nucleotide excision repair are mediated by the UvrABC nuclease complex. We have previously shown that the UvrABC nuclease specifically incises apyrimidinic (AP) sites less efficiently than o-benzylhydroxylamine-modified apyrimidinic (BA) sites. To investigate these differences, quantitative DNase I footprinting titration studies were performed. The UvrA binding isotherms were similar for both the AP site (Kd = 6 x 10(-9) M) and the bulkier BA lesion (Kd = 14 x 10(-9) M), despite the fact that the extent of incision differs for these two lesions. It was also found that the relative binding affinity of the preincision UvrA2B complex to the AP and BA substrates differs significantly with estimated apparent equilibrium dissociation constants (Kd) of 4 x 10(-9) M and 80 x 10(-9) to 120 x 10(-9) M, respectively. These results indicate that incision efficiency does not correlate to UvrA binding affinity, but is a direct result of interactions between the UvrA2B complex and the site of the DNA damage. It is also shown that high UvrA concentrations are inhibitory to the UvrABC nuclease reaction.

Base Sequence↗
PubMed1991

Crystallization and preliminary crystallographic analysis of a novel nuclease from Serratia marcescens.

Crystals have been obtained of the extracellular endonuclease from the bacterial pathogen Serratia marcescens. This magnesium-dependent enzyme is equally active against single and double-stranded DNA, as well as RNA, without any apparent base preference. The Serratia nuclease is not homologous with staphylococcal nuclease, the only other broad specificity endonuclease for which a structure exists, nor is it homologous with other nucleases that have been solved by X-ray diffraction. The structure of this enzyme should, therefore, provide new information about this class of enzyme. At present we have succeeded in obtaining large, high quality crystals using ammonium sulfate. They crystallize in the orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 106.7 A, b = 74.5 A, c = 68.9 A, and diffract to beyond 2 A. Low-resolution native data sets have been recorded and a search is under way for heavy-atom derivatives.

Endodeoxyribonucleases↗
PubMed1990

Nuclease sensitivity of estradiol-charged estrogen receptor binding sites in nuclei isolated from normal and neoplastic rat mammary tissues.

The interaction of partially purified calf uterine estradiol-charged estrogen receptor ([3H]ER) with rat nuclei was studied in vitro. We previously observed a significantly greater number of [3H]ER binding sites (at saturation) in nuclei of R3230AC mammary tumors from intact vs ovariectomized (ovex) rats with no difference in the affinity of [3H]ER binding for these nuclei. We now report on the nuclease sensitivity of [3H]ER binding sites in nuclei from these tumors and from normal rat tissues. Digestion of tumor nuclei with deoxyribonuclease I (DNase I) prior to incubation with [3H]ER in vitro resulted in a progressive loss of [3H]ER binding capacity, which was not accompanied by alterations in the affinity of [3H]ER for the nuclei (Kd = 1-3 nM). A significantly lower concentration (P less than 0.005) of DNase I eliminated 50% of the [3H]ER binding sites in nuclei of tumors from intact hosts (8 unit.min/ml) compared to tumors from ovex hosts (22 unit.min/ml). These results indicate that DNA regions capable of binding ER are more susceptible to DNase I digestion in tumors from intact rats than those from ovex hosts, suggesting that the endogenous hormonal milieu is responsible, at least in part, for maintenance of nuclease-sensitive DNA conformations in this hormone-responsive mammary tumor. The amount of DNase I required to eliminate 50% of [3H]ER binding to nuclei from lactating mammary gland, liver, and kidney ranged from 14 to 56 unit.min/ml. Therefore, accessibility of [3H]ER binding sites to nuclease digestion in normal rat tissue is generally less than that of R3230AC tumors.

Animals↗
PubMed1986

Single-stranded deoxyribonucleic acid nuclease induced by African swine fever virus and associated to the virion.

Infection of Vero cells with African swine fever (ASF) virus resulted in a marked increase of DNase active on single-stranded DNA (ss-DNase). No increase was observed for double-stranded DNA-specific nuclease activity. In contrast to uninfected cell ss-DNase, which has a pH optimum at pH range 8.5-9, virus-induced ss-DNase is most active at pH 7. Differences in sensitivity to several ions and other modifications of the reaction mixture and considerable difference in reaction kinetics suggest that the increase in nuclease activity is due to a new virus-induced enzyme. This is strengthened by the fact that anti-ASF virus antiserum inhibits the activity of ss-DNase from infected cells but not from uninfected cells. Exclusion chromatography of the digests shows that virus-specific ss-DNase is exclusively or predominantly an endonuclease. The increase in nuclease activity of infected cells is proportional to the multiplicity of infection. Virus-specific ss-DNase is synthesized at late times after infection and its synthesis is dependent on viral DNA replication since it is not induced when infected cells are treated with cytosine arabinoside. Most of ss-DNase activity in infected cells is associated to an insoluble cytoplasmic fraction, presumably virosomes. The enzyme can also be detected in partially stripped purified virions which hydrolyze 6.9 ng DNA per microgram viral protein.

African Swine Fever Virus↗