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PubMed1995

A triplet of nuclease proteins (NP42-50) is activated in human Jurkat cells undergoing apoptosis.

Activation of a triplet of nuclear proteins (NP42-50) was observed in human Jurkat T cell line following treatment with an antibody to CD95 (Fas/Apo-1), a cell surface molecule involved in apoptotic cell death. The nuclease activity, corresponding to a triplet of proteins observed at approximately 42, 45, and 50 kDa in size, was extractable, heat-stable, and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing deoxyribonucleic acids (SDS-PAGE-DNA) assay. The NP42-50 activity requires the presence of Mg2+/Ca2+ and is insensitive to inactivation by heating at 80 degrees C for 5 min. Zinc effectively inhibited the enzymatic activity of NP42-50 on SDS-PAGE-DNA and also protected Jurkat cells from the CD95-mediated apoptosis in cell cultures. The nuclease activation, however, was not a unique pathway for the CD95-mediated cell death. The apoptosis induced by arabinofuranosyl cytosine, a chemotherapeutic agent, also activated the NP42-50 nuclease activity in Jurkat cells, suggesting that a similar cascade of subsequent events in apoptosis may occur in most instances although many different signals can initiate apoptotic cell death in various cell types. The nuclease identified by this study appears to be distinguishable from DNase I or DNase II by its molecular characteristics and its enzymatic requirements. The NP42-50, with respect to the nuclease activity closely associated with apoptotic cell death, may serve as a candidate for the endonuclease(s) involved in the cleavage of DNA into fragments during apoptosis.

Amino Acid Sequence↗
PubMed1996

Regulation of the Serratia marcescens extracellular nuclease: positive control by a homolog of P2 Ogr encoded by a cryptic prophage.

The Serratia marcescens extracellular nuclease is a secreted protein that is subject to growth phase and SOS control. Regulatory mutants affecting nuclease expression have been isolated that define a new locus, nucC, essential for transcription of the nuclease gene nucA. The cloned nucC gene is able to activate efficient expression from the nucA promoter in Escherichia coli, where it normally is poorly expressed. NucC is very similar to the bacteriophage P2 Ogr protein, a transcriptional activator essential for P2 late gene expression. NucC is able to replace P2 Ogr to support the growth of P2 ogr- mutants in E. coli. Ogr is a poor activator of the nuclease promoter in E. coli, but the related delta gene product from satellite phage P4 is highly effective. The presence of genes encoding a lysozyme and a putative porin or holin in the nucC operon suggests that nucC may be part of a cryptic prophage genome. The putative holin-like membrane protein is required in E. coli for extracellular secretion of the S. marcescens nuclease.

Amino Acid Sequence↗
PubMed1998

Functions of the ATP hydrolysis subunits (RecB and RecD) in the nuclease reactions catalyzed by the RecBCD enzyme from Escherichia coli.

The RecBCD enzyme from Escherichia coli is an ATP-dependent nuclease and helicase. Two of its subunits, the RecB and RecD proteins, are DNA-dependent ATPases. We have purified RecB and RecD proteins with mutations in their consensus ATP binding sites to study the functions of these subunits in the ATP-dependent nuclease activities of RecBCD. Reconstituted heterotrimeric enzymes were prepared by mixing wild-type RecB or RecB-K29Q mutant protein (RecB*) with purified RecC protein, and with a histidine-tagged wild-type RecD (hD) or mutant hRecD-K177Q (hD*) protein. RecBCD and all four reconstituted enzymes (wild-type, two single mutants, and the double mutant) cleave a single-stranded DNA oligomer substrate (25-mer) in the absence of ATP at rates of 0.03 to 0.06 min-1. The nuclease reaction catalyzed by RecB*ChD* is not stimulated significantly by ATP, while the reactions catalyzed by RecBCD, RecBChD, RecBChD*, and RecB*ChD are 300 to 3000 fold faster in the presence of 0.5 mM ATP. RecB*ChD* also has very low ATP hydrolysis activity (approximately 10(3)-fold less than RecBCD), as do the individual mutant RecB* and hRecD* proteins (approximately 100-fold less than RecB or hRecD). The products from the ATP-stimulated nuclease reaction with the oligomer substrate suggest a mechanism where two DNA molecules bind to the enzyme in opposite orientations and are cleaved by the nuclease active site. Cleavage towards the 3'-end of one oligomer (observed with RecBChD*) depends on the wild-type RecB subunit, while RecD-dependent cleavage (observed with RecB*ChD) occurs towards the 5'-end of the second bound oligomer.

Adenosine Triphosphate↗
PubMed1990

Chloroplast DNA variability in the genus Helianthus: restriction analysis and S1 nuclease mapping of DNA-DNA heteroduplexes.

Chloroplast DNA (cpDNA) from 36 wild species of the genus Helianthus has been analysed with three restriction endonucleases (Bam HI, Hind III and Sst I). Out of the 71 restriction sites described on the reference cpDNA (sunflower cpDNA), three insertions/deletions and seven site modifications were detected during the survey of the other cpDNAs. Since restriction mapping showed only a very limited fraction of the DNA variability, we chose to adapt the S1 nuclease mapping technique to detect fine variations between chloroplast genomes. For this purpose, DNA-DNA heteroduplexes obtained between sunflower and wild-species DNAs were digested by S1 nuclease and the resulting mismatches were detected by classical endonuclease restriction and hybridization methods. The S1 nuclease mapping results were confirmed by sequencing one S1 nuclease-sensitive region detected between cultivated sunflower and two perennial wild-type species. As a result of these analyses, it appeared that the combination of restriction mapping and S1 nuclease mapping might be helpful to differentiate taxonomically close cytoplasms.

Base Sequence↗
PubMed1979

Isolation and genetic analysis of nuclease halo (nuh) mutants of Neurospora crassa.

Nuclease halo (nuh) mutants of the ascomycete Neurospora crassa have been isolated which are characterized reduced release of deoxyribonuclease (DNase) activities from colonies grown on sorbose-containing agar media. To identify nuh mutants, mutagenized isolates were transferred to commercial DNase test agar, or grown on minimal medium and then overlayed with agar that contained heat-denatured DNA. DNase activity was visualized by acid precipitation which produced clear rings of digestion (haloes) around the colonies. To identify the number of genes in which mutations lead to reduced release of nuclease activity, eleven nuh mutants were checked for close linkage and linked pairs were tested for complementation. These mutants were assigned to eight genes, and all except one were mapped in six small regions of the Neurospora linkage maps. In addition, among a large number of existing mutants which were tested for nuclease haloes, two mutants were found that showed the Nuh phenotype, namely uvs-3 and uvs-6. One of the isolated nuh mutants was also found to be sensitive to UV and was mapped close to uvs-3; it may represent a new allele of this gene. As a first step towards identification of genuine nuclease mutants, extensively backcrossed strains of mutants from different genes have been assayed for nuclease activity with denatured DNA in extracts. A pronounced reduction, compared to wild type at the same stage of growth, was found in uvs-3 and also in nuh-3, a mutant that is not UV-sensitive.

Chromosome Mapping↗
PubMed1996

Isolation and characterization of the nuclease O gene (nucO) from Aspergillus oryzae.

Nuclease O in the mycelia of Aspergillus oryzae has been purified 55-fold by successive steps of chromatography from the filtrate of the autolyzate. The molecular mass of nuclease O was 32 kDa, as estimated by SDS polyacrylamide-gel electrophoresis. The nuclease O gene (nucO) encoding this enzyme was cloned and sequenced. The open reading frame is interrupted by four introns with conserved splice sites and contains 328 amino-acid residues of the mature enzyme. A. nidulans transformants obtained by introduction of the cloned nucO gene produced 2.5-times as much nuclease O as the wild-type strain, showing that the cloned DNA fragment encodes nuclease O.

Amino Acid Sequence↗
PubMed1986

Use of sodium trichloroacetate and mung bean nuclease to increase sensitivity and precision during transcript mapping.

An improved method for mapping RNA transcript boundaries by the nuclease protection technique is presented. This method exploits the large (greater than 20 degrees C) difference in the thermal stability of RNA:DNA and DNA:DNA duplexes in concentrated chaotropic salt solutions. At 45 degrees C in 3.0 M sodium trichloroacetate RNA:DNA hybridization is very efficient but DNA:DNA duplexes remain completely denatured. For many applications, this solvent system can eliminate the need to prepare probes that are free of competing or irrelevant DNA molecules. Fifty- to 100-fold more RNA:DNA hybridization is observed when reassociation is performed in 3.0 M sodium trichloroacetate than in solutions containing high concentrations of formamide. A comparison of the use of S1 nuclease or mung bean nuclease suggests that mung bean nuclease can produce more precise and less ambiguous nuclease protection patterns.

DNA↗
PubMed1987

Putative nuclease-sensitive control element in unfertilized eggs of the sea urchin Lytechinus pictus.

Very limited nuclease-treatment of actively translating lysates from unfertilized eggs of the sea urchin Lytechinus pictus results in activation of the lysate. This activation is reflected by the ability of the nuclease-treated (or 'nuclease-activated') lysates to more efficiently utilize exogenous added RNAs. The activation may be reversed by adding small aliquots of untreated lysate to the nuclease-activated lysates. These results suggest that a nuclease-sensitive negative control element may be involved in the translational repression of unfertilized eggs, and that a deactivation of this control element is partially responsible for the dramatic activation of protein synthesis following fertilization in sea urchin eggs.

Ovum↗
PubMed1990

Kinetic studies of the hydrolysis of platinum-DNA complexes by nuclease S1.

The antitumor agent cis-diamminedichloroplatinum(II) (cis-DDP) reacts covalently with DNA and disrupts its secondary structure. Damaged DNA, but not native DNA, is readily digested by S1 nuclease, an endonuclease specific for single stranded polynucleotides. We have measured S1 nuclease digestion of platinated DNA by the release of platinum-DNA adducts and compared it with digestion of unplatinated DNA. The rate of hydrolysis of damaged substrate from platinum-DNA complexes was less than the overall rate of digestion of nucleotides. Similar results were observed for platinum-DNA complexes in native, denatured or renatured conformations. The hydrolysis of denatured platinum-DNA complexes, rb = 0.075 platinum per nucleotide, obeyed Michaelis-Menten kinetics. Taking into account the level of DNA damage, Vm, for the release of platinated adducts was 0.6 times smaller than for digestion of unplatinated DNA. Km values and competition experiments indicated that the enzyme bound equally well to platinated and unplatinated substrates. Similar results were obtained for denatured DNA complexes with trans-DDP while [PtCl(diethylenetriamine)]Cl had no influence on nuclease digestion. These results suggest that bifunctional platinum-DNA lesions have contradictory effects on the hydrolysis of double stranded DNA by S1 nuclease. On one hand they create nuclease sensitive substrate by disrupting DNA secondary structure. On the other, they inhibit digestion of the damaged strand by increasing the activation energy for hydrolysis.

Cisplatin↗
PubMed1976

Caffeine enhancement of digestion of DNA by nuclease S1.

The activity of Aspergillus orzae nuclease S1 on DNA has been investigated under varying pH and metal ion conditions. Nuclease S1 was found to preferentially digest denatured DNA. With native DNA as substrate the enzyme could only digest the DNA when caffeine was added to the reaction mixture. The enzyme was more active in sodium acetate buffer (pH 4.5), than in either standard saline citrate (PH 7.0) or sodium phosphate buffer (pH 6.8). Caffeine was also found to affect the thermal stability of DNA, resulting in a melting profile characterized by two transitions. The first transition (poorly defined) was below the normal melting temperature of the DNA, while the next transition was at the normal melting temperature of the DNA, while the next transition was at the normal melting temperature of the DNA. The susceptibility of caffeine-treated DNA to nuclease digestion seems to be a result of the local unwinding that caffeine causes in the regions of DNA that melt in the first transition. This selective destabilization presumably sensitizes the unwound regions to nuclease hydrolysis. The hydrolysates of the DNA digested by nuclease S1 were subjected first to ion exchange chromatography followed by paper chromatography. The results from this partial characterization of the digestion products showed that they contain mononucleotides as well as oligonucleotides of varying lengths. The base composition of the mononucleotide digests suggests that caffeine has greater preference for interacting with A-T base-pairs in DNA.

Animals↗
PubMed1994

Stable expression in rat glioma cells of sense and antisense nucleic acids to a human multifunctional DNA repair enzyme, APEX nuclease.

We have cloned mouse and human cDNAs for a multifunctional DNA repair enzyme (APEX nuclease) having apurinic/apyrimidinic (AP) endonuclease, 3',5' exonuclease, DNA 3' repair diesterase and DNA 3'-phosphatase activities. To investigate the biological role of APEX nuclease, sense or antisense APEX RNA was stably expressed at a high level in cultured rat glioma cells by introducing plasmids (pABWN-HAPX1F for expression of sense RNA or pABWN-HAPX2R for expression of antisense RNA) constructed from the human APEX cDNA and an expression vector pABWN. Multiple copies of the construct were integrated into the glioma cells transfected with pABWN-HAPX1F or pABWN-HAPX2R. These transfectants showed markedly high expression of RNA hybridizable to human APEX cDNA, indicating the expression of the sense or antisense RNA. Activity blotting analyses of salt extracts of these transfectants showed that the sense RNA-expressed cells had higher AP endonuclease activity and that the antisense RNA-expressed cells had extremely lower AP endonuclease activity than the control cells. The APEX nuclease-depressed glioma cells became more sensitive to methyl methanesulfonate and hydrogen peroxide than the control cells or the APEX nuclease-overexpressed cells. The results indicate that APEX nuclease plays an important role in repair of DNA damage caused by these genotoxic agents. The present stable expression systems for the sense and antisense APEX RNAs should be useful for analyzing the biological functions such as an antimutagenic function of the enzyme.

Animals↗
PubMed2005

Mre 11 p nuclease activity is dispensable for telomeric rapid deletion.

Telomeric rapid deletion (TRD) is an intrachromatid recombination process that truncates over-elongated telomeres to the genetically determined average telomere length. We have proposed that TRD is initiated by invasion of the 3' G-rich overhang into centromere-proximal telomere sequence, forming an intermediate that leads to excision of the distal telomere tract. TRD efficiency is dependent on Mre 11p and Rad50p, two members of the widely conserved Mre 11p/Rad50p/Xrs2p (MRX) complex. To investigate the role of Mre 11p in TRD, we conducted a structure/function analysis by testing the TRD rate and precision of mutations within known functional domains. We analyzed 12 alleles that disrupt different Mre 11p activities. Surprisingly, mutations in essential residues of the nuclease domain do not inhibit TRD, effectively ruling out nuclease activity as the source of the Mre 11p requirement. Interestingly, loss of Exo1p alone or loss of Exo1p in an Mre 11 nuclease deficient background does not eliminate TRD, suggesting the presence of an additional nuclease. Second, deletion of DNA binding sites A (residues 410--420) and B (residues 644--692) actually enhances the TRD rate. Even deletion of both DNA binding domains does not abrogate TRD, although its kinetics and precision are variable. This suggests altered DNA binding or a conformational defect in the MRX complex may affect the rate of TRD product formation and indicates that the DNA binding sites formally act as repressors of TRD. Remarkably, the H213Y allele (nuclease motif IV) confers an extraordinarily rapid kinetics, with the vast majority of elongated telomeres deleted imprecisely in a single round of subculturing. In striking contrast, the P162S allele that confers dissolution of the complex also exhibits the null phenotype. These data suggest that Mre 11p can act as a positive and negative regulator of TRD in context of the MRX complex that is essential for TRD.

Cell Cycle Proteins↗
PubMed2005

Ce(4+) induced down-regulation of ERK-like MAPK and activation of nucleases during the apoptosis of cultured Taxus cuspidata cells.

Ce(4+) (Ce(NH(4))(2)(NO(3))(6)) at 1mM induces apoptosis of suspension cultures of Taxus cuspidata cells; however, the underlying signal mechanisms are unknown. We show here that a 46-kDa ERK (extracellular signal-regulated kinase)-like MAPK appears to be down-regulated at 4h, and remains at low levels for up to 48 h. An inhibitor of superoxide anions (O(2)(-)) generation, diphenyl iodonium (DPI) successfully blocks down-regulation of ERK-like MAPK and degradation of DNA. Moreover, a 41-kDa p38-like MAPK activity remains unchanged from 0.5 to 48 h. The p38 inhibitor SB202190 effectively inhibits p38-like MAPK activity, however, SB202190 fails to modify the apoptotic rate at concentrations up to 100 microM. Three nuclease (34-kDa, 22-kDa and 20-kDa) activities are profoundly enhanced in Ce(4+)-induced T. cuspidata cells. They have an optimum pH at 6.8, and are stimulated by Ca(2+)/Mg(2+). Caspase-3 inhibitor, Ac-DEVD-CHO, does not attenuate the 34-kDa nuclease activity, but inhibits the 22-kDa and the 20-kDa nuclease activities. In addition, inhibition of O(2)(-) generation by DPI significantly reduces the three nuclease activities. In conclusion, the present study suggests that down-regulation of ERK-like MAPK, burst of O(2)(-), activation of caspase-3-like and induction of three nucleases as the key signaling events mediating apoptosis in Ce(4+)-induced cultured T. cuspidata cells.

Apoptosis↗
PubMed2004

Structural and functional characterization of mitochondrial EndoG, a sugar non-specific nuclease which plays an important role during apoptosis.

Combining sequence analysis, structure prediction, and site-directed mutagenesis, we have investigated the mechanism of catalysis and substrate binding by the apoptotic mitochondrial nuclease EndoG, which belongs to the large family of DNA/RNA non-specific betabetaalpha-Me-finger nucleases. Catalysis of phosphodiester bond cleavage involves several highly conserved amino acid residues, namely His143, Asn174, and Glu182 required for water activation and metal ion binding, as well as Arg141 required for proper substrate binding and positioning, respectively. These results indicate that EndoG basically follows a similar mechanism as the Serratia nuclease, the best studied representative of the family of DNA/RNA non-specific nucleases, but that differences are observed for transition state stabilisation. In addition, we have identified two putative DNA/RNA binding residues of bovine EndoG, Arg135 and Arg186, strictly conserved only among mammalian members of the nuclease family, suggesting a similar mode of binding to single and double-stranded nucleic acid substrates by these enzymes. Finally, we demonstrate by ectopic expression of active and inactive variants of bovine EndoG in HeLa and CV1-cells that extramitochondrial active EndoG by itself induces cell death, whereas expression of an enzymatically inactive variant does not.

Amino Acid Sequence↗
PubMed2004

Directed evolution of protein inhibitors of DNA-nucleases by in vitro compartmentalization (IVC) and nano-droplet delivery.

In vitro compartmentalization (IVC) uses water-in-oil emulsions to create artificial cell-like compartments in which genes can be individually transcribed and translated. Here, we present a new application of IVC for the selection of DNA-nuclease inhibitors. We developed a nano-droplets delivery system that allows the transport of various solutes, including metal ions, into the emulsion droplets. This transport mechanism was used to regulate the activity of colicin nucleases that were co-compartmentalized with the genes, so that the nucleases were activated by nickel or cobalt ions only after the potential inhibitor genes have been translated. Thus, genes encoding nuclease inhibitors survived the digestion and were subsequently amplified and isolated. Selection is therefore directly for inhibition, and not for binding of the nuclease. The stringency of selection can be easily modulated to give high enrichments (100-500-fold) and recoveries. We demonstrated its utility by selecting libraries of the gene encoding the cognate inhibitor of colicin E9 (immunity protein 9, or Im9) for inhibition of another colicin (ColE7). The in vitro evolved inhibitors show significant inhibition of ColE7 both in vitro and in vivo. These Im9 variants carry mutations into residues that determine the selectivity of the natural counterpart (Im7) while completely retaining the residues that are conserved throughout the family of immunity protein inhibitors. The in vitro evolution process confirms earlier hypotheses regarding the "dual recognition" binding mechanism and the way in which new colicin-immunity pairs diverged from existing ones.

Colicins↗
PubMed2006

RNases and nucleases in embryos and endosperms from naturally aged wheat seeds stored in different conditions.

Temperature and moisture content are particularly important factors influencing the longevity of seeds, and therefore the ageing of seeds is closely tied to storage conditions. The ageing process is characterised by many physiological and biochemical changes: membranes tend to leak, enzymes lose catalytic activity, and chromosomes accumulate mutations. Since viability loss is also associated with the breakdown of nucleic acids, the aim of the study was to determine whether the damage induced by ageing could be associated with changes in the activity of RNases and nucleases in embryos and endosperms of differently stored wheat seeds. In order to better characterise seed conditions, the damage to membranes during seed ageing was evaluated by measuring the conductivity of the soaking solution during imbibition, and by using the Evans Blue colorant; lipid peroxidation was also recorded. RNases and nucleases were studied by SDS-PAGE and activity staining. Ageing of seeds stored in a dry state involved a progressive loss of membrane integrity, which increased with the degree of ageing, while lipid peroxidation remained unchanged. Changes in nucleolytic enzyme activity were recorded in embryos: a decrease in RNases and an increase in nucleases. In the endosperm compartment there were no significant differences in ribonuclease and nuclease patterns during seed ageing. Moreover, neutral RNases were absent in endosperms of dry seeds and were activated following imbibition. Present studies reveal that embryos and endosperms have different enzymatic patterns, thus highlighting that the two seed compartments age independently. A different nucleolytic pattern was present in seeds of comparable viability and membrane damage, which were stored differently, and nuclease metabolism was subject to regulation according to both ageing and the length of the storage period.

Deoxyribonucleases↗
PubMed2003

Evaluation of the performance of LNA and MGB probes in 5'-nuclease PCR assays.

The aim of this study was to evaluate the use of Locked Nucleic Acids (LNA) probes in 5'-nuclease PCR, by comparison with Minor Groove Binder (MGB) probes routinely practiced in laboratories on ABI Prism 7700. The comparison was made using a collection of Staphylococcus aureus strains that have already been characterized by MGB 5'-nuclease PCR assays in a previous study [Mol Cell Probes, submitted for publication]. The sensitivity and specificity of 5'-nuclease PCR assays targeting the Staphylococcal enterotoxin genes sea to see were compared and showed that the LNA and MGB methods were equivalent. In conclusion, the LNA 5'-nuclease PCR assays developed in this work provide a specific and sensitive alternative to the well-established MGB 5'-nuclease PCR assays used for the rapid detection of bacterial pathogens genes on ABI Prism 7700.

DNA Primers↗
PubMed2005

Structural and functional insight into sugar-nonspecific nucleases in host defense.

In prokaryotes, sugar-nonspecific nucleases that cleave DNA and RNA in a sequence-independent manner take part in host defense, as well as site-specific restriction enzymes. Examples include the periplasmic nuclease Vvn and the secreted nuclease ColE7, which degrade foreign nucleic acid molecules in the host periplasm and in the cytoplasm of foreign cells, respectively. Recently determined crystal structures of Vvn and ColE7 in complex with double-stranded DNA provide structural insight into nonspecific DNA interactions and cleavage by sugar-nonspecific nucleases. Both nucleases bind DNA at the minor groove through a common 'betabetaalpha-metal' endonuclease motif and primarily contact the DNA phosphate backbone, probably to avoid sequence-dependent base recognition. In eukaryotes, several apoptotic endonucleases that are responsible for DNA degradation in programmed cell death also contain a betabetaalpha-metal fold at the active site, suggesting that they may recognize and cleave DNA in a comparable way.

DNA↗