Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “NUCLEASES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,027 records · Page 57Linked to original sources
PubMed2002

The nuclease A inhibitor represents a new variation of the rare PR-1 fold.

Nuclease A (NucA) from Anabaena sp. is a non-specific endonuclease able to degrade single and double-stranded DNA and RNA. The endonucleolytic activity is inhibited by the nuclease A inhibitor (NuiA), which binds to NucA with 1:1 stoichiometry and picomolar affinity. In order to better understand the mechanism of inhibition, the solution structure of NuiA was determined by NMR methods. The fold of NuiA is an alpha-beta-alpha sandwich but standard database searches by DALI and TOP revealed no structural homologies. A visual inspection of alpha-beta-alpha folds in the CATH database revealed similarities to the PR-1-like fold (SCOP nomenclature). The similarities include the ordering of secondary structural elements, a single helix on one face of the alpha-beta-alpha sandwich, and three helices on the other face. However, a major difference is in the IV helix, which in the PR-1 fold is short and perpendicular to the I and III helices, but in NuiA is long and parallel to the I and III helices. Additionally, a strand insertion in the beta-sheet makes the NuiA beta-sheet completely antiparallel in organization. The fast time-scale motions of NuiA, characterized by enhanced flexibility of the extended loop between helices III and IV, also show similarities to P14a, which is a PR-1 fold. We propose that the purpose of the PR-1 fold is to form a stable scaffold to present this extended structure for biological interactions with other proteins. This hypothesis is supported by data that show that when NuiA is bound to NucA significant changes in chemical shift occur in the extended loop between helices III and IV.

Anabaena↗
PubMed1997

Nuclease stability as dominant factor in the antiviral activity of oligonucleotides directed against HSV-1 IE110.

The anti-herpes simplex virus type 1 (anti-HSV-1) efficacy of a series of oligonucleotides was determined as a function of their chemical structure. All oligonucleotides consisted of the same sequence directed against the translation initiation codon of the HSV-1 immediate early gene. The oligonucleotides were modified with phosphorothioate linkage patterns according to various protection strategies against nucleolytic degradation. We show that nuclease resistance is the dominant factor that determines the antiviral efficacy in this series. A minimal protection strategy, consisting of end-capping and pyrimidine protection, has proven to be particularly useful, because it not only yields nuclease-resistant oligonucleotides but also minimizes non-sequence-specific effects.

Animals↗
PubMed1997

The interaction of plasmid DNA with polyamidoamine dendrimers: mechanism of complex formation and analysis of alterations induced in nuclease sensitivity and transcriptional activity of the complexed DNA.

The application of synthetic vectors for gene transfer has potential advantages over virus-based systems. However, little is known about the mechanisms involved in binding of synthetic materials to DNA and the nature of the DNA complexes that result from this interaction. Polyamidoamine (PAMAM) dendrimers are unique polymers with defined spherical structure. Dendrimers bind DNA to form complexes that efficiently transfect cells in vitro. We examined the formation of DNA/dendrimer complexes and found it based entirely on charge interaction. Electronmicroscopic examination of the complexes indicated that the majority of the plasmid DNA is contracted into isolated toroids, but also revealed larger, irregular aggregates of polymer and DNA. The binding of plasmid DNA to dendrimer appears to alter the secondary and tertiary structure, but does not fragment the DNA or alter its primary structure. Complexed DNA is protected against degradation by either specific nucleases or cellular extracts containing nuclease activity. While the initiation of transcription in vitro from promoters (for either T7 polymerase or eukaryotic RNA polymerase II) in dendrimer-complexed DNA is inhibited, elongation of the RNA transcript and translation do not appear to be affected. These resemble alterations of the DNA function when complexed with naturally-occurring polycations like non-acetylated histones. However, DNA complexed to dendrimer appears to maintain transcriptional activity while histone complexes at similar charge ratios do not. These results elucidate some aspects of the interaction between PAMAM dendritic polymers and DNA, and could lead to improvements in the design of polymers or formation of DNA complexes that will increase the efficiency of non-viral gene transfer.

DNA↗
PubMed2002

Factors affecting the performance of 5' nuclease PCR assays for Listeria monocytogenes detection.

The design and operating parameters affecting the performance of 5' nuclease PCR (TaqMan) assays for the detection of Listeria monocytogenes was investigated. A system previously developed and based on the hlyA gene was used as a model [Appl. Environ. Microbiol. 61 (1995) 3724]. A series of fluorogenic probes labeled with a reporter and a quencher dye was synthesized to explore the effect of probe position and sequence content on the efficiency of probe hydrolysis. In addition, a series of PCR primer pairs that altered the distance between the upstream primer and the interceding probe was examined. The effects of various assay parameters were evaluated by measuring the ratio of the fluorescence intensity of the reporter dye over the quencher dye (deltaRQ). For a given probe sequence, the deltaRQ was typically lower if the 5' terminus was a G residue. Decreasing the probe concentration increased the deltaRQ, although this was at the expense of reproducibility in the assay readout. The distance between the upstream primer and the interceding probe has a significant effect on probe hydrolysis. Reducing the primer-probe distance from, for example, 127 to 4 nt increased the deltaRQ from 2.87 to 5.00. These general rules were used to develop a 5' nuclease PCR (TaqMan) assay with enhanced signal output, providing higher and more reproducible deltaRQ values for L. monocytogenes detection.

Bacterial Toxins↗
PubMed1999

Detection and quantification of Salmonella in pure cultures using 5'-nuclease polymerase chain reaction.

Advances in detection and quantification assays based on nucleic acids conceivably will revolutionize the ability to quickly and specifically detect and quantify microorganisms in foods. Among these assays, the polymerase chain reaction (PCR) assay and the TaqMan PCR Detection System (Perkin-Elmer) probably are among the most promising. Since a 5'-nuclease PCR renders possible the automated and direct detection and quantification of PCR products (Holland et al., 1991. Proc. Natl. Acad. Sci. USA 88, 7276-7280), microorganisms in foods can be detected and quantified indirectly within a few hours through analysis of the microbial DNA or RNA sequences present. In the present report we have adapted a 5'-nuclease-based kit for the quantification of Salmonella.

Colony Count, Microbial↗
PubMed1999

Tumour suppressor protein p53 released by nuclease digestion increases at the onset of rat liver regeneration.

BACKGROUND/AIMS: Protein kinase CK2 (CK2) increases when cells are committed to proliferate, as in liver regeneration. This enzyme phosphorylates the tumour suppressor protein p53, whose expression controls the levels of many other cell cycle proteins. The aim of this study was to determine if CK2 was affected by p53. METHODS: Male Sprague-Dawley rats (200-250 g) were subjected to either partial hepatectomy or laparotomy and the levels and subcellular distribution of p53 were studied, following the approach used earlier for CK2. The levels of both proteins were also studied in the human cell lines HL-60 (devoid of p53) and HepG2 (with normal p53 levels) and in fibroblasts from transgenic p53-deficient mice (p53-/-) or homozygous for wild-type p53 (p53+/+). Computer-assisted search was used to detect p53 consensus sequences in genes for CK2 subunits Binding of p53 protein to some of these sequences was assayed by electrophoretic mobility shift assay. RESULTS: Rat liver p53 protein was present mainly in the fraction extracted from intact nuclei by nucleases (S1) and showed a transient increase at 6 h post partial hepatectomy, as observed previously with nuclear CK2. The human CK2a gene presents the consensus sequence for trans-activation by p53 and specific binding of p53 protein to some of these sequences was detected in vitro. Total CK2a was higher in HepG2 than in HL-60 cells but total CK2 and its cytosolic/ nuclear distribution was similar in mice (p53+/+) fibroblasts and (p53-/-) fibroblasts. CONCLUSIONS: p53 is present in the nuclease-extracted S1 fraction from liver cells, as described for CK2, and undergoes similar changes at the beginning of rat liver regeneration. However, the data on cultured cells suggest that the expression of CK2 and its subcellular localization are p53-independent events.

Animals↗
PubMed2000

Genetic engineering of Escherichia coli to produce a 1:1 complex of the anabaena sp. PCC 7120 nuclease NucA and its inhibitor NuiA.

A series of T7-promoter based bicistronic expression vectors was constructed in order to produce the complex of the Anabaena sp. PCC 7120 DNA/RNA non-specific nuclease NucA and its inhibitor NuiA. With all constructs, tandem expression of nucA and nuiA results in aggregation and inclusion body formation of NucA, independent of the order of the genes, the relative expression of the two proteins and the temperature applied during expression. Two constructs in which nuiA is the first and nucA the second cistron lead to an approximately one order of magnitude higher expression of nuiA compared with nucA. In these cells inclusion bodies are formed which contain NucA and NuiA in a 1:1 molar ratio. The complex can be solubilized with 6M urea after disruption of the cells by sonication, renatured by dialysis and purified to homogeneity. 2mg of the complex are obtained from 1l Escherichia coli culture. As shown by gel filtration and analytical ultracentrifugation, our system leads to a highly pure and homogeneous complex preparation, as required for biophysical and structural studies. Thus, our new method is a superior alternative for the production of the NucA/NuiA complex in which separately produced nuclease and inhibitor are mixed, and an excess of one or the other component, as well as aggregates of NucA, have to be removed from the preparation.

Algal Proteins↗
PubMed1999

Enhanced expression of a nuclease gene in leaves of barley plants under salt stress.

We isolated a cDNA clone, Bnuc1, encoding a nuclease I from leaves of salt-stressed barley (Hordeum vulgare L. cv. Haruna-nijyo) by the differential display method. Northern blot analysis revealed that the transcript of Bnuc1 gene was increased dramatically in barley leaves under salt stress. The expression of Bnuc1 gene was also increased by exogenously applied abscisic acid (ABA) in leaves, but not by gibberellic acid (GA) during seed germination. Furthermore, Bnuc1 gene was expressed more in old leaves than in young leaves during both salt stress and natural senescence. Salt-inducible nuclease activity possibly corresponding to the Bnuc1 gene was detected, and was much higher in old leaves than in young leaves under salt stress.

Amino Acid Sequence↗
PubMed1998

Detection of excision nuclease in cell-free extracts from the adult mammalian brain.

The autosomal recessive disorder xeroderma pigmentosum (XP) results from defects in the nucleotide excision repair (NER) pathway for DNA repair. NER normally repairs bulky DNA lesions, such as pyrimidine dimers resulting from UV radiation. XP patients have high rates of skin cancer, and some also develop progressive neurological degeneration. To better understand the mechanism of this neurodegeneration, I used a specific assay for the multicomponent excision nuclease of the NER pathway in cell-free extracts from the adult rat brain. Excision nuclease activity was detectable in whole-cell extracts prepared from the cerebellum, whereas extracts prepared from the forebrain, which has a lower density of cell nuclei, had much less activity. Nuclear extracts from both areas were equally capable of restoring activity to extracts from two different NER-deficient cell lines, despite large differences in the ratio of neurons to nonneuronal cells in the cerebellum and forebrain. These results indicate that the NER pathway is functional in neuronal cells in the adult brain. The implications of this finding for XP and other neurodegenerative diseases is discussed.

Animals↗
PubMed1999

Allelic discrimination using fluorogenic probes and the 5' nuclease assay.

Large-scale screening for known polymorphisms will require techniques with few steps and the ability to automate each of these steps. In this regard, the 5' nuclease, or TaqMan, PCR assay is especially attractive. A fluorogenic probe, consisting of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye, is included in a typical PCR. Amplification of the probe-specific product causes cleavage of the probe, generating an increase in reporter fluorescence. By using different reporter dyes, cleavage of allele-specific probes can be detected in a single PCR. The 5' nuclease assay has been successfully used to discriminate alleles that differ by a single base substitution. Guidelines have been developed so that an assay for any single nucleotide polymorphism (SNP) can be quickly designed and implemented. All assays are performed using a single reaction buffer and single thermocycling protocol. Furthermore, a standard method of analysis has been developed that enables automated genotype determination. Applications of this assay have included typing a number of polymorphisms in human drug metabolism genes.

5'-Nucleotidase↗
PubMed2003

Guarding the genome: electrostatic repulsion of water by DNA suppresses a potent nuclease activity of topoisomerase IB.

Type IB topoisomerases cleave and rejoin DNA strands through a stable covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate. The stability of the intermediate is a two-edged sword; it preserves genome integrity during supercoil relaxation, but it also reinforces the toxicity of drugs and lesions that interfere with the DNA rejoining step. Here, we identify a key determinant of the stability of the complex by showing that introduction of an Sp or Rp methylphosphonate linkage at the cleavage site transforms topoisomerase IB into a potent endonuclease. The nuclease reaction entails formation and surprisingly rapid hydrolysis of a covalent enzyme-DNA methylphosphonate intermediate. The approximately 30,000-fold acceleration in the rate of hydrolysis of a methylphosphonate versus phosphodiester suggests that repulsion of water by the DNA phosphate anion suppresses the latent nuclease function of topoisomerase IB. These findings expose an Achilles' heel of topoisomerases as guardians of the genome, and they have broad implications for understanding enzymatic phosphoryl transfer.

Binding Sites↗
PubMed2003

Complementary functions of the Saccharomyces cerevisiae Rad2 family nucleases in Okazaki fragment maturation, mutation avoidance, and chromosome stability.

Rad2 family nucleases, identified by sequence similarity within their catalytic domains, function in multiple pathways of DNA metabolism. Three members of the Saccharomyces cerevisiae Rad2 family, Rad2, Rad27, and exonuclease 1 (Exo1), exhibit both 5' exonuclease and flap endonuclease activities. Deletion of RAD27 results in defective Okazaki fragment maturation, DNA repair, and subsequent defects in mutation avoidance and chromosomal stability. However, strains lacking Rad27 are viable. The expression profile of EXO1 during the cell cycle is similar to that of RAD27 and other genes encoding proteins that function in DNA replication and repair, suggesting Exo1 may function as a back up nuclease for Rad27 in DNA replication. We show that overexpression of EXO1 suppresses multiple rad27 null mutation-associated phenotypes derived from DNA replication defects, including temperature sensitivity, Okazaki fragment accumulation, the rate of minichromosome loss, and an elevated mutation frequency. While generally similar findings were observed with RAD2, overexpression of RAD2, but not EXO1, suppressed the MMS sensitivity of the rad27 null mutant cells. This suggests that Rad2 can uniquely complement Rad27 in base excision repair (BER). Furthermore, Rad2 and Exo1 complemented the mutator phenotypes and cell cycle defects of rad27 mutant strains to differing extents, suggesting distinct in vivo nucleic acid substrates.

Cell Cycle↗
PubMed2002

Ngl2p is a Ccr4p-like RNA nuclease essential for the final step in 3'-end processing of 5.8S rRNA in Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains three nonessential genes (NGL1, NGL2, and NGL3) that encode proteins containing a domain with similarity to a Mg(2+)-dependent endonuclease motif present in the mRNA deadenylase Ccr4p. We have investigated a possible role of these proteins in rRNA processing, because for many of the pre-rRNA processing steps, the identity of the responsible nuclease remains elusive. Analysis of RNA isolated from cells in which the NGL2 gene has been inactivated (ngl2delta) demonstrates that correct 3'-end formation of 5.8S rRNA at site E is strictly dependent on Ngl2p. No role in pre-rRNA processing could be assigned to Ngl1p and Ngl3p. The 3'-extended 5.8S rRNA formed in the ngl2delta mutant is slightly shorter than the 6S precursor previously shown to accumulate upon combined deletion of the 3' --> 5' exonuclease-encoding REX1 and REX2 genes or upon depletion of the exosomal subunits Rrp40p or Rrp45p. Thus, our data add a further component to the set of nucleases required for correct 3'-end formation of yeast 5.8S rRNA.

Amino Acid Sequence↗
PubMed2003

Structure elucidation of DNA interstrand cross-link by a combination of nuclease P1 digestion with mass spectrometry.

DNA interstrand cross-link reagents are among the most powerful agents for cancer treatment. Here we report a combined nuclease P1 digestion/mass spectrometry method for the structure elucidation of duplex oligodeoxynucleotides (ODNs) containing an interstrand cross-link. Our results demonstrate that nuclease P1 digestion of a double-stranded ODN containing an interstrand cross-link (ICL) of 4,5',8-trimethylpsoralen or mitomycin C gives a tetranucleotide bearing the cross-linked nucleobase moiety. Product ion spectra of the deprotonated ions of the tetranucleotides provide information about the structure of the cross-link. Furthermore, product-ion spectra of tetranucleotides containing two orientation isomers of mitomycin C interstrand cross-link are distinctive. We believe that the method described in this paper can be generally applicable for investigating the structures of other DNA ICLs.

Base Sequence↗
PubMedDate not supplied

Nuclease activity of 1,10-phenanthroline-copper. New conjugates with low molecular weight targeting ligands.

The chemical nuclease activity of 1,10-phenanthroline-copper depends on DNA sequence because the coordination complex has affinity for DNA. In order to target this efficient nucleolytic activity, it is essential to override its inherent specificity. The minimal size of ligands capable of redirecting the specificity has been investigated. A conjugate (HOP) prepared by alkylating Hoechst dye 33258 with 5-(iodoacetamido)-1,10-phenanthroline has a greater preference for A-T-rich regions than the unsubstituted 1,10-phenanthroline-copper complex, reflecting the specificity of this A-T-specific minor-groove binder. However, since quaternizing the dye with 5-(iodoacetamido)-1,10-phenanthroline increases its affinity for DNA, the specificity of cleavage by the conjugate is less than the binding selectivity of the dye. Linking 1,10-phenanthroline with the peptide of the helix-turn-helix domain of the Trp repressor specificity results in a conjugate with greater reactivity for the operator sequence than the unsubstituted complex. The intrinsic affinity of the 1,10-phenanthroline-Cu can only be partially overridden by the conformationally unstable peptide. Attachment of 1,10-phenanthroline to a deoxyoligonucleotide complementary to a single-stranded loop of RNA successfully targets the scission of the chemical nuclease. Cleavage sites are observed not only contiguous to the site of hybridization but also at nonadjacent sequence positions. The latter set of sites must be close in space to the 5' end of the hybridized deoxyoligonucleotide.

Alkylation↗
PubMedDate not supplied

Synthesis, hybridization properties, nuclease stability, and cellular uptake of the oligonucleotide--amino-beta-cyclodextrins and adamantane conjugates.

Synthesis of the oligonucleotides conjugated with amino derivatives of beta-cyclodextrin and adamantane, at the 3'-end of host oligonucleotide, has been described. The oligonucleotide conjugates were examined for their nuclease stability, hybridization properties, and cellular uptake. The oligonucleotide conjugates had increased nuclease resistance compared to their parent oligonucleotides. Conjugation of adamantane to the oligonucleotides did not adversely affect the ability of the oligonucleotides to hybridize with their complementary RNA. Conjugation with amino derivatives of beta-cyclodextrin, however, significantly destabilized the duplex formation. In the cellular uptake studies, we found that amino derivatives of beta-cyclodextrin attached at 3'-end of the oligonucleotides did not help to increase the uptake by cells. Cellular uptake of oligonucleotide-adamantane conjugates in association with 2-(hydroxypropyl)-beta-cyclodextrin (HPCD) as a "carrier" was significantly higher than that of control oligonucleotides.

Adamantane↗
PubMedDate not supplied

Optimizing the targeted chemical nuclease activity of 1,10-phenanthroline-copper by ligand modification.

Our interest in improving the efficiency of targeted scission reagents has prompted us to study the influence of ring substituents on the nuclease activity of 1,10-phenanthroline-copper conjugated to oligonucleotides and DNA-binding proteins. Since methyl substitution at all but the 2 and 9 positions enhances the copper-dependent chemical nuclease activity of 1,10-phenanthroline, we have compared the activity of conjugates prepared from 5-(aminomethyl)-1,10-phenanthroline (MOP) to those of conjugates prepared from 5-amino-1,10-phenanthroline (amino-OP). Tethering MOP derivatives to the Escherichia coli Fis protein enhances DNA scission several-fold at the weaker cleavage sites initially observed with conjugates prepared from amino-OP. However, scission efficiency is not increased at the stronger cleavage sites, or when scission is targeted to single-stranded DNA by a complementary oligonucleotide. These results are consistent with a change in the rate-determining step for cleavage associated with the differential accessibility of the DNA-bound coordination complex to solvent and reductant. Although the free bis cuprous complex of 2,9-dimethyl-1,10-phenanthroline (neocuproine) is redox-inactive, an oligonucleotide tethered to neocuproine through C5 of the phenanthroline ring efficiently cleaves a complementary DNA sequence. These results establish that the nucleolytic species in targeted scission is the 1:1 cuprous complex and suggest that the oxidative reaction proceeds through a copper-oxo intermediate rather than a metal-coordinated peroxy species. However, substituents at the 2 and 9 positions of the ligand will often hinder close approach of the phenanthroline-copper moiety to the oxidatively sensitive ribose as shown by the preference of the oligonucleotide-targeted chimera for cleavage of single-stranded regions and the failure of neocuproine-DNA-binding protein chimeras and a C2-tethered chimera to cleave DNA.

Amino Acid Transport Systems, Neutral↗
PubMedDate not supplied

Nucleosides and nucleotides. 160. Synthesis of oligodeoxyribonucleotides containing 5-(N-aminoalkyl)carbamoyl-2'-deoxyuridines by a new postsynthetic modification method and their thermal stability and nuclease-resistance properties.

Heptadecadeoxynucleotides containing 5-(N-aminoethyl- or N-aminohexyl)carbamoyl-2'-deoxyuridines (E or H) were synthesized using a newly developed postsynthetic modification method. As a convertible nucleoside unit, 5-methoxycarbonyl-2'-deoxyuridine (1) was initially incorporated into oligodeoxynucleotides (ODNs) according to the phosphoramidite method at various positions using a DNA synthesizer. Fully protected ODNs attached to a solid support were treated with alkyldiamines such as ethylenediamine and 1,6-hexanediamine to give the above modified ODNs. The thermal stability, resistance toward nuclease digestion, and stability in fetal calf serum of the modified ODNs were studied. An increase in the number of 5-(N-aminohexyl)carbamoyl-2'-deoxyuridines (H) in the ODNs was found to effectively stabilize duplex formation with both the corresponding complementary DNA and RNA and protect against nucleolytic hydrolysis by snake venom phosphodiesterase. In particular, the half-life of ODN 19, which contained four H residues, was about 162 h in the presence of the nuclease. Furthermore, 19 was also stable in medium containing 10% fetal calf serum with a t1/2 of about 48 h, while t1/2 for the corresponding unmodified ODN was 13 min.

Deoxyuridine↗