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PubMed1977

The problem of nuclease activity in nucleic acid hybridization reactions. Theoretical considerations.

The presence of nuclease during DNA-DNA or DNA-RNA hybridization reactions alters the kinetics of hybrid formation. Unfortunately, while the effect (even of small amounts of nucleas) on the Cot curve may be large, it may not be readily detectable. The effect of various types of nuclease is shown. As many nucleases cause a shift in the position rather than a change in shape of the curve, all studies involving nucleic acid hybridization should assay for the presence of nucleases and care must be taken to avoid their presence as contaminants.

DNA↗
PubMed1987

The extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli.

We are studying exoproteins of the enteric bacterium Serratia marcescens as a model system for the release of extracellular proteins from the cell. In this work we report the cloning of the gene for a secreted nuclease from S. marcescens and its complete nucleotide sequence. Following expression of the nuclease gene in both S. marcescens and Escherichia coli we were able to demonstrate the presence of the nuclease extracellularly in both organisms. Cell lysis did not occur and there was no concurrent release of cytoplasmic or periplasmic proteins. No accessory genes appeared to be required for extracellular secretion of the nuclease from E. coli. We can conclude that E. coli is capable of secreting certain proteins extracellularly, and may be a suitable host organism for the genetic analysis of extracellular protein secretion when provided with a suitable protein to export.

Amino Acid Sequence↗
PubMed1991

Alteration of a nuclease in Fanconi anemia.

Fanconi anemia is a cancer-prone disease characterized by progressive loss of blood cells, skeletal defects and stunted growth. Studies of a nuclease acting on double-stranded DNA have revealed an enzyme alteration in cells derived from Fanconi patients. A particulate fraction isolated from cultured human lymphoblasts and fibroblasts was solubilized with detergent and subjected to isoelectric focusing. Nuclease activity observed in four normal cell lines bands in a pH gradient with a pI of 6.3. Four cell lines belonging to complementation group A exhibit an increase in the pI of that nuclease to 6.8. These observations provide a new diagnostic for this disorder. Analysis of this enzyme in tetraploid cultures derived from fusion of normal and Fanconi cells suggest that the normal phenotype is dominant. That observation supports the hypothesis that the Fanconi A gene is required for modification of the nuclease pI. Definition of the molecular basis of this enzyme alteration should provide insight into the primary genetic lesion in this disorder.

Cell Fractionation↗
PubMed2006

Nuclease P1 digestion/high-performance liquid chromatography, a practical method for DNA quantitation.

We have developed a practical method for quantifying DNA. The method is practical in two ways. First, a single enzyme is used to digest the DNA to nucleotides that are then quantified by HPLC under ordinary conditions. Second, the method quantifies DNA even when it is impure. In our method, "nuclease P1/HPLC," the DNA is hydrolyzed by nuclease P1 and the resulting 2'-deoxynucleoside 5'-monophosphates are quantified by HPLC with UV detection. This method was applied to several kinds of genomic DNA in terms of origin and method by which it had been purified. Calf thymus DNA (purified by salt precipitation by the supplier), pig liver DNA (purified by phenolic extraction or by anion-exchange chromatography using a Genomic Tip from Qiagen) and mouse skin DNA (similarly purified) were tested. In some cases a given sample was purified by two of these methods. The values for the amount of DNA by our method were compared with those by three other methods: acid hydrolysis/HPLC (selected as a reference procedure), UV absorbance, and dye binding. Agreement for all DNA samples between the values by our method versus those provided by acid hydrolysis/HPLC was within 10% for amounts of DNA in the 19-54 microg range. In contrast, UV absorbance and the dye-binding assay gave differences up to 30-40% relative to the consistent values furnished by acid hydrolysis and our method. Overall, normalizing the concentrations of the DNA (thymus, liver, skin) by acid hydrolysis/HPLC in 10 samples to values of 1.0 gave the following, relative values and standard deviations: 1.01+/-.07 (nuclease P1/HPLC), 0.8+/-0.17 (dye binding), and 1.1+/-0.1 (UV). Since one cannot assume that any sample of DNA is pure, and determining purity of DNA is difficult, then nuclease P1/HPLC or acid hydrolysis/HPLC is recommended rather than the UV absorbance or dye binding for quantifying DNA whenever an accurate value is important.

Animals↗
PubMed2004

Molecular characterization of a novel amastigote stage specific Class I nuclease from Leishmania major.

Leishmania parasites like other kinetoplastids are unable to synthesize purines de novo and so are reliant on a salvage pathway for recycling ribonucleotides. A stage specific class one nuclease enzyme, 3'-Nucleotidase/nuclease, has been implicated in salvage of preformed purines in Leishmania insect stage promastigote via hydrolysis of 3'-nucleotides and nucleic acids. Although a similar activity is known to exist in amastigotes which reside in infected mammalian cells, the homologous gene and the corresponding protein responsible for carrying out this function have not been well characterized. Using primers specific for conserved regions of trypanosomatid class one nucleases, a gene encoding a novel class one nuclease from amastigotes of Leishmania major (LmaC1N) was cloned and sequenced. The coding sequence consists of 951 bp encoding a 316 amino acid protein with a predicted molecular mass of 35,300 Da. Analysis of the deduced amino acid sequence showed that LmaC1N is highly homologous to other class I nucleases and contains all five conserved regions reported for promastigotes 3'-Nucleotidase/nuclease. Analysis by reverse transcriptase polymerase chain reaction and Western blotting demonstrated that expression of LmaC1N gene is regulated in a stage-specific manner. Whereas the gene appeared to be silenced in promastigotes, high level expression in amastigotes implied an important function in support of parasite survival and multiplication in the mammalian cells.

Amino Acid Sequence↗
PubMed2006

Kinetics of ATP-stimulated nuclease activity of the Escherichia coli RecBCD enzyme.

The RecBCD enzyme is an ATP-dependent nuclease on both single-stranded and double-stranded DNA substrates. We have investigated the kinetics of the RecBCD-catalyzed reaction with small, single-stranded oligodeoxyribonucleotide substrates under single-turnover conditions using rapid-quench flow techniques. RecBCD-DNA complexes were allowed to form in pre-incubation mixtures. The nuclease reactions were initiated by mixing with ATP. The reaction time-courses were fit to several possible reaction mechanisms and quantitative estimates were obtained for rate constants for individual reaction steps. The relative rates of forward reaction versus dissociation from the DNA, and the fact that inclusion of excess non-radiolabeled single-stranded DNA to trap free RecBCD has no effect on the nuclease reaction, indicates that the reaction is processive. The reaction products show that the reaction begins near the 3'-end of the [5'-32P]DNA substrates and the major cleavage sites are two to four phosphodiester bonds apart. The product distribution is unchanged as the ATP concentration varies from 10 microM to 100 microM ATP, while the overall reaction rate varies by about tenfold. These observations suggest that DNA cleavage is tightly coordinated with movement of the enzyme along the DNA. The reaction time-courses at low concentrations of ATP (10 microM and 25 microM) have a significant lag before cleavage products appear. We propose that the lag represents ATP-dependent movement of the DNA from an initial binding site in the helicase domain of the RecB subunit to the nuclease active site in a separate domain of RecB. The extent of reaction of the substrate is limited (approximately 50%) under all conditions. This may indicate the formation of a non-productive RecBCD-DNA complex that does not dissociate in the 1-2 s time-scale of our experiments.

Adenosine Triphosphate↗
PubMed2004

Detection by 5'-nuclease PCR of Shiga-toxin producing Escherichia coli O26, O55, O91, O103, O111, O113, O145 and O157:H7, associated with the world's most frequent clinical cases.

This paper describes 5'-nuclease PCR assays for detecting eight O-serogroups, H7 flagellar antigen and stx genes from the Shiga toxin-producing Escherichia coli (STEC) associated with the world's most frequent clinical cases. A single set of primers was used to detect the genes stx1 and stx2 in the same reaction by 5'-nuclease PCR. Serotyping by 5'-nuclease PCR of STEC was based on the selection of primers and probes targeting the O-antigen gene clusters of E. coli O26, O55, O91, O111, O113, O157, the eae gene of E. coli O103, the O-island 29 of E. coli O145, and the flagellar H7 antigen gene. Results obtained on a collection of 190 strains indicate that the 5'-nuclease PCR assays used here could serve as a basis for rapid specific stx, O and H7 typing of these major pathogenic serogroups of E. coli. This work provides sensitive and specific tests for the rapid, reliable detection of the main pathogenic E. coli O-serogroups of major public health concern.

DNA Primers↗
PubMed2006

Structure-specific DNA nucleases: structural basis for 3D-scissors.

Structure-specific DNA nucleases play important roles in various DNA transactions such as DNA replication, repair and recombination. These enzymes recognize loops and branched DNA structures. Recent structural studies have provided detailed insights into the functions of these enzymes. Structures of Holliday junction resolvase revealed that nucleases are broadly diverged in the way in which they fold, however, are required to form homodimers with large basic patches of protein surfaces, which are complementary to DNA tertiary structures. Many nucleases maintain structure-specific recognition modes, which involve particular domain arrangements through conformal changes of flexible loops or have a separate DNA binding domain. Nucleases, such as FEN-1 and archaeal XPF, are bound to proliferating cell nuclear antigen through a common motif, and thereby actualize their inherent activities.

Archaea↗
PubMed1998

Comparison of immunoaffinity chromatography enrichment and nuclease P1 procedures for 32P-postlabelling analysis of PAH-DNA adducts.

32P-postlabelling analysis for detecting DNA adducts formed by polycyclic aromatic compounds is one of the most widely used techniques for assessing genotoxicity associated with these compounds. In cases where the formation of adducts is extremely low, a crucial step in the analysis is an enrichment procedure for adducts prior to the radiolabelling step. The nuclease P1 enhancement procedure is the most established and frequently used of these methods. An immunoaffinity procedure developed for class specific recognition for polycyclic aromatic hydrocarbon (PAH)-DNA adducts has therefore been compared with the nuclease P1 method for a range of DNA adducts formed by PAHs. The evaluation was carried out with skin DNA from mice treated topically with benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 5-methylchrysene or chrysene. The immobilised antibody had the highest affinity for adducts structurally similar to the BPDE-I-deoxyguanosine adduct ([+/-]-N2-(7r,8t,9r-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-1 0t-yl)-2'-deoxyguanosine) against which the antibody had been raised. Of the PAH-modified DNAs evaluated, the maximum adduct recovery was obtained for DNA containing the BPDE I-deoxyguanosine adduct. With DMBA-modified DNA, the profiles of adducts recovered from the column were similar when the column material was treated either with a digest of DMBA-modified DNA or with 32P-labelled DMBA adducts. I-compounds (endogenous adducts in tissue DNA of unexposed animals), which had similar chromatographic properties to PAH-DNA adducts, were not enriched by the immunoaffinity procedure. Compared to the simple nuclease P1 enhancement procedure, the immunoaffinity methods were lengthier and more labour intensive. Advantages of the immunoaffinity procedure include: specificity, allowing the selective detection of a certain class of adducts: efficient adduct enrichment, providing a viable alternative to other enrichment procedures; adequate sensitivity for model studies and the potential to purify adducts for further characterisation. However, as a general screen for detecting the formation of DNA adducts, the nuclease P1 procedure was viewed as the initial method of choice since it was capable of detecting a wider range of PAH-DNA adducts.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1990

Transcription initiation and nuclease-sensitive sites upstream of the epsilon-globin gene in K562 cells are related to poly (dA).poly (dT) sequences.

Previous studies have documented transcription initiation sites and nuclease hypersensitive sites upstream of the epsilon-globin canonical cap site in K562 cells. The upstream transcription initiation sites coincide with some of the nuclease hypersensitive sites. Comparison of the positions of the upstream transcription initiation and the nuclease hypersensitive sites with the nucleotide base order shows that these upstream sites fall significantly closer to poly (dA).poly (dT) tracts than can reasonably be accounted for by chance. It is concluded that these sites are related to the occurrence of poly (dA).poly (dT) tracts of at least five base pairs. Other studies have related some particular functional properties to poly (dA).poly (dT) tracts. Additionally, poly (dA).poly (dT) tracts have been shown to have unusual physical characteristics and to produce an intrinsic bending of the DNA molecules in which they are located. This study indicates that poly (dA).poly (dT) tracts can provide access to DNA for RNA polymerases and induce a DNA conformation recognized by DNase I or S1 nuclease.

Base Sequence↗
PubMed2000

Apoptotic nuclease assays.

One of the defining biochemical characteristics of apoptosis is the degradation of chromatin into regularly sized (oligonucleosomal and approximately 30- to 50-kb) fragments. Because destruction of the genome represents a clear commitment to death, considerable interest has focused on this component of apoptosis and numerous assays have been developed to assess the relevant nucleases involved. These assays fall into two major categories: (1) those independent of chromatin structure and (2) those dependent on chromatin structure. The chromatin-independent assays (plasmid degradation assay and radioactive gel assay) examine the ability to degrade naked DNA and are advantageous because of their simplicity and speed and ability to analyze single nucleases or mixtures of nucleases. However, these assays do not mimic the conditions present in normal cells and consequently do not assess the ability of an enzyme to function in apoptosis. In contrast, chromatin structure-dependent assays (nuclear autodigestion and HeLa nuclei assay) present intact chromatin to either endogenous or exogenous enzymes and assess the ability to degrade chromatin in a manner that recapitulates the genomic destruction seen in vivo. Detailed protocols are discussed for both classes of assays. These assays have been instrumental in the identification of several apoptotic nucleases.

Animals↗
PubMed2001

Immobilization of sugar-non-specific nucleases by utilizing the streptavidin--biotin interaction.

Due to their high enzymatic activity, the sugar-non-specific endonucleases from Serratia marcescens and Anabaena can be used for a number of applications, such as the removal of contaminating genetic material from biological preparations, footprinting studies, and the determination of nucleic acids in biochemical samples. These methods would benefit from immobilized nucleases. For this purpose, a single cysteine residue was added at the N-terminus of the Serratia and Anabaena nucleases and subsequently modified with a maleimide-biotin conjugate. Alternatively, a biotin acceptor domain was fused to the Anabaena nuclease, allowing biotinylation during expression in E. coli without a further chemical step. The attachment of biotin-modified nucleases to streptavidin-coated paramagnetic beads and to streptavidin-coated surface plasmon resonance sensor chips (to study interactions with substrate and inhibitor) worked well when aggregates present in the protein preparations were removed by ultrafiltration. These methods should be of general use for similar enzyme systems.

Amino Acid Sequence↗
PubMed1999

Extracellular nuclease from Rhizopus stolonifer: purification and characteristics of - single strand preferential - deoxyribonuclease activity.

An extracellular nuclease from Rhizopus stolonifer (designated as nuclease Rsn) was purified to homogeneity by chromatography on DEAE-cellulose followed by Blue Sepharose. The M(r) of the purified enzyme determined by native PAGE was 67¿ omitted¿000 and it is a tetramer and each protomer consists of two unidentical subunits of M(r) 21¿ omitted¿000 and 13¿ omitted¿000. It is an acidic protein with a pI of 4.2 and is not a glycoprotein. The purified enzyme showed an obligate requirement of divalent cations like Mg(2+), Mn(2+) and Co(2+) for its activity but is not a metalloprotein. The optimum pH of the enzyme was 7.0 and was not influenced by the type of metal ion used. Although, the optimum temperature of the enzyme for single stranded (ss) DNA hydrolysis in presence of all three metal ions and for double stranded (ds) DNA hydrolysis in presence of Mg(2+) was 40 degrees C, it showed higher optimum temperature (45 degrees C) for dsDNA hydrolysis in presence of Mn(2+) and Co(2+). Nuclease Rsn was inhibited by divalent cations like Zn(2+), Cu(2+) and Hg(2+), inorganic phosphate and pyrophosphate, low concentrations of SDS, guanidine hydrochloride and urea, organic solvents like dimethyl sulphoxide, dimethyl formamide and formamide but not by 3'- or 5'-mononucleotides. The studies on mode and mechanism of action showed that nuclease Rsn is an endonuclease and cleaves dsDNA through a single hit mechanism. The end products of both ssDNA and dsDNA hydrolysis were predominantly oligonucleotides ending in 3'-hydroxyl and 5'-phosphoryl termini. Moreover, the type of metal ion used did not influence the mode and mechanism of action of the enzyme.

Cations, Divalent↗
PubMed2000

The C. elegans apoptotic nuclease NUC-1 is related in sequence and activity to mammalian DNase II.

The Caenorhabditis elegans nuc-1 gene has previously been implicated in programmed cell death due to the presence of persistent undegraded apoptotic DNA in nuc-1 mutant animals. In this report, we describe the cloning and characterization of nuc-1, which encodes an acidic nuclease with significant sequence similarity to mammalian DNase II. Database searches performed with human DNase II protein sequence revealed a significant similarity with the predicted C. elegans C07B5.5 ORF. Subsequent analysis of crude C. elegans protein extracts revealed that wild-type animals contained a potent endonuclease activity with a cleavage preference similar to DNase II, while nuc-1 mutant worms demonstrated a marked reduction in this nuclease activity. Sequence analysis of C07B5.5 DNA and mRNA also revealed that nuc-1(e1392), but not wild-type animals contained a nonsense mutation within the CO7B5.5 coding region. Furthermore, nuc-1 transgenic lines carrying the wild-type C07B5.5 locus demonstrated a complete complementation of the nuc-1 mutant phenotype. Our results therefore provide compelling evidence that the C07B5.5 gene encodes the NUC-1 apoptotic nuclease and that this nuclease is related in sequence and activity to DNase II.

Amino Acid Sequence↗
PubMed2003

A strategy based on 5' nuclease multiplex PCR to detect enterotoxin genes sea to sej of Staphylococcus aureus.

We describe the development of a strategy based on 5' nuclease multiplex PCR for the rapid detection of nine enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej) of Staphylococcus aureus. The genotyping scheme consists in identifying these nine enterotoxin genes by three 5' nuclease Triplex-PCR assays. The strategy was evaluated using a collection of S. aureus reference strains previously examined with conventional PCR assays, and by testing previously characterized food S. aureus field strains. The 5' nuclease Triplex-PCR assays correctly detected the se genes in all the reference strains. In tests with field strains there was generally excellent agreement with the results obtained by conventional PCR, except for some strains harbouring variant se genes. The detection limits of the Triplex-PCR assays evaluated using fivefold dilution of recombinant plasmids for each se gene ranged from 16 to 2000 copies of target se genes in the PCR tube. The 5' nuclease Triplex-PCR assays developed are fast and specific, and provide a useful diagnostic tool for the detection and genotyping of se genes. The development of this method is an improvement that should facilitate epidemiological investigations of staphylococcal food poisoning outbreaks.

Base Sequence↗
PubMed2003

X-ray and biochemical anatomy of an archaeal XPF/Rad1/Mus81 family nuclease: similarity between its endonuclease domain and restriction enzymes.

The XPF/Rad1/Mus81-dependent nuclease family specifically cleaves branched structures generated during DNA repair, replication, and recombination, and is essential for maintaining genome stability. Here, we report the domain organization of an archaeal homolog (Hef) of this family and the X-ray crystal structure of the middle domain, with the nuclease activity. The nuclease domain architecture exhibits remarkable similarity to those of restriction endonucleases, including the correspondence of the GDX(n)ERKX(3)D signature motif in Hef to the PDX(n)(E/D)XK motif in restriction enzymes. This structural study also suggests that the XPF/Rad1/Mus81/ERCC1 proteins form a dimer through each interface of the nuclease domain and the helix-hairpin-helix domain. Simultaneous disruptions of both interfaces result in their dissociation into separate monomers, with strikingly reduced endonuclease activities.

Amino Acid Sequence↗
PubMed1998

Scission of DNA at a preselected sequence using a single-strand-specific chemical nuclease.

BACKGROUND: We were interested in developing a protocol for cleaving large DNAs specifically. Previous attempts to develop such methods have failed to work because of high levels of nonspecific background scission. RESULTS: R-loop formation was chosen for sequence-specific targeting, a method of hybridization whereby an RNA displaces a DNA strand of identical sequence in 70% formamide using Watson-Crick base-pairing, leading to a three-stranded structure. R-loops are stabilized in aqueous solution by modifying the bases with chemical reagents. The R-loop was cleaved using a novel nuclease prepared from the Thr48-->Cys mutant of the single-strand-specific M-13 gene V protein (GVP), which was alkylated with 5-(iodoacetamido-beta-alanyl)1,10-phenanthroline. The cleavage products of the pGEM plasmid were cloned in to the pCR 2.1-TOPO vector. Adenovirus 2 DNA (35.8 kb; tenfold larger than the pGEM plasmid) was also cleaved quantitatively at a preselected sequence. CONCLUSIONS: A new method for cleaving duplex DNA at any preselected sequence was developed. The cleavage method relies on the chemical conversion of M-13 GVP into a nuclease, reflecting GVP's specificity for single-stranded DNA. The GVP chimera is the first example of a semisynthetic secondary structure specific nuclease. The chemical nuclease activity of 1,10-phenanthroline-copper is uniquely suited to this technique because it oxidizes the deoxyribose moiety without generating diffusible intermediates, providing clonable DNA fragments. The protocol could be useful in generating large DNA fragments for mapping the contiguity of probes or defining the exon-intron structure of transcription units.

Adenoviridae↗
PubMedDate not supplied

Nucleosides and nucleotides. 204. Synthesis of oligodeoxynucleotides containing 6'alpha-[N-(Aminoalkyl)carbamoyloxy]-carbocyclic-thymidines and the thermal stability of the duplexes and their nuclease-resistance properties.

To construct the nuclease-resistant oligodeoxynucleotides (ODNs) with natural phosphodiester linkages, we synthesized ODNs that contain 6'alpha-[N-(aminoalkyl)carbamoyloxy]-carbocyclic-thymidines (4, 5, and 6). The stability of these ODNs to nuclease hydrolysis was examined by using snake venom phosphodiesterase (3'-exonuclease) and nuclease S1 (endonuclease). It was found that the ODNs containing 4, 5, or 6 were more resistant to both the enzymes than the unmodified ODN. These nuclease-resistant properties are noteworthy, since they have natural phosphodiester linkages. Next, the thermal stabilities of duplexes consisting of these ODNs and either the complementary DNA or RNA were studied by thermal denaturation. The ODNs that contain 4 were found to enhance the thermal stability of the duplexes with the complementary DNA, while those containing 5 or 6 decreased the thermal stability of the ODN-DNA duplexes. On the other hand, all ODNs that contained 4, 5, or 6 decreased the thermal stability of the ODN-RNA duplexes.

Base Sequence↗