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PubMedDate not supplied

Ca2+-sensitive cytosolic nucleases prevent efficient delivery to the nucleus of injected plasmids.

BACKGROUND: Efficient gene delivery by synthetic vectors is a major challenge in gene therapy. However, inefficient nuclear delivery of cDNA is thought to be a major limiting step in gene transfer using non-viral vectors. It is commonly thought that, in the cytosol, cDNA has to be released from its vector before importation to the nucleus. The stability of naked cDNA in the cytoplasm is not well established. METHODS: cDNA plasmids, either free or complexed with poly(ethyleneimine) (PEI), were microinjected into the cytoplasm of mammalian cells and their turnover was assessed by fluorescence in situ hybridization (FISH). Incubations of cDNA plasmids in cytosolic extracts were also performed. RESULTS: FISH experiments showed that naked cDNA rapidly fade with time when injected into the cytosol. Fading was not observed when naked cDNA plasmids were injected into the nucleus. Incubation of naked cDNA in a cytosolic fraction isolated from mammalian cells reproduced cDNA degradation as observed in microinjection experiments. Nuclease inhibitors, including aurin tricarboxylic acid or Zn2+, prevented in vitro cDNA degradation. The cytosolic nuclease activity was optimal at physiological pH and physiological Ca2+ concentration. By contrast, it was insensitive to Mg2+ or Na+ concentrations. Finally, cDNA complexation with PEI or addition of oligonucleotides prevented in vitro cDNA degradation. CONCLUSION: Altogether, these experiments suggest that cDNA digestion by cytosolic nucleases occur when the decomplexed transgene is present in the cytosol. We propose that the inefficient transfer of cDNA into the nucleus during transfection with synthetic vectors may result from rapid digestion of naked cDNA by a Ca2+-sensitive cytosolic nuclease.

Animals↗
PubMed1999

Detection of anticodon nuclease residues involved in tRNALys cleavage specificity.

The tRNALys-specific anticodon nuclease exists in latent form in Escherichia coli strains containing the optional prr locus. The latency is a result of a masking interaction between the anticodon nuclease core-polypeptide PrrC and the Type IC DNA restriction-modification enzyme EcoprrI. Activation of the latent enzyme by phage T4-infection elicits cleavage of tRNALys 5' to the wobble base, yielding 5'-OH and 2', 3'-cyclic phosphate termini. The N-proximal half of PrrC has been implicated with (A/G) TPase and EcoprrI interfacing activities. Therefore, residues involved in recognition and cleavage of tRNALys were searched for at the C-half. Random mutagenesis of the low-G+C portion encoding PrrC residues 200-313 was performed, followed by selection for loss of anticodon nuclease-dependent lethality and production of full-sized PrrC-like protein. This process yielded a cluster of missense mutations mapping to a region highly conserved between PrrC and two putative Neisseria meningitidis MC58 homologues. This cluster included two adjacent members that relaxed the inherent enzyme's cleavage specificity. We also describe another mode of relaxed specificity, due to mere overexpression of PrrC. This mode was shared by wild-type PrrC and the other mutant alleles. The additional substrates recognised under the promiscuous conditions had, in general, anticodons resembling that of tRNALys. Taken together, the data suggest that the anticodon of tRNALys harbours anticodon nuclease identity elements and implicates a conserved region in PrrC in their recognition.

Amino Acid Sequence↗
PubMed1999

The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates.

A steady-state kinetic analysis of the cleavage of the oligonucleotides d(CGCTTTTTTGC) (d(y)), d(GCAAAAAAGCG) (d(r)), r(CGCUUUUUUGC) (r(y)) and r(GCAAAAAAGCG) (r(r)) in single and double-stranded form by the extracellular Serratia marcescens endonuclease, in conjunction with structural data from a circular dichroism spectroscopic analysis of these substrates, suggests that oligonucleotides adopting the A-conformation are preferred over those adopting the B-conformation as substrates. Relative catalytic efficiencies (kcat/KM) for the cleavage of the homo- and heteroduplexes follow the order r(r).r(y) (1.0)>r(r).d(y) (0.9)>d(r). r(y) (0.7)>d(r).d(y) (0.3). The purine-rich single-stranded oligonucleotides r(r) and d(r), are cleaved more efficiently than the pyrimidine-rich oligonucleotides, r(y) and d(y), presumably because they adopt helical structures with pronounced base stacking. Except for the double-stranded oligodeoxynucleotide substrate, the individual strands are cleaved more efficiently when incorporated into a duplex, than in a single-stranded form. Cleavage experiments with various polynucleotides, including a viroid RNA and a specifically designed 167 bp DNA, confirm that double-stranded A-form nucleic acids are preferentially attacked by Serratia nuclease. In an attempt to analyze the basis of these preferences, we have mutated the amino acid residues Tyr76 and Trp123 of Serratia nuclease. These residues are located close to the active site and are conserved in all members of the Serratia nuclease family, suggesting that they could be involved in substrate binding, e.g. by stacking interactions with the bases, which could lead to the cleavage preferences observed. However, only effects on the activity, but no change of the sequence or substrate preferences, were detected upon substitution of these amino acid residues, ruling out any involvement of these residues in the A-form preference of Serratia nuclease.

Amino Acid Sequence↗
PubMed1993

Herpes simplex virus 1 alkaline nuclease is required for efficient egress of capsids from the nucleus.

We previously described the isolation of AN-1, a null mutant of the HSV-1 alkaline nuclease gene, which is able to synthesize near wild-type levels of viral DNA and late viral proteins under nonpermissive growth conditions; these results lead us to conclude that the alkaline nuclease is not essential for viral DNA synthesis (Weller, S. K., Seghatoleslami, R. M., Shao, L., Rowse, D., and Carmichael, E. P., 1990, J. Gen. Virol. 71, 2941-2952). AN-1 was found to be deficient in the production of infectious virions suggesting that the nuclease may play a role in processing or packaging of viral DNA into infectious virions. In this report we demonstrate that the defect is distinct from that observed in other late HSV-1 mutants which make but fail to process viral DNA under nonpermissive growth conditions. Following restriction enzyme digestion, specific terminal fragments are observed in DNA from AN-1-infected Vero cells, indicating that specific cleavage has occurred; moreover, the efficiency of cleavage is at near wild-type levels. Also in contrast to cells infected with previously described late mutants, DNase I or staphylococcal nuclease resistant DNA is observed in these cells further indicating that encapsidation has occurred. Three lines of evidence suggest, however, that maturation of DNA-containing AN-1 capsids is defective in some ways. First, in contrast to wild-type, very small amounts of protected DNA is detected in cytoplasmic extracts from AN-1-infected cells. Second, very few if any mature, DNA-containing C capsids are observed in the cytoplasm when analyzed by electron microscopy or sucrose gradient sedimentation. Finally, analysis of nuclei by sucrose gradient sedimentation indicates an elevated ratio of A to B capsids. These data indicate that AN-1 may be defective for the production of capsids competent to mature into the cytoplasm. Possible models for the nature of the defect in AN-1 will be discussed.

Animals↗
PubMed1980

Nucleases and their control in wild-type and nuh mutants of Neurospora.

A review of all of the work on Neurospora nucleases strongly suggests that five nucleases, originally isolated on the basis of markedly different properties, may actually be derived from a single inactive precursor polypeptide via different routes of proteolysis. One of these nucleases may be involved in DNA repair and/or recombination. Two repair-deficient mutants of Neurospora, uvs-3 and nuh-4, may have a lesion in protease(s) which control the level of this nuclease or in some function which regulates the protease(s). Both of these mutants map in the same gene region and they may be defective in recombination, since they are sensitive to various mutagens and to mitomycin C and they show high frequency of spontaneous, but not radiation-induced, recessive lethal mutations and/or deletions.

DNA Repair↗
PubMed1990

Parameters influencing the flow cytometric analysis of DNA sensitivity to nuclease S1.

Some parameters that influence the analysis in situ of DNA sensitivity to digestion with nuclease S1 have been studied in isolated HeLa nuclei with flow cytometry. DNA staining with the intercalating fluorochrome propidium iodide allowed the nucleolytic activity on double-stranded (ds) DNA to be determined by monitoring the relative reduction in nuclear fluorescence intensity. Nuclei isolated in buffer at low ionic strength in order to decondense chromatin fibres, showed a lower fluorescence intensity than nuclei with native chromatin, after digestion with nuclease S1 under identical conditions. Nuclei prepared with dispersed chromatin and digested with increasing amounts of enzyme showed a decrease in fluorescence intensity that reached a limit value at about 50% of the value of undigested control samples. On the other hand, in nuclei with native chromatin, fluorescence intensity decreased only about 18%. The NaCl concentration in the reaction buffer strongly influenced the DNA sensitivity to S1 nuclease. By increasing salt molarity from 5 mM to 200 mM, the digestion of dsDNA was significantly reduced as also shown by the amount of released nucleotides from purified calf thymus DNA. The detection of DNA sensitivity to nuclease S1, as assessed by the cytometric method, was shown to be more sensitive than a biochemical technique involving hydrolysis of purines. These results indicate that both the procedure for nuclei isolation and the digestion conditions have to be carefully controlled when evaluating in situ the presence of S1-sensitive sites.

Cell Nucleus↗
PubMed1985

The accessibility of phage MS2 RNA to structure specific nucleases in various conditions.

The accessibility of ds- and ss-segments of phage MS2 RNA to ds- and ss-specific nucleases (RNase III, nuclease SV and nuclease S1) was studied. The results show that the RNA has hydrolysis sites for all the nucleases used. These sites are unvariable in a wide range of the conditions (ionic strength, pH, bivalent cations and temperature) and are not changed also after denaturation-renaturation of the RNA. This testifies that the distribution and interactions of ds- and ss-segments in the whole molecule are very specific and stable.

Bacteriophages↗
PubMed2005

Early zygote-specific nuclease in mitochondria of the true slime mold Physarum polycephalum.

The active, selective digestion of mtDNA from one parent is a possible molecular mechanism for the uniparental inheritance of mtDNA. In Physarum polycephalum, mtDNA is packed by DNA-binding protein Glom, which packs mtDNA into rod-shaped mt-nucleoids. After the mating, mtDNA from one parent is selectively digested, and the Glom began to disperse. Dispersed Glom was retained for at least 6 h after mtDNA digestion, but disappeared completely by about 12 h after mixing two strains. We identified two novel nucleases using DNA zymography with native-PAGE and SDS-PAGE. One is a Ca2+-dependent, high-molecular-weight nuclease complex (about 670 kDa), and the other is a Mn2+-dependent, high-molecular-weight nuclease complex (440-670 kDa); the activity of the latter was detected as a Mn2+-dependent, 13-kDa DNase band on SDS-PAGE. All mitochondria isolated from myxamoebae had mt-nucleoids, whereas half of the mitochondria isolated from the zygotes at 12 h after mixing had lost the mt-nucleoids. The activity of the Mn2+-dependent nuclease in the isolated mitochondria was detected at least 8 h after mixing of two strains. The timing and localization of the Mn2+-dependent DNase activity matched the selective digestion of mtDNA.

Animals↗
PubMed1979

Purification of an alkaline nuclease from Physarum polycephalum.

An alkaline nuclease was purified from microplasmodia of Physarum polycephalum. The nuclease, active on denatured DNA and RNA and free of contamination by other nucleolytic activities, appeared to be a zinc-metallo protein. The enzyme was only active under conditions, where Zn2+ were retained in the enzyme. Loss of zinc occurred by the chelating action of EDTA, EGTA or ampholines, by acid of highly alkaline pH conditions or by high ionic strength. The addition of ZnCl2 to compensate losses, restored all activity, while all other divalent cations caused inhibition. The nuclease, with a molecular weight of 32 000, was stable at neutral pH at high temperatures with a half-life of 20 min at 80 degrees C. It was inhibited by any salt of buffer concentration above the level of zero ionic strength and showed a special sensitivity towards phosphate ions. The possible similarity of this enzyme to nuclease S1 from Aspergillus oryzae is pointed out.

Deoxyribonucleases↗
PubMed1976

Random cleavage of superhelical SV 40 DNA S1 nuclease.

SV40 DNA FO I is randomly cleaved by S1 nuclease both at moderate (50 mM) and higher salt concentrations (250 mM NaC1). Full length linear S1 cleavage products of SV40 DNA when digested with various restriction endonucleases revealed fragments that were electrophoretically indistinguishable from the products found after digestion of superhelical SV40 DNA FO I with the corresponding enzyme. Concordingly, when the linear S1 generated duplexes were melted and renatured, circular duplexes were formed in addition to complex larger structures. This indicated that cleavage must have occurred at different sites. The double-strand-cleaving activity present in S1 nuclease preparations requires circular DNA as a substrate, as linear SV40 DNA is not cleaved. With regard to these properties S1 nuclease resembles some of the complex type I restriction nucleases from Escherichia coli which also cleave SV40 DNA only once, and, completely at random.

Centrifugation, Density Gradient↗
PubMed1984

The DNase activity of an endoplasmic reticulum nuclease and its effect on DNA synthesis in vitro.

An endoplasmic reticulum nuclease which was isolated previously in this laboratory from rat liver ( Kouidou et al. (1981) Eur.J. Bioch . 120, 9-14) was found to degrade linear and circular single stranded DNA but not double stranded DNA. The DNA fragments resulting from this cleavage were longer than 20 nucleotides. In addition the nuclease was found to improve the efficiency of DNA template used by DNA polymerase I in DNA synthesis in vitro. The results were the same whether incubation of the template with the nuclease was prior to addition of DNA polymerase I or simultaneously with polymerization. When nuclease was added after the completion of polymerization by DNA polymerase I it was ineffective unless the product was denatured. These data further corroborate the observation that double stranded DNA is not cleaved by this enzyme.

Animals↗
PubMed1992

32P-postlabeling of acrolein-deoxyguanosine adducts in DNA after nuclease P1 digestion.

In order to study the relationship between the level of acrolein-DNA adducts and their biological effects, sensitive methods are needed to quantitate DNA adducts. 32P-postlabeling is one such method that has been widely used and we have adapted the technique to detect acrolein-deoxyguanosine adducts. Adducts formed by the reaction of acrolein and deoxyguanosine-3'-monophosphate were isolated by HPLC. Based on their UV spectra and cochromatography with standards after dephosphorylation with acid phosphatase, these adducts were identified as the nucleotide equivalents of cyclic 1,N2-propanodeoxyguanosine adducts formed by acrolein that have been described by Chung et al. [15]. As nucleotides, the adducts were good substrates for polynucleotide kinase-mediated transfer of phosphate from ATP and were able to be detected by 32P-postlabeling. These adducts were resistant to the activity of nuclease P1 and dinucleoside monophosphates in the form d(G*pN) where G* is the acrolein-guanine adduct also resisted digestion by nuclease P1. Digestion of DNA by nuclease P1 and acid phosphatase resulted in the conversion of normal nucleotides to nucleosides and selective enrichment of the adducts as dinucleoside monophosphates. Using nuclease P1/acid phosphatase digestion, followed by 32P-postlabeling and TLC separation, levels of the two adducts in acrolein-treated DNA were found to be about 6185 and 19,222 nmol/mol.

Acid Phosphatase↗
PubMed1991

Cellular and tissue distribution of a single-strand-specific nuclease.

1. Specific antibodies which were raised against a single-strand-specific nuclease isolated from rat liver microsomes were used for characterizing this enzyme and determining its cellular and tissue distribution. 2. The single-strand-specific nuclease does not show any homology with other known nucleolytic enzymes. 3. It is mainly localized in microsomes and cytosol; traces of it are also found in nuclei, but it could not be detected in mitochondria. 4. Using the same specific antibodies we attempted to detect this nuclease in some other tissues which exhibit high metabolic rates, namely kidneys, heart and spleen. 5. Thus, with the aid of immunological techniques we were able to determine that at least part of the total poly(U) nucleolytic activity observed in kidney and heart is due to a nuclease immunologically identical to the enzyme under study. Kidneys were the best source for this enzyme.

Animals↗
PubMed1986

Mapping of endogenous nuclease-sensitive regions and of putative topoisomerase sites of action along the chromatin of Dictyostelium ribosomal RNA genes.

Indirect end-labelling and the digestion patterns of endogenous and exogenous nucleases were used to analyse chromatin organization along the ribosomal RNA genes of Dictyostelium discoideum cells. A zone just upstream from the 5' end of the coding region was particularly sensitive to endogenous nucleases. In exponentially growing cells, this hypersensitive zone extended from -350 to -1600 bp relative to the transcription start. In sharp contrast, the DNA between 0 and -350 bp was strongly protected. In differentiating cells, in which the ribosomal RNA transcription rate is low, the 5' hypersensitive zone was more diffuse than in exponentially growing cells, and the protected region at the 5' end of the transcribed region was less pronounced. It is known that where DNA topoisomerase is acting on DNA, the addition of sodium dodecyl sulphate will result in cleavage of the DNA and covalent attachment of the enzyme to the cut DNA end. Treatment of nuclei from both exponentially growing cells and differentiating cells with SDS caused double-stranded cleavages at -200 (i.e. within the protected region), at -2200, and at two sites at about -17 kb. A fraction of the cleavage products appeared to be strongly associated with protein. Novobiocin, a DNA topoisomerase II inhibitor, did not inhibit the SDS-induced cleavages in vegetative cells. However, it significantly reduced the extent of nuclease cleavage within the -350 to -1600 bp hypersensitive zone. The possibility is discussed that there are two DNA topoisomerase-like activities on the ribosomal genes. One is site-specific and novobiocin-insensitive. We speculate that the other is responsible for maintaining DNA at the 5' end of the gene in a torsionally strained, nuclease-hypersensitive state.

Chromatin↗
PubMed1993

Mutant H-ras over-expression inhibits a random apoptotic nuclease in myeloid leukemia cells.

Cell suicide, or apoptosis, is now recognized as an essential regulatory step in such diverse developmental processes as embryogenesis, thymocyte restriction, and hematopoiesis. One of the major features of apoptosis is the activation of an endogenous nuclease that cleaves DNA into nucleosomal fragments. Little is known about the activation or specificity of the apoptotic endonuclease. In this study, we investigated signalling pathways and the specificity of the apoptotic nuclease. We found that forced over-expression of activated H-ras inhibited activation of the apoptotic endonuclease. Since a high percentage of myelodysplasias and leukemias have mutations that activate ras, this finding lends insight into how ras might be leukemogenic. In addition, the phorbol ester TPA and a cyclic AMP analogue also slowed activation of this endonuclease. Interestingly, protein synthesis inhibition stimulated the endonuclease activity. In addition, by cloning and sequencing apoptotic fragments we found that the apoptotic nuclease has no sequence specificity. Thus, the apoptotic nuclease inhibited by H-ras over-expression was random in nature.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Alkaline nuclease activity in cells infected with herpes simplex virus type 1 (HSV-1) and HSV-1 temperature-sensitive mutants.

An in situ assay has been adapted to the herpes simplex virus type 1 (HSV-1) system which can detect alkaline nuclease activity in infected cell lysates following sodium dodecylsulfate polyacrylamide gel electrophoresis. Lysates of cells infected with HSV-1 temperature-sensitive (ts) mutants possessing mutations in the genes for an immediate-early transcriptional regulatory protein (ICP4), viral DNA polymerase (pol), and the major HSV-1 DNA binding protein (ICP8) exhibited altered alkaline nuclease profiles relative to that of wild-type virus-infected cells at 39 degrees C. Infections with a control mutant defective in the gene for glycoprotein B yielded wild-type nuclease profiles. The diverse effects on alkaline nuclease expression of mutants with lesions in different viral proteins involved directly in viral DNA synthesis provides evidence for the cooperative interaction between HSV-encoded viral DNA replication components.

Animals↗
PubMed1988

Circular intermediates with missing nucleotides in the conversion of supercoiled or nicked circular to linear duplex DNA catalyzed by two species of BAL 31 nuclease.

The extracellular nucleases from Alteromonas espejiana BAL 31 can catalyze the endonucleolytic and/or exonucleolytic hydrolysis of duplex DNA in response to a variety of alterations, either covalent or noncovalent, in DNA structure. The nuclease can exist as at least two kinetically and molecularly distinct protein species. The two species that have been studied, called the 'fast' (F) and 'slow' (S) nucleases, both readily convert negatively supercoiled DNAs to linear duplex molecules and accomplish this conversion through the formation of a circular duplex intermediate containing usually a single interruption in one strand. It is further shown that most of these intermediates contain gaps arising from the removal in a processive manner of one or more nucleotide residues after the introduction of the initial strand break (nick). Considering only the intermediates with gaps, the average number of missing residues is 6.3 +/- 0.5 and 2.8 +/- 0.3, respectively, for DNA acted upon by the F and S enzymes independently of the extent of conversion of supercoiled DNA. The nicks and gaps are bounded by 3'-hydroxyl and 5'-phosphoryl termini. When singly nicked circular DNA is used as the substrate, conversion to the linear duplex form occurs predominantly through a gapped circular intermediate with the same average numbers, within experimental error, of missing nucleotides for the respective nuclease species as found when supercoiled DNA is the substrate. The conversion to linear duplex DNA is much slower when nicked circular DNA is the substrate compared to that found when supercoiled DNA is the starting material.

DNA, Circular↗
PubMed1993

The differential lysis of phosphoester bonds by nuclease P1.

The hydrolysis by nuclease P1 of the 16 common deoxydinucleoside monophosphates was examined. The rates of hydrolysis of phosphodiester bond differ by more than two orders of magnitude; dinucleotide monophosphates of the type d(TpN) being most resistant and d(GpN) being next most resistant. The profiles of a mixture of the 16 common dinucleoside monophosphates and of DNA after partial hydrolysis by nuclease P1 and simultaneous treatment with acid phosphatase were compared. The resultant profiles are very similar, except for the appearance of 5-methyldeoxycytidine in the latter. Similar profiles are also obtained from a mixture of dinucleoside monophosphates and from DNA exposed to ionizing radiation beforehand. The 8-hydroxyguanine lesion and a formamido remnant of thymine appear in both profiles as a modified nucleoside and as modified dinucleoside monophosphate respectively. These results suggest that certain radiation induced DNA lesions can be selectively postlabelled based on their resistance to hydrolysis by nuclease P1. The nature of the nuclease P1-substrate interaction is discussed.

Animals↗