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PubMed1981

Sequence specific cleavage of DNA by micrococcal nuclease.

Micrococcal nuclease is shown to cleave DNA under conditions of partial digestion in a specific manner. Sequences of the type 5'CATA and 5'CTA are attacked preferentially, followed by exonucleolytic degradation at the newly generated DNA termini. GC-rich flanking sequences further increase the probability of initial attack. Unexpectedly, long stretches containing only A and T are spared by the nuclease. These results, which were obtained with spared by the nuclease. These results, which were obtained with mouse satellite DNA and two fragments from the plasmid pBR22, do not support the previous contention that it is the regions of high At-content which are initially cleaved by micrococcal nuclease. This specificity of micrococcal nuclease complicates its use in experiments intended to monitor the nucleoprotein structure of a DNA sequence in chromatin.

Animals↗
PubMed1989

Structure and dynamics of staphylococcal nuclease mutants as studied by fluorescence quenching techniques.

Fluorescence techniques have been used to investigate the effect of mutations on the structure and dynamics of staphylococcal nuclease. An estimate of the accessibility to acrylamide of the enzyme's single tryptophan residue (Trp140) was obtained from the Stern-Volmer constant for fluorescence quenching. This was indicative of a partially buried tryptophan in the wild-type nuclease. Five single-site mutant nucleases (H124L, V66L, G88V, G79S and F76V) and one double mutant (V66L + G88V), with widely differing stabilities to denaturants, gave Stern-Volmer constants which were very similar to that of their parent enzyme. Studies of the temperature- and viscosity-dependence of quenching suggest that access by acrylamide to Trp140 is limited by diffusion rather than by protein structural fluctuations. Lifetime-resolved fluorescence anisotropy studies using steady-state instrumentation suggest that there is very little segmental motion of the Trp140; most of the anisotropy therefore decays due to protein rotation in the solution. Rotational correlation times for several nuclease mutants have been determined and these are very similar to that of the native nuclease. Thus it appears that these substitutions in the primary amino acid sequence, which have significant effects on the stability of the folded proteins, do not cause a significant change in the protein structure or dynamics around Trp140.

Escherichia coli↗
PubMed2002

Signal-sequence-independent secretion of the staphylococcal nuclease in Mycobacterium smegmatis.

Staphylococcus aureus nuclease is a small, secreted protein which has been successfully used as a reporter system to identify exported products in Lactococcus lactis. Here, biochemical evidence is provided that the nuclease is exported by Mycobacterium smegmatis in the presence, but also in the absence of a signal sequence, and thus probably independently of the Sec translocation pathway. This implies that the nuclease should not be used as a reporter system in mycobacteria for the identification of exported products, despite what has been reported previously in the literature. The nuclease can be extended to create hybrid proteins that remain compatible with its secretion, whereas some other shorter fusions are not tolerated. This suggests that correct folding is required for efficient export. Extensive mutational analysis did not identify a specific secretion pathway. This suggests that the nuclease may be exported by different redundant systems or that components of this alternative Sec pathway are essential for bacterial survival.

Base Sequence↗
PubMed2001

Targeted histone acetylation and altered nuclease accessibility over short regions of the pea plastocyanin gene.

The chromatin structure of the pea plastocyanin gene (PetE) was examined at three different transcriptional states by investigating the acetylation states of histones H3 and H4 and the nuclease accessibility of the gene in pea roots, etiolated shoots, and green shoots. The acetylation states of histones associated with different regions of PetE were analyzed by chromatin immunoprecipitation with antibodies specific for acetylated or nonacetylated histone H3 or H4 tails, followed by polymerase chain reaction quantification. Comparison of pea tissues indicated that histone hyperacetylation was associated with increased PetE transcription in green shoots. Moreover, hyperacetylation of both histones H3 and H4 was targeted to the enhancer/promoter region in green shoots, suggesting that only specific nucleosomes along the gene were modified. Time-course digestions of nuclei with micrococcal nuclease and DNaseI indicated that the enhancer/promoter region was more resistant to digestion in the inactive gene in pea roots than was the same region in the active gene in shoots, whereas the transcribed region of PetE was digested similarly among the tissues. This finding indicates that transcription is accompanied by changes in the nuclease accessibility of the enhancer/promoter region only. Moreover, these results indicate that the changes in nuclease accessibility are organ specific, whereas histone hyperacetylation is light dependent, and they suggest that changes in nuclease accessibility precede histone hyperacetylation during PetE activation.

Acetylation↗
PubMed1990

Enrichment of ubiquitinated histone H2A in a low salt extract of micrococcal nuclease-digested myotube nuclei.

We recently demonstrated that ubiquitinated histone H2A (uH2A) declines 2.5-fold during terminal skeletal muscle differentiation, coincident with reductions in transcriptional activity (Wunsch, A. M., Haas, A. L., and Lough, J. (1987) Dev. Biol. 119, 85-93). To assess whether this indicates an association of uH2A with transcriptionally active genes, we have used micrococcal nuclease digestion and salt extraction to fractionate myotube nuclei. An oligonucleosomal fraction obtained by micrococcal nuclease digestion and extraction in low salt (100 mM NaCl) comprising only 25% of the nuclear DNA contained 90% of the total uH2A, as revealed by Western blotting. Further fractionation of this 100 mM salt extract by sucrose gradient centrifugation revealed that virtually all of the uH2A was localized in monomer to heptamer-sized oligonucleosomes. A second ubiquitinated species of 57-kDa (u57) was also localized in the 100 mM salt extract. In contrast, an 18-kD band (u18) was associated with fractions that were resistant to micrococcal nuclease digestion and salt extraction. Although micrococcal nuclease recognized a unique structural feature of active myotube chromatin, as evidenced by the appearance of hybridized skeletal alpha-actin sequences as a smear rather than the nucleosomal ladder exhibited by inactive and bulk sequences, neither the skeletal alpha-actin gene nor the inactive alpha D-globin gene was exclusively localized in the 100 mM salt fraction. Moreover, further fractionation of the 100 mM salt extract on a sucrose gradient failed to separate active from inactive genes. These findings suggest that uH2A is localized in a fraction of myotube chromatin which, although nuclease-sensitive and relatively soluble, is not enriched in active or inactive genes.

Animals↗
PubMed1987

Tissue-specific and periodic changes in the nuclease sensitivity of the fibroin gene chromatin in the silkworm Bombyx mori.

Nuclei of substantial purity were isolated from the middle or posterior silk glands of the silkworm Bombyx mori larvae. Both the fibroin H- and L-chain gene sequences in the isolated nuclei from the posterior silk glands of the fifth instar larvae, where the genes are transcribed actively, are extremely sensitive to the digestion with DNaseI; on the other hand, these sequences in the middle silk gland nuclei from the same larvae, where the genes are not expressed, are markedly resistant to the digestion. The H-chain gene sequences in the posterior silk gland nuclei from the fifth instar larvae are also highly susceptible to the digestion with micrococcal nuclease, HinfI, and HhaI. The digestion products with micrococcal nuclease show a continuous size distribution. The H-chain gene sequences in the middle silk gland nuclei or the posterior silk gland nuclei from the fourth molting stage are cleaved partially into nucleosome dimer to oligomer sizes upon digestion with higher concentrations of micrococcal nuclease, suggesting that the inactive forms of the H-chain gene chromatin are constructed by folding of the chromatin fiber containing a regular array of nucleosomes. Hypersensitive sites to micrococcal nuclease are present near both ends of the second exon, a major body of the fibroin H-chain gene, in both the active and inactive forms of the chromatin. The DNaseI or micrococcal nuclease sensitivity of the H-chain gene chromatin in the posterior silk gland nuclei shows periodical changes corresponding to the intermolt-molt-intermolt cycle.

Animals↗
PubMed1979

Superstructural differences between chromatin in nuclei and in solution are revealed by kinetics of micrococcal nuclease digestion.

Digestion of chromatin in nuclei by micrococcal nuclease, measured as the change in the concentration of monomer-length DNA with time, displays Michaelis-Menten kinetics. Redigestion of soluble chromatin prepared from nuclei by micrococcal nuclease treatment, however, is apparently first order in enzyme and independent of chromatin concentration. This qualitative difference results from an increase in the apparent second order rate constant, kcat/Km, for liberation of monomer DNA: the apparent Km for soluble chromatin is lower by close to 3 orders of magnitude than that for chromatin in nuclei, whereas kcat decreases by less than 1 order of magnitude. Neither the integrity of the nuclear membrane nor the presence of histone H1 contributes to the high Michaelis constant characteristic of chromatin in nuclei. Moreover, differences due to the buffers used for digestion and redigestion are minimal. Low catalytic efficiency is, however, correlated with the presence of higher order chromatin superstructure. Micrococcal nuclease added to soluble chromatin under nondigesting conditions at low ionic strength (I = 0.002) co-sediments with chromatin in sucrose gradients. In 0.15 M NaCl, added nuclease no longer sediments with chromatin and redigestion kinetics become first order in both enzyme and substrate. Kinetic analysis of this type may afford an assay for native, higher order structures in chromatin. Our results suggest that micrococcal nuclease binds to soluble chromatin through additional interactions not present in nuclei, which may be partly ionic in nature.

Animals↗
PubMed1984

Effect of irradiation and endogenous nucleases on rat liver chromatin.

These assessment of the consequences of irradiation on chromatin is complicated by endogenous nucleases. Isolation and prolonged storage of rat liver nuclei in buffers containing divalent metal ions activates these enzymes and promotes the degradation of chromatin. Irradiation of rat liver nuclei to dose levels of 20,000 rad under conditions in which endogenous nucleases are inhibited and analysis of the irradiated chromatin by sucrose density gradient centrifugation gave no evidence for monosomes or oligosomes. When chromatin from irradiated nuclei was digested with micrococcal nuclease, the levels of monosomes and oligosomes were identical to those of micrococcal nuclease, the levels of monosomes and oligosomes are identical to suggest that irradiation results in neither a direct fragmentation of linkers nor the sensitization of linkers for subsequent cleavage by micrococcal nuclease. Histones isolated from monosomes of irradiated and unirradiated nuclei were intact, showing no fragmentation or loss of residues, as judged by sodium dodecyl sulfate-polyacrylamide electrophoresis.

Animals↗
PubMed1982

Chromatin structure of the chicken beta-globin gene region. Sensitivity to DNase I, micrococcal nuclease, and DNase II.

We have examined in some detail the chromatin structure of a 6.2 kilobase pair (kbp) chromosomal region containing the chicken beta-globin gene. The chromatin structure was probed with three nucleases, DNase I, micrococcal nuclease, and DNase II, and the rate of digestion of specific subfragments of the region was compared with the rate of bulk DNA digestion. We have characterized the rate of digestion of each fragment in terms of a sensitivity factor which measures the sensitivity of a fragment to a particular nuclease relative to bulk DNA. The sensitivity factors were determined by a least squares curve fitting method based on target analysis. In nuclei isolated from 14-day-old chicken embryo red blood cells, the entire 6.2-kbp region shows approximately a 10- to 20-fold increase in sensitivity to DNase I, a 3-fold increased sensitivity to micrococcal nuclease, and a 6-fold increased sensitivity to DNase II. In addition to the adult beta-globin gene, this region contains 5' and 3' flanking sequences, the 5' half of the inactive, embryonic globin gene, epsilon, and some repeated sequences. There is no obvious correlation between these genetic elements and the overall chromatin structure as measured by the nuclease sensitivity. This same region shows little or no special sensitivity in nuclei isolated from 14-day-old chicken embryo brain. Furthermore, fragments of the inactive ovalbumin gene show little or no sensitivity in either red blood cells or brain. These results support the conclusion that the entire 6.2-kbp region is largely packaged as active chromatin in 14-day-old chicken embryo red blood cells.

Animals↗
PubMed1994

High-level expression of staphylococcal nuclease A in Escherichia coli.

The staphylococcal nuclease A gene has been successfully cloned and overexpressed in E. coli under the transcriptional control of the bacteriophage lambda PRPL promoters regulated by the temperature sensitive repressors. The SDS-PAGE analysis demonstrates that the nuclease A is produced to the extent of as much as 60% of the total cellular protein. The N-terminal analysis of the nuclease A shows that the amino terminal formyl methionine residue of the enzyme is precisely processed. The recombinant nuclease A with full activity is finally obtained after appropriate solubilization--denaturation and renaturation treatment. The conformational identity of the renatured nuclease A in different conditions is also studied by using hydrophobic interaction chromatography on a phenyl-superose HR5/5 column.

Base Sequence↗
PubMed2007

Effect of siRNA nuclease stability on the in vitro and in vivo kinetics of siRNA-mediated gene silencing.

Small interfering RNA (siRNA) molecules achieve sequence-specific gene silencing through the RNA interference (RNAi) mechanism. Here, live-cell and live-animal bioluminescent imaging (BLI) is used to directly compare luciferase knockdown by unmodified and nuclease-stabilized siRNAs in rapidly (HeLa) and slowly (CCD-1074Sk) dividing cells to reveal the impact of cell division and siRNA nuclease stability on the kinetics of siRNA-mediated gene silencing. Luciferase knockdown using unmodified siRNAs lasts approximately 1 week in HeLa cells and up to 1 month in CCD-1074Sk cells. There is a slight increase in the duration of luciferase knockdown by nuclease-stabilized siRNAs relative to unmodified siRNAs after cationic lipid transfection, but this difference is not observed after electroporation. In BALB/cJ mice, a fourfold increase in maximum luciferase knockdown is observed after hydrodynamic injection (HDI) of nuclease-stabilized siRNAs relative to unmodified siRNAs, yet the overall kinetics of the recovery after knockdown are nearly identical. By using a mathematical model of siRNA-mediated gene silencing, the trends observed in the experimental data can be duplicated by changing model parameters that affect the stability of the siRNAs before they reach the cytosolic compartment. Based on these findings, we hypothesize that the stabilization advantages of nuclease-stabilized siRNAs originate primarily from effects prior to and during internalization before the siRNAs can interact with the intracellular RNAi machinery.

Animals↗
PubMed1991

Nuclease-induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide.

A flow cytometric analysis of DNA structural changes induced by cleavage with nucleases was performed on isolated HeLa nuclei by assessing changes in stainability with the DNA-specific fluorochrome propidium iodide (PI). After mild digestion with DNAse I, micrococcal nuclease, or with the single-strand-specific S1 and Neurospora crassa nucleases, fluorescence intensity of nuclei stained with PI increased by about 15-30% above the value of undigested control samples. No significant modifications were observed with the restriction enzymes Eco RI, Alu I, and Not I. The DNAse I-induced increase in fluorescence intensity was also observed with the non-intercalating dye Hoechst 33258, but not with mithramycin. Nuclease-induced fluorescence intensity changes as determined with PI were found to be dependent on the dye concentration. A constant increase (about 20%) was measured at dye/DNA-P ratios greater than 0.11. Below this value (2 micrograms/ml PI), the fluorescence intensity of digested samples was 15-30% lower than that of undigested controls. This behaviour towards intercalating dyes is similar to that of the relaxed (nicked) vs. the supercoiled (intact) form of circular DNA. These results suggest that conformation- but not sequence-specific nucleases induce a relaxation of DNA supercoils.

Coloring Agents↗
PubMed1993

Increased sensitivity of damaged DNA to digestion with nuclease S1 as assessed in single cells by flow cytometry.

DNA sensitivity to digestion with nuclease S1 was investigated in cells irradiated with gamma rays, or treated with the antitumor drug adriamycin (Adr). The nuclease-resistant DNA fraction was determined by propidium iodide staining. Treated cells were found to be more sensitive to nuclease digestion than the undamaged controls. Gamma ray-induced strand breaks were detectable at doses up to 10 Gy; an increase in the reaction temperature, from 37 degrees to 63 degrees C, was necessary in order to detect higher levels of damage. Nuclease S1 sensitivity in Adr-treated cells showed a single-peak, concentration-dependent relationship, in agreement with the known self-inhibitory effect exerted by high drug doses. Determination of DNA digestion could be performed in combination with other cellular parameters (e.g., protein content). Detection of drug-resistant cells in a heterogeneous population of small-cell lung carcinoma was achieved on the basis of the different sensitivity of the cells to enzymatic digestion. These results indicate that nuclease S1 may be a useful probe for studying in single cells DNA alterations induced by drugs or radiation.

Cell Separation↗
PubMed1992

HSD restriction-modification proteins partake in latent anticodon nuclease.

Phage T4-induced anticodon nuclease triggers cleavage-ligation of the host tRNA(Lys). The enzyme is encoded in latent form by the optional Escherichia coli locus prr and is activated by the product of the phage stp gene. Anticodon nuclease latency is attributed to the masking of the core function prrC by flanking elements homologous with type I restriction-modification genes (prrA-hsdM and prrD-hsdR). Activation of anticodon nuclease in extracts of uninfected prr+ cells required synthetic Stp, ATP and GTP and appeared to depend on endogenous DNA. Stp could be substituted by a small, heat-stable E. coli factor, hinting that anticodon nuclease may be mobilized in cellular situations other than T4 infection. Hsd antibodies recognized the anticodon nuclease holoenzyme but not the prrC-encoded core. Taken together, these data indicate that Hsd proteins partake in the latent ACNase complex where they mask the core factor PrrC. Presumably, this masking interaction is disrupted by Stp in conjunction with Hsd ligands. The Hsd-PrrC interaction may signify coupling and mutual enhancement of two prokaryotic restriction systems operating at the DNA and tRNA levels.

DNA Restriction-Modification Enzymes↗
PubMedDate not supplied

DNA transfection of macaque and murine respiratory tissue is greatly enhanced by use of a nuclease inhibitor.

BACKGROUND: Nuclease activity present within respiratory tissues contributes to the rapid clearance of injected DNA and therefore may reduce the transfection activity of directly injected transgenes. Most gene transfer technologies transduce or transfect murine tissues more efficiently than corresponding primate tissues. Therefore, it is prudent to assess the utility of novel gene transfer strategies in both rodent and primate models before proceeding with further development. METHODS: This study analyzed the effects of ATA (a nuclease inhibitor) on the direct transfection of macaque and murine lung tissue; compared the levels of DNase activity in murine, primate, and human lung fluids; and tested the inhibitory activity of ATA on the DNase activity present in these samples. Fluorescent microspheres were used to detect areas of transfection in lung. RESULTS: Intratracheal administration of a nuclease inhibitor (ATA) with naked DNA (0.5 microg ATA/g body weight) enhanced direct transfection efficacy in macaque lung by over 86-fold and by over 54-fold in mouse lung. Hematoxylin and eosin staining showed no apparent tissue toxicity. Moreover, macaque, human, and mouse lung fluids were found to possess similar levels of DNase activity and this activity was inhibited by similar concentrations of ATA. The authors also successfully pioneered the use of carboxylate-modified microsphere tracers to identify areas of transfection and/or treatment. CONCLUSION: This work provides evidence that using direct nuclease inhibitors will enhance lung transfection and that nuclease activity is present in all lung fluids tested, which can be inhibited by the use of direct DNase inhibitors.

Animals↗
PubMed2006

Identification of a new subfamily of HNH nucleases and experimental characterization of a representative member, HphI restriction endonuclease.

The restriction endonuclease (REase) R. HphI is a Type IIS enzyme that recognizes the asymmetric target DNA sequence 5'-GGTGA-3' and in the presence of Mg(2+) hydrolyzes phosphodiester bonds in both strands of the DNA at a distance of 8 nucleotides towards the 3' side of the target, producing a 1 nucleotide 3'-staggered cut in an unspecified sequence at this position. REases are typically ORFans that exhibit little similarity to each other and to any proteins in the database. However, bioinformatics analyses revealed that R.HphI is a member of a relatively big sequence family with a conserved C-terminal domain and a variable N-terminal domain. We predict that the C-terminal domains of proteins from this family correspond to the nuclease domain of the HNH superfamily rather than to the most common PD-(D/E)XK superfamily of nucleases. We constructed a three-dimensional model of the R.HphI catalytic domain and validated our predictions by site-directed mutagenesis and studies of DNA-binding and catalytic activities of the mutant proteins. We also analyzed the genomic neighborhood of R.HphI homologs and found that putative nucleases accompanied by a DNA methyltransferase (i.e. predicted REases) do not form a single group on a phylogenetic tree, but are dispersed among free-standing putative nucleases. This suggests that nucleases from the HNH superfamily were independently recruited to become REases in the context of RM systems multiple times in the evolution and that members of the HNH superfamily may be much more frequent among the so far unassigned REase sequences than previously thought.

Amino Acid Sequence↗
PubMed2005

A conditional suicide system for Saccharomyces cerevisiae relying on the intracellular production of the Serratia marcescens nuclease.

A conditional lethal system for biological containment of genetically modified strains of Saccharomyces cerevisiae is described. This suicide system is based on the intracellular production of the Serratia marcescens nuclease in the yeast cell, aiming at the destruction of the host genetic material. The S. marcescens nuclease, encoded by the nucA gene, is normally secreted by the bacterium into the medium. In the present work, the nucA gene, devoid of its signal peptide coding sequence, was cloned in a yeast expression vector, under control of the glucose-repressed S. cerevisiae alcohol dehydrogenase 2 gene (ADH2) promoter. When transformed into S. cerevisiae, the recombinant plasmid proved to be effective in killing the host cells upon glucose depletion from the medium, and the nuclease activity was found in lysates prepared from the transformants. In addition, the nuclease degrading effect was shown to reach chromosomal DNA in the yeast host. The killing effect of the nucA plasmid was also demonstrated in soil microcosm assays, indicating that whenever the GMM escapes into the environment where glucose is scarce, the nucA gene will be expressed and the resulting nuclease will destroy the genetic material and kill the cells. In contrast to other suicide systems that target the cell envelope, the advantage of the one described here is that it disfavours horizontal gene transfer from recombinant yeast cells to other microorganisms found in the environment.

Blotting, Western↗
PubMed1994

Purification and characterization of fungal nuclease composed of heterogeneous subunits.

A nuclease was purified from the extract of wheat bran culture of Aspergillus sp. isolated from "Katsuobushi" by a series of column chromatographies. The purified nuclease showed a single protein band on nondenaturing polyacrylamide gel electrophoresis. The enzyme showed three protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their molecular weights were estimated to be 80,000, 50,000, and 25,000. The molecular weight of the nuclease was estimated to be 125,000 by gel permeation chromatography. The enzyme showed maximum activity around pH 8.0 for DNA and RNA. The enzyme required Mg2+, Mn2+, or Co2+ for the appearance of activity. The enzyme was stable until 40 degrees C and in pH range of 5-9. The stability of the enzyme for temperature increased until 50 degrees C by Ca2+. The enzyme exonucleolytically degraded DNA and RNA by 3'-->5' mode to produce 5'-mononucleotides. The fungal nuclease acted on heat-denatured DNA and native DNA and RNA, but not bis(p-nitrophenyl)phosphate, p-nitrophenyl thymidine 5'-phosphate, and p-nitrophenyl thymidine 3'-phosphate. The enzyme did not show strict base specificity for DNA and RNA, while the affinity for substrate was affected by 3'-terminal bases. The enzyme preferentially degraded poly(C) and poly(U), but hardly degraded poly(A) and poly(G). The nuclease acted on closed circular double-stranded DNA to produce open circular DNA and then linear form DNA by single-strand scission. The nicking activity was intrinsic to the enzyme.

Animals↗