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PubMed1995

Capillary electrophoresis of S. nuclease mutants.

The electrophoretic migration behavior of 12 S. nuclease variants from Staphylococcus aureus with small but well defined structural differences from site directed mutation was investigated in free solution capillary electrophoresis at pH 2.8 to 9.5. The nucleases are basic proteins; the pI and the M(r) of the wild type are 10.3 and 16.811 kd, respectively. With specially selected oligoamino buffers and with an inert, hydrophilic wall coating in 75 microns I.D. quartz capillary tubes, most of the proteins could be separated by CZE without interference by wall adsorption even at pH 9.5 where the selectivity was the highest. At pH 2.8, 4.1 and 7.0, S. nucleases are known to be in the random coil, "swollen" and the tight native state. Assuming that in a given state, i.e., at a certain pH, the molecular radii of the nucleases are the same, their hydrodynamic radii were calculated from their pertinent electrophoretic mobilities. The respective radii of 50.1, 26.8, and 25.0 Angstrum thus obtained agreed very well with the corresponding radii of gyration obtained from X-ray scattering. In fact, from the electrophoretic mobilities at pH 9.5, the existence of a hitherto unknown swollen basic state of the nuclease having a hydrodynamic radius of 30.5 Angstrum was postulated. In addition, a method was described to evaluate the valence of the protein at different pH from their pertinent electrophoretic mobilities. A general advantage of this method is that only the differences between the valences of the mutants and the wild type are needed; and for none of the proteins is required the knowledge of the actual valence. The results of the methods allowed the construction of a pH profile of the protein's valence. For the wild type, this profile was compared to the H+ titration curve and the agreement was excellent. Both methods employed some novel structure-electrophoretic mobility relationships and the predicted protein properties compared remarkably well to the values obtained by exoelectrophoretic methods such as pH titration and X-ray scattering. Surprisingly, certain S. nucleases having the same valence could also be readily separated by CZE in some cases under the same conditions used for the others. Close examination of appropriate X-ray crystallography and/or NMR data indicated subtle differences in the molecular structure of these proteins that could be responsible for slight alteration in their hydrodynamic radii.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1985

Neurospora crassa and S1 nuclease cleavage in hsp 83 gene chromatin.

We have examined the distribution of Neurospora crassa and S1 nuclease cleavage products in the chromatin of the hsp 83 heat shock gene from the Drosophila melanogaster cytogenetic locus 63 BC. Both of these nucleases generate double strand breaks in chromatin at specific sites close to the 5' end of the hsp 83 gene. With N. crassa nuclease we observe one major 5' fragment which is derived from nuclease cleavage in a DNA segment mapping approximately 120 base-pairs from the beginning of the transcription unit. With S1 nuclease we observe one major fragment which overlaps the transcription start site. In addition to the major hypersensitive sites at the beginning of the gene, the hsp 83 transcription unit is also sensitive to attack by these nucleases both before and after heat shock; however, the yield of cleavage products from within the gene is considerably greater after heat induction.

Animals↗
PubMed1982

The intranuclear distribution of rat uterine estrogen receptors determined after nuclease treatment and chromatin fractionation.

The intranuclear locations at which rat uterine estrogen receptors interact with chromatin have been probed using digestions performed with DNAase I and micrococcal nuclease. Exposure to nuclease has been controlled to effect limited to extensive digestion of nuclear DNA under conditions which maintain the integrity of the [3H] estradiol-receptor complex. The effect of divalent cation concentration on the release of estrogen receptors fron nuclease-treated chromatin was examined and found to be of consequence above 2 mM. Exposure to nuclease released nuclear estrogen receptors from chromatin, with DNAase I being more efficient than micrococcal nuclease in mediating this release. The release of the bulk of nuclear estrogen receptors closely paralleled the nuclease-mediated digestion of chromatin DNA. At 1 h after exposure to estrogen, substantial quantities of uterine estrogen receptors (80-90%) were distributed in chromatin fractions which, on the basis of fractionation terminology, have been termed 'transcriptionally inactive' by convention. Enrichment of estrogen receptors in chromatin which has been termed ' transcriptionally active' only occurred with 10-20% of the estrogen receptors. Hence, our findings support a model where, at early times after estrogen exposure, receptors from the rat uterus are enriched to only a minor extent in chromatin to which 'transcriptional activity' is generally assigned while the bulk of receptors are localized in chromatin which is generally considered 'transcriptionally inactive'.

Animals↗
PubMed2005

Effect of N-terminal deletions on the foldability, stability, and activity of staphylococcal nuclease.

The effect of N-terminally successive deletions on the foldability, stability, and activity of staphylococcal nuclease was examined. The structural changes in the nuclease caused by the deletions follow a hierarchical pattern: N-terminal truncation of the nuclease by up to nine residues clearly perturbs the conformation of the N-terminal beta-subdomain but does not affect the C-terminal alpha-subdomain; deletion of 11 or 12 residues perturbs the C-terminal alpha-subdomain, resulting in formation of a molten globule state; deletion of 13 residues causes the nuclease to become highly unfolded. N-terminally deleted nuclease delta11 retains the ability to fold but delta12 is not able to fold into an enzymatically active conformation, suggesting that 11 residues is the maximum length that can be deleted from the N-terminus while still retaining the folding competence of the nuclease. Further, the results suggest that proper folding of the C-terminal alpha-subdomain probably relies on the integrity of the N-terminal beta-subdomain.

Amino Acid Sequence↗
PubMedDate not supplied

Strand invasion by oligonucleotide--nuclease conjugates.

Conjugates consisting of staphylococcal nuclease crosslinked to oligonucleotides hybridize to supercoiled duplex DNA by Watson--Crick base-pairing. Here we describe this strand invasion. Affinity cleavage by these conjugates provides a probe for the local topology of the DNA duplex and is most efficient at a target DNA sequence known to form a cruciform. Additional supercoiling of the substrate DNA increases selective cleavage at other sequences. Hybridization of the conjugate to duplex DNA is temperature dependent and is stable over time. Affinity cleavage is not substantially inhibited by a 200-fold excess of the analogous unmodified oligonucleotide, demonstrating that hybridization of the unmodified oligonucleotide must be less favored and that the nuclease is involved in substrate binding. Surprisingly, affinity cleavage is also not effectively inhibited by complementary oligonucleotides unless they contain an extended 5'-sequence capable of separate interactions with the nuclease domain of the conjugate. These results suggest that the oligonucleotide-nuclease conjugate prefers to hybridize to target sequences which will allow interactions with both the oligonucleotide and the nuclease domains. Affinity cleavage by oligonucleotide-nuclease conjugates provides general insights for the design of oligonucleotides and their conjugates for strand invasion and affords a convenient competition assay for their hybridization.

Amino Acid Sequence↗
PubMed1989

Staphylococcal nuclease active-site amino acids: pH dependence of tyrosines and arginines by 13C NMR and correlation with kinetic studies.

The pH and temperature dependence of the kinetic parameters of staphylococcal nuclease (EC 3.1.4.7) have been examined with three p-nitrophenyl phosphate containing DNA analogues that vary as to 3'-substituent. With wild-type (Foggi variant) (nuclease wt) and the substrates thymidine 3'-phosphate 5'-(p-nitrophenyl phosphate) (PNPdTp), thymidine 3'-methylphosphonate 5'-(p-nitrophenyl phosphate) (PNPdTp*Me), and thymidine 5'-(p-nitrophenyl phosphate) (PNPdT), kcat remains nearly constant at 13 min-1. However, kcat/Km with nuclease wt varies considerably: 413, 13, and 0.52 mM-1 min-1 with PNPdTp, PNPdTp*Me, and PNPdT, respectively. When tyrosine-85 is changed to phenylalanine (nuclease Y85F) by site-directed mutagenesis, kcat is unchanged at about 13 min-1, except with PNPdTp where it drops to 1 min-1. With nuclease Y85F, kcat/Km is 19.5 and 25 mM-1 min-1 with PNPdTp and PNPdTp*Me, respectively. With PNPdTp as the substrate, a bell-shaped kcat/Km vs pH profile is seen with pKa values at 8.94 and 9.67 in 0.3 M KCl and H2O. The pKa at 9.67 disappears, and a new pKa appears at 10.1 when tyrosine-85 is changed to phenylalanine (nuclease Y85F) or when the substrate 3'-phosphomonoester is changed to a 3'-methylphosphonate (PNPdTp*Me). This suggests that the inflection in kcat/Km with pKa at 9.67 arises from ionization of tyrosine-85, which hydrogen bonds to the divalent 3'-phosphomonoester of substrates with this substituent.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine↗
PubMed1990

Coupling between local structure and global stability of a protein: mutants of staphylococcal nuclease.

Staphylococcal nuclease exists in solution as a mixture of two folded (N and N') and two unfolded (U and U*) forms. Earlier workers [Evans et al. (1989) Biochemistry 28, 362] have proposed that the N'/N and U/U* structural differences involve cis/trans isomerization about the Lys116-Pro117 peptide bond with N and U cis and N' and U* trans. The present results show that residue changes throughout the nuclease structure have large effects on the distribution of the N and N'forms. The N'/N ratios at 313 K for nuclease H124L (N'/N = 0.07) and nuclease G79S (N'/N = 12) differ by 2 orders of magnitude. Thermodynamic parameters for equilibria linking the two folded and two unfolded substates were evaluated for seven mutants of nuclease which were found by kinetic assays to have similar enzymatic activities but by NMR spectroscopy to have a wide dispersion of thermal stabilities. Our results indicate that mutational perturbations of the N'/N equilibrium in folded nuclease (delta G for the N in equilibrium N' reaction) are strongly coupled to changes in the stability of the N form (delta G for the N in equilibrium U reaction), but much less so to the stability of the N' form (delta G for the N' in equilibrium U* reaction).

Amino Acid Sequence↗
PubMed1975

Evaluation of quantitative affinity chromatography by comparison with kinetic and equilibrium dialysis methods for the analysis of nucleotide binding to staphylococcal nuclease.

The elution of staphylococcal nuclease on thymidine 3'-(p-Sepharose-aminophenyl phosphate) 5'-phosphate (nucleotide ligand of nuclease covalently bound to Sepharose 4B) was studied in the presence of a variety of soluble nucleotide ligands. The elution volumes of nuclease vary proportionally with matrix-bound ligand concentration (at constant soluble ligand concentration), inversely with soluble ligand concentration (at constant matrix-bound ligand concentration), and inversely with dissociation constant of soluble ligand (at constant concentrations of soluble and matrix-bound ligand). The variation of elution volume was related to an expression which described the competition of soluble and matrix-bound ligand for nuclease binding. Using this expression, values for dissociation constants were derived for nucleotide ligands in both the soluble and bound form. The values for soluble ligand were found to correspond closely to those obtained by either equilibrium dialysis or kinetics of inhibition of nuclease activity. Furthermore, a close correspondence was found between the values of dissocation constants for matrix-bound and soluble thymidine 3'-(p-aminophenyl phosphate) 5'-phosphate, thus defining the interaction of nuclease with the matrix-bound ligand as a process quite similar to that occurring in solution.

Chromatography, Affinity↗
PubMed2003

A micrococcal nuclease homologue in RNAi effector complexes.

RNA interference (RNAi) regulates gene expression by the cleavage of messenger RNA, by mRNA degradation and by preventing protein synthesis. These effects are mediated by a ribonucleoprotein complex known as RISC (RNA-induced silencing complex). We have previously identified four Drosophila components (short interfering RNAs, Argonaute 2 (ref. 2), VIG and FXR) of a RISC enzyme that degrades specific mRNAs in response to a double-stranded-RNA trigger. Here we show that Tudor-SN (tudor staphylococcal nuclease)--a protein containing five staphylococcal/micrococcal nuclease domains and a tudor domain--is a component of the RISC enzyme in Caenorhabditis elegans, Drosophila and mammals. Although Tudor-SN contains non-canonical active-site sequences, we show that purified Tudor-SN exhibits nuclease activity similar to that of other staphylococcal nucleases. Notably, both purified Tudor-SN and RISC are inhibited by a specific competitive inhibitor of micrococcal nuclease. Tudor-SN is the first RISC subunit to be identified that contains a recognizable nuclease domain, and could therefore contribute to the RNA degradation observed in RNAi.

Animals↗
PubMed2001

Probing the conformational state of a truncated staphylococcal nuclease R using time of flight mass spectrometry with limited proteolysis.

The conformational state of C-terminally truncated staphylococcal nuclease R (SNR135), with and without bound ligands, has been studied by performing limited proteolysis with a specific endoproteinase Glu-C followed by electrophoresis and mass spectrometry. Comparison of the accessibility of the cleavage sites shows that the C-terminal truncation of 14 amino-acid residues causes significant unfolding of the C-terminal part of alpha helix 1 and the center of alpha helix 2, but there is little effect on other regions of the nuclease, in particular the N-terminal subdomain, which includes the active site of the nuclease. The truncation also makes the overall conformation of the nuclease more loose and flexible. Binding of ligands makes helices 1 and 2 more resistant to protease Glu-C attack and converts the partially unfolded state to a native-like state, although the conformational stability of the SNR135 complex is still much lower than that of the full-length enzyme. The results suggest that the amino-acid residues around the active site in the truncated nuclease are arranged in a similar topology to those in the full-length nuclease. The study shows that there is a clear-cut correlation between protease susceptibility and conformational stability of the protein, and the initial proteolytic events are the most critical for evaluating the conformational features of the protein. This study demonstrates how mass spectrometry can be combined with limited proteolysis to observe conformational changes induced by ligand binding.

Binding Sites↗
PubMed1999

Reduction of cell lysate viscosity during processing of poly(3-hydroxyalkanoates) by chromosomal integration of the staphylococcal nuclease gene in Pseudomonas putida.

Poly(3-hydroxyalkanoates) (PHAs) are biodegradable thermoplastics which are accumulated by many bacterial species in the form of intracellular granules and which are thought to serve as reserves of carbon and energy. Pseudomonas putida accumulates a polyester, composed of medium-side-chain 3-hydroxyalkanoic acids, which has excellent film-forming properties. Industrial processing of PHA involves purification of the PHA granules from high-cell-density cultures. After the fermentation process, cells are lysed by homogenization and PHA granules are purified by chemical treatment and repeated washings to yield a PHA latex. Unfortunately, the liberation of chromosomal DNA during lysis causes a dramatic increase in viscosity, which is problematic in the subsequent purification steps. Reduction of the viscosity is generally achieved by the supplementation of commercially available nuclease preparations or by heat treatment; however, both procedures add substantial costs to the process. As a solution to this problem, a nuclease-encoding gene from Staphylococcus aureus was integrated into the genomes of several PHA producers. Staphylococcal nuclease is readily expressed in PHA-producing Pseudomonas strains and is directed to the periplasm, and occasionally to the culture medium, without affecting PHA production or strain stability. During downstream processing, the viscosity of the lysate from a nuclease-integrated Pseudomonas strain was reduced to a level similar to that observed for the wild-type strain after treatment with commercial nuclease. The nuclease gene was also functionally integrated into the chromosomes of other PHA producers, including Ralstonia eutropha.

Culture Media↗
PubMed1985

Secretion of staphylococcal nuclease by Bacillus subtilis.

The staphylococcal nuclease (nuc) gene from Staphylococcus aureus has been cloned and expressed in Bacillus subtilis. The nuclease protein was expressed either from its own promoter and translation start signals, or from a combination of a B. subtilis promoter, ribosome binding site, and a signal peptide sequence. Greater than 80% of the active gene product was secreted into the medium, whereas, when a signal peptide sequence was absent, as little as 4% of the nuclease activity was found in the culture medium. Intracellular (or cell-bound) nuclease, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, was shown to have the molecular weight of the predicted precursor protein with the signal peptide. Levels of nuclease reached 50 mg per liter in the culture medium, depending on the growth medium and the strain used. These findings indicate the prospective use of nuclease as a model system for studying secretion of heterologous proteins in B. subtilis.

Bacillus subtilis↗
PubMed1982

Intracellular forms of simian virus 40 nucleoprotein complexes. IV. Micrococcal nuclease digestion.

The structures of DNAs present in various intracellular forms of simian virus 40 (SV40) nucleoprotein complexes were analyzed by micrococcal nuclease digestion. The results showed that the 70S SV40 chromatin was completely sensitive to nuclease digestion, whereas CsCl gradient-purified mature virion was completely resistant. Virion assembly intermediates with different degrees of virion maturation showed intermediate resistance, and three products were found: nucleosomal DNA fragments, representing the fraction of intermediates that were sensitive to nuclease; linear SV40 genome-sized DNA, representing the more mature intermediates that contained one or limited defects in the capsid shell; and supercoiled SV40, which was derived from mature virions. These digestion products, however, remained associated with capsid shells after nuclease digestion. These results were consistent with the model in which maturation of the SV40 virion is achieved through the organization of capsid proteins that accumulate around SV40 chromatin. Mild digestion of SV40 nucleoprotein complexes with micrococcal nuclease revealed the difference in nucleosome repeat length between SV40 chromatin and virion assembly intermediates. A novel DNA fragment of about 75 nucleotides was observed early in nuclease digestion.

Animals↗
PubMed1982

Effect of thyrotropin on the sensitivity of thyroid nuclear deoxyribonucleic acid to digestion by micrococcal nuclease.

Little is known about the mechanisms of action of polypeptide hormones on chromatin structure and nuclear function. We have employed micrococcal nuclease to examine the effect of TSH on the accessibility of DNA in thyroid nuclei. Brief digestion of nuclear suspensions with 0.05-0.2 U/ml micrococcal nuclease at 26-28 C decreased their opacity at 600 nm. The decrease in opacity was linear with increasing nuclear concentration up to 0.2 mg/ml DNA. This response to nuclease was enhanced in nuclear suspensions prepared from thyroid slices that had been incubated with TSH (50 mU/ml) for 5 h (P less than 0.001). To determine whether TSH also increased the digestion of DNA, we measured the amount of DNA released into 1200 X g supernatants by nuclease treatment of nuclei prepared from control and TSH-treated slices. When TSH-treated nuclei (110 micrograms/100 microliters) were digested with 0.2 U micrococcal nuclease/ml at 37 C for 30 sec, a mean of 12.6 micrograms +/- 3.6 (SD) DNA appeared in the supernatant, as compared to 8.4 micrograms +/- 1.98 DNA from control nuclei (P less than 0.05). This increase in the insensitivity of nuclear DNA to micrococcal nuclease may reflect some conformational change in chromatin in response to TSH. Since micrococcal nuclease sensitivity may reflect transcriptional competence of DNA, we speculate that polypeptide hormones may enhance the accessibility of DNA to RNA polymerase or to endogenous stimulators of transcription.

Animals↗
PubMed1984

Effects of cycloheximide, alpha-amanitin, and alpha-difluoromethylornithine on thyrotropin-induced increases in the micrococcal nuclease sensitivity of thyroid nuclear chromatin.

Treatment of calf thyroid slices with TSH increases the nuclease sensitivity of nuclear chromatin, i.e. the amount of DNA released from nuclei by mild digestion with DNase I and micrococcal nuclease. Cycloheximide and alpha-amanitin were used to investigate the roles played by protein and RNA synthesis in mediating this effect of TSH; alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, was used to investigate the possible involvement of polyamines. Calf thyroid slices were incubated with or without TSH (50 mU/ml) for 5 h, in the presence or absence of inhibitors. Nuclei were then prepared, subjected to mild digestion with micrococcal nuclease, and centrifuged at 1200 X g. The amount of DNA in 1200 X g supernatants was increased by TSH; this was inhibited by cycloheximide (100 micrograms/ml) and alpha-amanitin (4 micrograms/ml) when these agents were present throughout incubations with TSH. In contrast, alpha-amanitin failed to inhibit the TSH effect when it was added to incubations 30 min or 2 h after the addition of TSH. These results indicate that RNA and protein synthesis play a part in mediating the effect of TSH on the micrococcal nuclease sensitivity of chromatin, and that the RNA synthesis involved takes place within the first 30 min of exposure of thyroid slices to TSH. alpha-Difluoromethylornithine (5 mM) inhibited the TSH-dependent development of micrococcal nuclease sensitivity; however, it also inhibited nuclease digestion when it was added directly to nuclei prepared from fresh thyroid tissue. This observation should serve as a warning against uncritical acceptance of the notion that all effects of alpha-difluoromethylornithine are the result of inhibition of ornithine decarboxylase.

Amanitins↗
PubMed1985

Nuclease S1-sensitive sites in multigene families: human U2 small nuclear RNA genes.

We show here that human U2 small nuclear RNA genes contain a 'strong nuclease S1 cleavage site' (SNS1 site), a sequence that is very sensitive to digestion by nuclease S1. This site is located 0.50-0.65 kb downstream of the U2 RNA coding region. It comprises a 0.15-kb region in which (dC-dT)n:(dA-dG)n co-polymeric stretches represent greater than 90% of the sequence. Nuclease S1 is able to excise unit length repeats of the human U2 RNA genes both from cloned fragments and total human genomic DNA. The precise locations of the cleavage sites are dependent on the superhelicity of the substrate DNA. In negatively supercoiled substrates, cleavages are distributed over the entire 0.15-kb region, but in linearized substrates, they occur within a more limited region, mainly at the boundary of the SNS1 site closest to the human U2 RNA coding region. Nuclease S1 cleavage of negatively supercoiled substrates occurs at pHs as high as 7.0; in contrast, cleavage of linearized substrates requires a pH less than 5.0, indicating that supercoiling contributes to the sensitivity of this site. Mung bean nuclease gives results similar to that observed with nuclease S1.

Base Sequence↗
PubMedDate not supplied

An endogenous nuclease in hamster, mouse, and human spermatozoa cleaves DNA into loop-sized fragments.

Recent work from our laboratory provided evidence for the existence of a nuclease in hamster spermatozoa. This endogenous nuclease cleaves sperm chromatin at the bases of DNA loop domains into large fragments with an average size of roughly 50 kb. Here, we demonstrate that this sperm nuclease is present in the sperm nucleus and that it is activated by the presence of both calcium and magnesium much more efficiently than with either ion alone, resulting in DNA degradation in 30 minutes. We also show that similar nucleases are present in mouse and human spermatozoa. The human nuclease can be activated by freeze-thawing spermatozoa in noncryoprotective media. The activity of the sperm nuclease in all 3 species resembles that of a group of somatic cell DNAses that also require both calcium and magnesium and that digest the chromatin into loop-sized fragments during apoptosis.

Animals↗
PubMedDate not supplied

S1 nuclease: immunoaffinity purification and evidence for the proximity of cysteine 25 to the substrate binding site.

A simple procedure, involving heat treatment, gel filtration on Sephadex-G 100 followed by chromatography on anti-S1 nuclease antibodies bound to Sepharose, was developed for purification of S1 nuclease to homogeneity with an overall yield of 72%. S1 nuclease was rapidly inactivated, at pH 6.0 and 37 degrees C, in presence of o-phthalaldehyde. Kinetic analysis of o-phthalaldehyde medicated inactivation showed that the reaction followed pseudo-first-order kinetics and the loss of enzyme activity was due to the formation of a single isoindole derivative per molecule of the enzyme. Absorbance and fluorescence spectrophotometric data also gave similar results. The isoindole derivative formation, as a result of o-phthalaldehyde treatment is known to occur through crosslinking of the thiol group of cysteine and the epsilon-amino group of lysine, situated in close proximity in the native enzyme. Since, modification of the only available cysteine residue (Cys25) did not affect the catalytic activity of the enzyme, the o-phthalaldehyde mediated inactivation of S1 nuclease is due to the modification of lysine. Substrates of S1 nuclease, namely ssDNA, RNA, 3'AMP, could protect the enzyme against o-phthalaldehyde mediated inactivation. Moreover, the modified enzyme (having very little catalytic activity) showed a significant decrease in its ability to bind 5'AMP, a competitive inhibitor of S1 nuclease, suggesting that the modification has occurred at the substrate binding site. The above results point towards the presence of cysteine 25 in close proximity to the substrate binding site.

Animals↗