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PubMed1975

Circular dichroism and absorbance properties of nitrotyrosyl chromophores in staphylococcal nuclease and in a model diketopiperazine.

An active derivative of staphylococcal nuclease, in which only tyrosine residue 115 has been nitrated with use of tetranitromethane, has been characterized using absorbance, circular dichroism, and fluorescence spectroscopy. The results show that nitrotyrosine-115 nuclease is indistinguishable from native nuclease with regard to the average secondary structure of the folded polypeptide chain, the susceptibility of the enzyme to heat denaturation, and the local tertiary structure around tryptophan residue 140. Inasmuch as optical properties of nitrotyrosine-115 nuclease from 300 to 500 nm can be unambiguously assigned to nitrotyrosine residue 115 in the active site region, this modified enzyme presents a good model system for studying the circular dichroism properties of this aromatic amino acid in a protein. The spectral properties of nitrotyrosine-115 nuclease have been compared to those of the model compounds, cyclo-(-Gly-Tyr(3NO2)-) and Tyr(3NO2). Circular dichroism spectral changes in nitrotyrosine-115 nuclease due to the binding of deoxythymidine 3',5'-diphosphate and Ca-2+ have been compared to the corresponding nitrotyrosyl-115 absorption spectral changes. This comparison shows that the circular dichroism difference spectrum exhibits an over-all change in the intensity of the observed Cotton effects, whereas the absorption difference spectrum exhibits a blue shift. This finding supports the suggestion that perturbations of aromatic amino acid chromophores in proteins due to ligand binding result in red or blue shifts in absorption difference spectra, but in over-all changes of intensity in circular dichroism difference spectra.

Amino Acid Sequence↗
PubMed1985

[Aggregation of fragmented chromatin associated with the synthesis of products of its treatment with nuclease].

Isolated cell nuclei were incubated with nucleases followed by extraction of chromatin with a low salt buffer. With an increase of nuclear chromatin degradation with DNAse I or micrococcal nuclease, solubilization of deoxyribonucleoprotein (DNP) by a low salt buffer increases, reaching a maximum upon hydrolysis with 2-4% nuclear DNA and then decreases appreciably after extensive treatment with nucleases. Soluble fragmented chromatin aggregates in the course of treatment with DNAase. I. Addition to gel chromatin preparations of exogenous products of nuclease treatment of isolated nuclei leads to its aggregation. Pretreatment of nuclear chromatin with RNAase prevents solubilization of DNP by low ionic strength solutions. Some experimental data obtained with the use of severe nuclease treatment are discussed; for a correct interpretation of these data the aggregation of fragmented chromatin by products of its nuclease degradation should be taken into consideration.

Animals↗
PubMed1995

[Nuclease isoforms of natural and recombinant strains of Serratia marcescens. Comparative characteristics of plasma desorption mass spectrometry].

The primary structure of isoforms of natural nuclease secreted by Serratia marcescens as well as of recombinant nuclease produced by Escherichia coli has been characterized by plasma desorption mass spectrometry. The isoforms were isolated from the purified nuclease on a DEAE-cellulose anion-exchange column and digested with endoproteinase Lys-C. The peptides generated were isolated by reversed phase IIPLC and their molecular masses determined by plasma desorption mass spectrometry. Among the nuclease isoforms secreted by parent cells, a new isoform, Sm3, was found. Comparison of the peptides yielded by nuclease Sm2 and two isoenzymes, Sm1 and Sm3, demonstrated their distinction only in the N-termini; three amino acids are lacking in Sm1 and one residue in Sm3. Identity was demonstrated between nucleases Sm1 and Sm2 produced by S. marcescens and the isoforms rSm1 and rSm2 secreted by the recombinant E. coli strain.

Amino Acid Sequence↗
PubMedDate not supplied

[Activity of intracellular and extracellular nuclease according to the phases of growth of pigment and pigment-free strains of Serratia marcescens].

Changes in the activity of intracellular and extracellular nuclease of pigment and pigment-free strains of Serratia marcescens were studied. The activity of intra- and extracellular nuclease of the pigment-free strain was higher than that of the pigment strain at all growth stages of the microorganism. The activity of intracellular nuclease in the lag-phase was higher than in the phase of exponential growth of both strains. Prior to cell division the enzyme activity declined in both strains. At the beginning of the stationary phase the activity of intracellular nuclease was relatively stable in both strains. By the end of the stationary phase the activity of intracellular nuclease of the pigment-free strain increased 4--6 fold and that of the pigment strain remained unchanged. Simultaneously the activity of extracellular nuclease of the pigment-free strain increased and that of the pigment strain grew but slightly.

Bacterial Proteins↗
PubMed1993

Stress and strain in staphylococcal nuclease.

Protein molecules generally adopt a tertiary structure in which all backbone and side chain conformations are arranged in local energy minima; however, in several well-refined protein structures examples of locally strained geometries, such as cis peptide bonds, have been observed. Staphylococcal nuclease A contains a single cis peptide bond between residues Lys 116 and Pro 117 within a type VIa beta-turn. Alternative native folded forms of nuclease A have been detected by NMR spectroscopy and attributed to a mixture of cis and trans isomers at the Lys 116-Pro 117 peptide bond. Analyses of nuclease variants K116G and K116A by NMR spectroscopy and X-ray crystallography are reported herein. The structure of K116A is indistinguishable from that of nuclease A, including a cis 116-117 peptide bond (92% populated in solution). The overall fold of K116G is also indistinguishable from nuclease A except in the region of the substitution (residues 112-117), which contains a predominantly trans Gly 116-Pro 117 peptide bond (80% populated in solution). Both Lys and Ala would be prohibited from adopting the backbone conformation of Gly 116 due to steric clashes between the beta-carbon and the surrounding residues. One explanation for these results is that the position of the ends of the residue 112-117 loop only allow trans conformations where the local backbone interactions associated with the phi and psi torsion angles are strained. When the 116-117 peptide bond is cis, less strained backbone conformations are available. Thus the relaxation of the backbone strain intrinsic to the trans conformation compensates for the energetically unfavorable cis X-Pro peptide bond. With the removal of the side chain from residue 116 (K116G), the backbone strain of the trans conformation is reduced to the point that the conformation associated with the cis peptide bond is no longer favorable.

Enzyme Stability↗
PubMed1997

The kinetic basis for the stabilization of staphylococcal nuclease by xylose.

The effect of xylose on the rates of folding and unfolding of staphylococcal nuclease (nuclease) have been investigated using fluorescence-detected pressure-jump relaxation kinetics in order to establish the kinetic basis for the observed stabilization of nuclease by this sugar (Frye KJ, Perman CS, Royer CA, 1996, Biochemistry 35:10234-10239). The activation volumes for both folding and unfolding and the equilibrium volume change for folding were all positive. Their values were within experimental error of those reported previously (Vidugiris GJA, Markley JL, Royer CA, 1995, Biochemistry 34:4909-4912) and were independent of xylose concentration. The major effect of xylose concentration was to increase significantly the rate of folding. The large positive activation volume for folding was interpreted previously as indicating that the rate-limiting step in nuclease folding involves dehydration of a significant amount of surface area. A large effect of xylose on the rate constant for folding provides strong support for this interpretation, because xylose, an osmolyte, stabilizes the folded state of proteins through surface tension effects. These studies further characterize the transition state in nuclease folding as lying closer to the folded, rather than the unfolded state along the folding coordinate in terms of the degree of burial of surface area. The image of the transition state that emerges is consistent with a dry molten globule.

Enzyme Stability↗
PubMed1993

Mutational tests of the NMR-docked structure of the staphylococcal nuclease-metal-3',5'-pdTp complex.

In the X-ray structure of the staphylococcal nuclease-Ca(2+)-3',5'-pdTp complex, the conformation of the inhibitor 3',5'-pdTp is distorted by Lys-70* and Lys-71* from an adjacent molecule of staphylococcal nuclease (Loll, P.J., Lattman, E.E. Proteins 5:183-201, 1989). In order to correct this crystal packing problem, the solution conformation of enzyme-bound 3',5'-pdTp in the staphylococcal nuclease-metal-pdTp complex determined by NMR methods was docked into the X-ray structure of the enzyme [Weber, D.J., Serpersu, E.H., Gittis, A.G., Lattman, E.E., Mildvan, A.S. (preceding paper)]. In the NMR-docked structure, the 5'-phosphate of 3',5'-pdTp overlaps with that in the X-ray structure. However, the 3'-phosphate accepts a hydrogen bond from Lys-49 (2.89 A) rather than from Lys-84 (8.63 A), and N3 of thymine donates a hydrogen bond to the OH of Tyr-115 (3.16 A) which does not occur in the X-ray structure (5.28 A). These interactions have been tested by binding studies of 3',5'-pdTp, Ca2+, and Mn2+ to the K49A, K84A, and Y115A mutants of staphylococcal nuclease using water proton relaxation rate and EPR methods. Each mutant was fully active and structurally intact, as found by CD and two-dimensional NMR spectroscopy, but bound Ca2+ 9.1- to 9.9-fold more weakly than the wild-type enzyme. While the K84A mutation did not significantly weaken 3',5'-pdTp binding to the enzyme (1.5 +/- 0.7 fold), the K49A mutation weakened 3',5'-pdTp binding to the enzyme by the factor of 4.4 +/- 1.8-fold. Similarly, the Y115A mutation weakened 3',5'-pdTp binding to the enzyme 3.6 +/- 1.6-fold. Comparable weakening effects of these mutations were found on the binding of Ca(2+)-3',5'-pdTp. These results are more readily explained by the NMR-docked structure of staphylococcal nuclease-metal-3',5'-pdTp than by the X-ray structure.

Binding Sites↗
PubMed1987

Nuclease sensitivity of alpha-fetoprotein, metallothionein-1, and immunoglobulin gene sequences in mouse during development.

The production of alpha-fetoprotein (AFP) and metallothionein-1 (MT-1) in mouse tissues follows a well-defined developmental pattern. The genes for these proteins are highly transcribed in embryo liver but transcribed at a very low rate in adult liver and in brain at all stages of development. A dot hybridization procedure was defined for quantitative screening for AFP, MT-1, immunoglobulin, and satellite DNA sequences to determine the relative degree of micrococcal nuclease sensitivity of these DNA sequences in fetal, newborn, and adult liver and brain, and the visceral yolk sac of the embryo. It was found that, for the DNA sequences assayed, three distinct chromatin conformations exist. DNA that does not code for protein (satellite DNA) was highly resistant to nuclease cleavage. DNA that codes for protein, but is not available for transcription (unrearranged immunoglobulin (C mu) genes in brain, liver, and yolk sac) was fourfold more sensitive to cleavage than were satellite DNA sequences. A further sevenfold increase in nuclease sensitivity was detected in genes actively being transcribed (MT-1 and AFP genes in embryo liver). Quiescent MT-1 and AFP genes were intermediate in nuclease-sensitivity between active genes and unrearranged C mu genes. These data indicate that MT-1 and AFP genes are permanently established in a nuclease-sensitive chromatin conformation early in liver development, and that conformation is maintained regardless of the degree of transcription of the genes. A second, reversible change in chromatin structure occurs in step with changes in the degree of developmentally regulated expression of AFP and MT-1 genes.

Animals↗
PubMed1986

Protein-DNA interactions and nuclease-sensitive regions determine nucleosome positions on yeast plasmid chromatin.

To study mechanisms of nucleosome positioning, small circular plasmids were constructed, assembled into chromatin in vivo in Saccharomyces cerevisiae, and their chromatin structures were analysed with respect to positions of nucleosomes and nuclease-sensitive regions. Plasmids used include insertions of the URA3 gene into the TRP1 gene of the TRP1ARS1 circular plasmid in the same (TRURAP) or opposite (TRARUP) orientation. The URA3 gene has six precisely positioned, stable nucleosomes flanked by nuclease-sensitive regions at the 5' and 3' ends of the gene. Three of these nucleosome positions do not depend on the flanking nuclease-sensitive regions, since they are formed at similar positions in a derivative plasmid (TUmidL) that contains the middle of the URA3 sequence but not the 5' and 3' ends. These positions are probably due to protein-DNA interactions. In both TRURAP and TRARUP, the positions of the nucleosomes on the TRP1 gene were, however, shifted compared with the positions on the parental TRP1ARS1 circle and TUmidL. These changes are interpreted to be due to changes in the positions of flanking nuclease-sensitive regions that might act as boundaries to position nucleosomes. Thus, two independent mechanisms for nucleosome positioning have been demonstrated in vivo. The ARS1 region contains the 3' end of the TRP1 gene and the putative origin of replication. Since in TRURAP and TRARUP the TRP1 gene is interrupted, but the ARS1 region remains nuclease sensitive, this non-nucleosomal conformation of the ARS1 region probably reflects a chromatin structure important for replication.

Base Sequence↗
PubMed1982

The small chromatin fragments released by micrococcal nuclease from hepatoma tissue cultured cell nuclei are strongly enriched in coding DNA sequences and are related to an actively transcribed single-stranded DNA fraction.

It was shown with the use of specific probes that mild micrococcal nuclease digestion released from chromatin actively-transcribed genes as small nucleosome oligomers. In the present work we demonstrate that most if not all of the active genes are accessible to the nuclease. It was found that the short released fragments are greatly enriched in transcribed DNA sequences, the most enriched being the dimers of nucleosomes since 35% of their DNA could be hybridized to cytoplasmic RNA. The results of cDNA-DNA hybridizations indicate that the monomers and dimers of nucleosomes contain most of the DNA sequences which encode poly(A+) RNAs, however larger released fragments include some transcribed sequences, while the nuclease resistant chromatin is considerably impoverished in coding sites. These evidences are the finding that about 25% of the DNA from the dimers of nucleosomes are exclusively located in this class of fragments, tend to prove that the active chromatin regions are attacked in a non-random way by micrococcal nuclease. We have previously isolated, without using exogenous nuclease, an actively transcribed genomic fraction amounting to 1.5-2% of the total nuclear DNA, formed of single-stranded DNA. In the present study we show that all or nearly all the single-stranded DNA sequences could be reassociated with the DNA fragments present in the released monomers and dimers of nucleosomes. Our observations confirmed our previous finding that the greatest part of single-stranded DNA selectively originates from the coding strand of genomic DNA.

Animals↗
PubMed1992

High-level expression of staphylococcal nuclease R gene in Escherichia coli.

Staphylococcal nuclease R, an analogue of nuclease A, was overproduced under the transcriptional control of the bacteriophage lambda PRPL promoters regulated by temperature sensitive repressors. The expression level reached 200-300 mg l-1 and showed little host dependence in different strains. The investigations of the recombinant nuclease R have revealed that the amino terminal formyl methionine residue of the nuclease is precisely processed, the protein consists of 155 amino acid residues. The experiment shows that the pBV221-DH5 alpha is a quite suitable vector-host system for high-level expression and precise processing of heterologous genes in Escherichia coli. The comparative studies between the codons used in the staphylococcal nuclease R gene and the optimal codon usage in E. coli indicate that high level expression of heterologous genes in E. coli may not always require a high degree of codon usage bias.

Amino Acid Sequence↗
PubMed2003

A modified Escherichia coli protein production strain expressing staphylococcal nuclease, capable of auto-hydrolysing host nucleic acid.

The large-scale production of recombinant biotherapeutics, particularly recombinant proteins, provides significant process and regulatory challenges to the biotechnology industry in order to meet the regulatory agencies stringent requirements in a cost-effective manner. Host cell derived nucleic acid causes problems from both a process and a regulatory perspective, as high molecular weight chromosomal DNA is responsible both for the viscosity of cell lysates, and it is a source of heterologous DNA sequences whose inclusion in the final product must be prevented. We have constructed a modified Escherichia coli JM107 expression host (JMN), containing a staphylococcal nuclease expression cassette, integrated into the host chromosome at the dif locus. The nuclease is expressed as a fusion to the ompA signal peptide, and is translocated to the periplasm of the cell, protecting the cytoplasmic nucleic acid from any toxic activity. The nuclease is released during cell lysis, where it subsequently acts to hydrolyse host nucleic acid present in the lysate. Results with this strain show that sufficient levels of nuclease activity are produced to completely auto-hydrolyse the host's chromosomal DNA to a size non-visible on 1% agarose gel, generating a markedly lower lysate viscosity. This provides a suitable methodology to remove heterologous DNA sequences early in the product stream and decrease lysate viscosity, improving the efficiency of downstream processing and product yield, whilst avoiding the addition of exogenous nuclease and its prohibitive costs at large-scale.

Base Sequence↗
PubMed1980

Subnuclear fractionation by mild micrococcal-nuclease treatment of nuclei of different transcriptional activities causes a partition of expressed and non-expressed genes.

Extremely mild treatment with micrococcal nuclease of isolated nuclei yields subnuclear fractions in which the majority of RNA polymerase II transcriptional complexes formed in vivo are segregated [Tata & Baker (1978) J. Mol. Biol. 118, 249-272]. We now describe different approaches followed to established whether or not the nuclei are thus resolved into transcribed and non-transcribed DNA. First, we have compared the sensitivity to deoxyribonuclease I, which is known to digest preferably expressed genes as present in nuclei or chromatin, of three micrococcal-nuclease-derived fractions from nuclei of different transcriptional activities. In transcriptionally active nuclei (rat liver, hen liver and oviduct, and Xenopus liver), the DNA in a polynucleosomal fraction comprising 6-15% of DNA and the majority of template-engaged RNA polymerase II (fraction P2) was 10-50 times as sensitive to deoxyribonuclease I as the DNA in the other two fractions (fractions P1 and S, comprising 78-88% of total nuclear DNA as large polynucleosomal aggregates and 2-6% of DNA mostly as mononucleosomes, respectively). In transcriptionally inactive nuclei obtained from hen erythrocytes, micrococcal nuclease did not separate DNA into fractions exhibiting such differential sensitivities. Second, we have monitored the partition of an expressed gene. Hybridization of complementary DNA to Xenopus albumin mRNA revealed a 5-10-fold enrichment of the albumin (but not the globin) gene in the P2 fraction of nuclei from Xenopus liver in which this gene is fully expressed. Third, a large part of the nascent rapidly labelled RNA synthesized in vivo in rat liver nuclei was recovered in the micrococcal-nuclease-derived fraction that is more susceptible to digestion with deoxyribonuclease I. It is concluded that mild micrococcal-nuclease treatment of nuclei causes their separation into transcribed and non-transcribed DNA as determined by a number of very different criteria.

Animals↗
PubMed1982

Nucleosome phasing and micrococcal nuclease cleavage of African green monkey component alpha DNA.

The micrococcal nuclease cleavage of intact nuclear chromatin from African green monkey cells and of the completely deproteinized sequences was studied by using high-resolution analytical and DNA sequencing gels and secondary restriction enzyme analysis. When deproteinized component alpha DNA was used as substrate, not all phosphodiester bonds in the 172-base-pair repeat units were cleaved with equal frequency by the nuclease. A distinct preference for the cleavage of A-T rather than G-C bonds was observed; however, A + T-richness in itself did not confer susceptibility to cleavage by micrococcal nuclease. The results suggested that, in deproteinized DNA, nuclease cleavage at particular dinucleotides may be influenced more by the effect of adjacent sequences than by the composition of the dinucleotide. In contrast to complex cleavage patterns of the deproteinized component alpha DNA which arose because of multiple cleavage sites in the repeat unit, micrococcal nuclease cleaved component alpha nuclear chromatin at one site per nucleosome repeat, near position 126 in the nucleotide sequence. This simple chromatin cleavage pattern is consistent with the discrete nucleosomal structure of component alpha in chromatin and a direct phase relationship between the component alpha DNA sequence repeats and the nucleosome protein structural repeats.

Animals↗
PubMed1982

Micrococcal nuclease cleavage of nucleotide linked to glutamine synthetase yields phosphotyrosine at the ligation site.

The activity of micrococcal nuclease was studied on a novel substrate, denatured adenylylated glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2], which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP. The products of the digestion were adenosine and O-phosphotyrosylglutamine synthetase. The Km of the macromolecular substrate with the nuclease was 1/40 that of the synthetic substrate, nitrophenyl-pdT, which is commonly used for assay of the enzyme. Native adenylylated glutamine synthetase was not deadenosylated by micrococcal nuclease under the conditions that permit rapid deadenosylation of denatured glutamine synthetase. Failure to attack native glutamine synthetase is probably not due to steric factors because the native enzyme is deadenylylated by snake venom phosphodiesterase under identical conditions. The inability of micrococcal nuclease to deadenosylate native glutamine synthetase may be due to the formation of an inactive complex because the native protein inhibited the nuclease activity on the denatured protein.

Adenosine Monophosphate↗
PubMed1985

Specific regions of the intervening sequences of beta-globin RNA are resistant to nuclease in 50S heterogeneous nuclear RNA-protein complexes.

The specific assembly of heterogeneous nuclear RNA-protein complexes (hnRNPs) containing precursor beta-globin RNA was investigated by using the 50S hnRNP released from chicken reticulocyte nuclei by endogenous nuclease. The nuclease-resistant regions were mapped on adult beta-globin intervening sequences (IVS) at the resolution of nucleotides with an RNA mapping method [Patton, J. R. and Chae, C.-B. (1983) J. Biol. Chem. 258, 3991-3995]. We found that there is one 28-nucleotide-long nuclease-resistant region in the first IVS and there are four nuclease-resistant regions in the second IVS. Of particular interest is the presence in 50S hnRNP of a nuclease-resistant region (24-28 nucleotides long) in both IVS immediately upstream from the putative lariat branch site in an RNA splicing intermediate. Our results demonstrate that hnRNPs containing precursor beta-globin RNA are, like those containing mature beta-globin RNA, assembled in a site-specific manner.

Animals↗
PubMed1983

Sequence specific cleavage of African green monkey alpha-satellite DNA by micrococcal nuclease.

The sequence specificity of micrococcal nuclease complicates its use in experiments addressed to the still controversial issue of nucleosome phasing. In the case of alpha-satellite DNA containing chromatin from African green monkey (AGM) cells cleavage by micrococcal nuclease in the nucleus was reported to occur predominantly at only one location around position 126 of the satellite repeat unit (Musich et al. (1982) Proc. Natl. Acad. Sci. USA 79, 118-122). DNA control experiments conducted in the same study indicated the presence of many preferential cleavage sites for micrococcal nuclease on the 172 bp long alpha-satellite repeat unit. This difference was taken as evidence for a direct and simple phase relationship between the alpha-satellite DNA sequence and the position of the nucleosomes on the DNA. We have quantitatively analyzed the digestion products of the protein-free satellite monomer with micrococcal nuclease and found that 50% of all cuts occur at positions 123 and 132, 5% at position 79, and to a level of 1-3% at about 20 other positions. We also digested high molecular weight alpha-satellite DNA from AGM nuclei with micrococcal nuclease. Again cleavage occurred mostly at positions 123 and 132 of the satellite repeat unit. Thus digestion of free DNA yields results very similar to those reported by Musich et al. for the digestion of chromatin. Therefore no conclusions on a possible phase relationship can be drawn from the chromatin digestion experiments.

Animals↗
PubMed1980

Nuclease sensitivity of active chromatin.

The active regions of chicken erythrocyte nuclei were labeled using the standard DNase I directed nick translation reaction. These nuclei were then used to study the characteristics and, in particular, the nuclease sensitivity of active genes. Although DNase I specifically attacks active genes, micrococcal nuclease solubilizes these regions to about the same degree as the total DNA. On the other hand micrococcal nuclease does selectively cut the internucleosomal regions of active genes resulting in the appearance of mononucleosomal fraction which is enriched in active gene DNA. A small percentage of the active chromatin is also released from the nucleus by low speed centrifugation following micrococcal nuclease treatment. The factors which make active genes sensitive to DNase I were shown to reside on individual nucleosomes from these regions. This was established by showing that isolated active mononucleosomes were preferentially sensitive to DNase I digestion. Although the high mobility group proteins are essential for the maintenance of DNase I sensitivity in active regions, these proteins are not necessary for the formation of the conformation which makes these genes preferentially accessible to micrococcal nuclease. The techniques employed in this paper enable one to study the chromatin structure of the entire population of actively expressed genes. Previous studies have elucidated the structure of a few special highly prevalent genes such as ovalbumin and hemoglobin. The results of this paper show that this special conformation is a general feature of all active genes irregardless of the extent of expression.

Animals↗