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At least 19 recordsLinked to original sources

PubMed1977

Nuclease B. A possible precursor of nuclease A, an extracellular nuclease of Staphylococcus aureus.

During purification of nuclease (redisignated as nuclease A in the present studies) from the culture media of Staphylococcus aureus strain Foggi, three enzymacally active second species (nucleases B1, B2, and B3) were isolated as a mixture by ion exchange chromatography. Examination of the amino acid sequence of these second species indicates that nucleases B1, B2, and B3 apparently contain the same sequence as that of nuclease A with an extra sequence Ser-Gln-Thr-Asp-Asx-Gly-Val-Asx-Arg-Ser-Gly-Ser-Glu-Asp-Pro-Thr-Val-Tyr-Ser linked through a peptide bond to the NH2 terminus of the nuclease A portion. In nuclease B1 the 2 residues indicated by Asx are aspartic acid, in nuclease B2 the first and the second Asx from the NH2 terminus are aspartic acid and asparagine, respectively, and in nuclease B3 both Asx are asparagine. These second species do not contain a significant amount of carbohydrate. The extra amino acid sequence appears to be flexible and does not interfere with the ordered structure and function of the nuclease A portion. The nuclease A portion was recovered, in part, from a mixture of these nuclease B species after digestion with staphylococcal protease in the presence of ligands, deoxythymidine 3',5'-diphosphate, and calcium ion. Thus, these nuclease B species may be closely related to, if not identical with, a precursor of nuclease A. Similar second species of nuclease have been found in strain V8.

Amino Acid Sequence↗

PubMed1985

Genetic control of immune response to staphylococcal nuclease. XII: Analysis of nuclease antigenic determinants using anti-nuclease monoclonal antibodies.

SJL mice, which are high responders to Staphylococcal nuclease (nuclease), were immunized and used to produce hybridoma cell lines secreting anti-nuclease monoclonal antibodies (mAb). Ten stable clones were derived from a single fusion. Seven of these produced antibodies of the IgG1, kappa isotype and were more precisely characterized for antigenic specificity. Only one hybridoma cell line (54-10-4) produced anti-nuclease antibodies capable of inhibiting enzymatic activity of nuclease. Binding inhibition analyses strongly suggest that the other monoclonal antibodies, which failed to inhibit nuclease activity detect two different antigenic regions, or epitopes, of the molecule: epitope cluster 1 domain is defined by hybridomas 54-2-7, 54-5-2, 54-9-8, and 54-10-8; epitope cluster 2 by 54-5-1 and 54-1-9. Because of its capacity to inhibit nuclease enzymatic activity mAb 54-10-4 was considered specific for a third epitope of the nuclease molecule called epitope 3. Binding studies of these monoclonal antibodies were extended to peptide fragments of the nuclease molecule in order to examine possible cross-reactions with such fragments, as has previously been reported for antibodies purified from polyclonal antisera. Monoclonal antibodies specific for epitope cluster 1 on the native molecule also bound to the fragments 1-126 and 49-149 but failed to bind to fragment 99-149, suggesting that the corresponding epitope(s) is determined by amino acids localized between residues 49 and 99. The epitope clusters 2 and 3 appeared to be expressed only on the native molecule. Monoclonal antibodies of different clusters exhibited very different migration patterns on isoelectric focusing while monoclonal antibodies of the same cluster were indistinguishable, which suggests that they may have originated from the same B cell precursor. Taken together these data suggest that this panel of monoclonal antibodies detects at least three distinct epitopes of the nuclease molecule, one of which could be involved in the determination of the enzymatic site.

Animals↗

PubMed1994

Crystallization and preliminary X-ray analysis of an anti-staphylococcal nuclease-staphylococcal nuclease complex and of a second anti-staphylococcal nuclease antibody.

The Fab fragments of several monoclonal antibodies that bind Staphylococcal nuclease have been screened for crystallization conditions. Two of these, N10 and N25, have been crystallized in forms suitable for X-ray structural analysis. The anti-Staphylococcal nuclease antibody complex N10 Fab-nuclease crystallizes with symmetry consistent with space group C2 and cell parameters of a = 234.7 A; b = 43.5 A; c = 74.4 A; beta = 106.4 degrees. A second anti-Staphylococcal nuclease antibody, N25, although crystallized starting with the Fab-nuclease complex, apparently crystallizes as uncomplexed N25 Fab with symmetry consistent with space group P3(1)21 (or its enantiomorph P3(2)21) and cell parameters of a = b = 80.9 A; c = 138.4 A.

Antibodies, Bacterial↗

PubMed1995

Induction of double-strand breaks by S1 nuclease, mung bean nuclease and nuclease P1 in DNA containing abasic sites and nicks.

Defined DNA substrates containing discrete abasic sites or paired abasic sites set 1, 3, 5 and 7 bases apart on opposite strands were constructed to examine the reactivity of S1, mung bean and P1 nucleases towards abasic sites. None of the enzymes acted on the substrate containing discrete abasic sites. Under conditions where little or no non-specific DNA degradation was observed, all three nucleases were able to generate double-strand breaks when the bistranded abasic sites were 1 and 3 base pairs apart. However, when the abasic sites were further apart, the enzymes again failed to cleave the DNA. These results indicate that single abasic sites do not cause sufficient denaturation of the DNA to allow incision by these single-strand specific endonucleases. The reactivity of these enzymes was also investigated on DNA substrates that were nicked by DNasel or more site-specifically by endonuclease III incision at the discrete abasic sites. The three nucleases readily induced a strand break opposite such nicks.

Base Composition↗

PubMed2002

Amino acid sequence and characterization of a nuclease (nuclease Le3) from Lentinus edodes.

The fruit body of shiitake (Lentinus edodes) produces two acid nucleases, nuclease Le1 and nuclease Le3, both of which are thought to be candidates for the enzyme that produces a flavorful substance, 5'-GMP, and the primary structure of one of the nucleases, nuclease Le1, has been analyzed by both protein chemistry and gene cloning [Biosci. Biotechnol. Biochem. 64, 948-957 (2000)]. In this study the amino acid sequence of nuclease Le3 was analyzed by protein chemistry and gene cloning. Nuclease Le3 is a glycoprotein that contains 280 amino acid residues, and the molecular mass of the protein moiety of nuclease Le3 is 31,045. The nucleotide sequence of the cDNA and genomic DNA encoding nuclease Le3 revealed the presence of an 18-residue putative signal peptide. Nuclease Le3 contains 170, 108, and 98 amino acid residues that are identical to residues of nuclease Le1, nuclease P1, and nuclease S, respectively. The amino acid residues involved in coordination with Zn2+ atoms in nuclease P1 are all conserved in nuclease Le3. Nuclease Le3 contains 9 half-cystine residues, and 7 of them are located in the same positions as in nuclease Le1.

Amino Acid Sequence↗

PubMed2000

The nuclease activity of the yeast DNA2 protein, which is related to the RecB-like nucleases, is essential in vivo.

Saccharomyces cerevisiae Dna2 protein is required for DNA replication and repair and is associated with multiple biochemical activities: DNA-dependent ATPase, DNA helicase, and DNA nuclease. To investigate which of these activities is important for the cellular functions of Dna2, we have identified separation of function mutations that selectively inactivate the helicase or nuclease. We describe the effect of six such mutations on ATPase, helicase, and nuclease after purification of the mutant proteins from yeast or baculovirus-infected insect cells. A mutation in the Walker A box in the C-terminal third of the protein affects helicase and ATPase but not nuclease; a mutation in the N-terminal domain (amino acid 504) affects ATPase, helicase, and nuclease. Two mutations in the N-terminal domain abolish nuclease but do not reduce helicase activity (amino acids 657 and 675) and identify the putative nuclease active site. Two mutations immediately adjacent to the proposed nuclease active site (amino acids 640 and 693) impair nuclease activity in the absence of ATP but completely abolish nuclease activity in the presence of ATP. These results suggest that, although the Dna2 helicase and nuclease activities can be independently affected by some mutations, the two activities appear to interact, and the nuclease activity is regulated in a complex manner by ATP. Physiological analysis shows that both ATPase and nuclease are important for the essential function of DNA2 in DNA replication and for its role in double-strand break repair. Four of the nuclease mutants are not only loss of function mutations but also exhibit a dominant negative phenotype.

Amino Acid Sequence↗

PubMed2000

Amino acid sequence of a nuclease (nuclease Le1) from Lentinus edodes.

The fruit bodies of Lentinus edodes produce two acid nucleases, nucleases Le1 and Le3, both of which are thought to be candidates for the enzymes producing a tasty substance, 5'-GMP. To obtain the basic information on the mechanism of production of 5'-GMP, and structure-function relationship of these nucleases, the primary structure of nuclease Le1 was estimated by both protein chemistry and gene cloning. Nuclease Le1 is a glycoprotein and consists of 290 amino acid residues, and about 2 and 6 residues of hexosamine and neutral sugar, respectively. The nucleotide sequence of cDNA and genomic DNA encoding nuclease Le1 indicated the presence of 20 amino acid residues of a signal peptide. Nuclease Le1 has 115 and 108 residues of identical amino acid residues with nucleases P1 and S, respectively. The amino acid residues concerning the coordination with Zn2+ in nuclease P1 are all conserved in nuclease Le1. Nuclease Le1 contains 8 half-cystine residues and 4 of them are located at the same places as those of nucleases P1 and S.

Amino Acid Sequence↗

PubMed1991

Fluorescence and conformational stability studies of Staphylococcus nuclease and its mutants, including the less stable nuclease-concanavalin A hybrids.

We report steady-state and time-resolved fluorescence studies with the single tryptophan protein, Staphylococcus aureus A, and several of its site-directed mutants. A couple of these mutants, nuclease-conA and nuclease-conA-S28G (which are hybrid proteins containing a six amino acid beta-turn substitute from concanavalin A), are found to have a much lower thermodynamic stability than the wild type. The thermal transition temperatures for nuclease-conA and S28G are 32.8 and 30.5 degrees C, which are about 20 degrees C lower than the Tm for wild-type nuclease A. These mutant proteins also are denatured by a much lower concentration of the denaturants urea and guanidine hydrochloride. We also show that an unfolding transition in the structure of the nuclease-conA hybrids can be induced by relatively low hydrostatic pressure (approximately 700 bar). The free energy for unfolding of nuclease-conA (and nuclease-conA-S28G) is found to be only 1.4 kcal/mol (and 1.2 kcal/mol) by thermal, urea, guanidine hydrochloride, and pressure unfolding. Time-resolved fluorescence intensity and anisotropy measurements with nuclease-conA-S28G show the temperature-, urea-, and pressure-perturbed states each to have a reduced average intensity decay time and to depolarize with a rotational correlation time of approximately 1.0 ns (as compared to a rotational correlation time of 11 ns for the native form of nuclease-conA-S28G at 20 degrees C).

Acrylamides↗

PubMed1997

Solution structures of staphylococcal nuclease from multidimensional, multinuclear NMR: nuclease-H124L and its ternary complex with Ca2+ and thymidine-3',5'-bisphosphate.

The solution structures of staphylococcal nuclease (nuclease) H124L and its ternary complex, (nuclease-H124L).pdTp.Ca2+, were determined by ab initio dynamic simulated annealing using 1925 NOE, 119 phi, 20 chi 1 and 112 hydrogen bond constraints for the free protein, and 2003 NOE, 118 phi, 20 chi 1 and 114 hydrogen bond constraints for the ternary complex. In both cases, the final structures display only small deviations from idealized covalent geometry. In structured regions, the overall root-mean-square deviations from mean atomic coordinates are 0.46 (+/- 0.05) A and 0.41 (+/- 0.05) A for the backbone heavy atoms of nuclease and its ternary complex, respectively. The backbone conformations of residues in the loop formed by Arg81-Gly86, which is adjacent to the active site, are more precisely defined in the ternary complex than in unligated nuclease. Also, the protein side chains that show NOEs and evidence for hydrogen bonds to pdTp (Arg35, Lys84, Tyr85, Arg87, Tyr113, and Tyr115) are better defined in the ternary complex. As has been observed previously in the X-ray structures of nuclease-WT, the binding of pdTp causes the backbone of Tyr113 to change from an extended to a left-handed alpha-helical conformation. The NMR structures reported here were compared with available X-ray structures: nuclease-H124L [Truckses et al. (1996) Protein Sci., 5, 1907-1916] and the ternary complex of wild-type staphylococcal nuclease [Loll and Lattman (1989) Proteins Struct. Funct. Genet., 5, 183-201]. Overall, the solution structures of nuclease-H124L are consistent with these crystal structures, but small differences were observed between the structures in the solution and crystal environments. These included differences in the conformations of certain side chains, a reduction in the extent of helix 1 in solution, and many fewer hydrogen bonds involving side chains in solution.

Calcium↗

PubMed1977

Genetic control of the immune response to staphylococcal nuclease. III. Time-course and correlation between the response to native nuclease and the response to its polypeptide fragments.

The progression of the Ir gene-controlled antibody response to staphylococcal nuclease in mice with repeated immunizations has been examined. H-2-linked control of the response to a single immunization with 100 mug of nuclease in complete Freund's adjuvant was confirmed. However, among strains of the high responder H-2a haplotype, the response of the A/J mice was about 10-fold higher than that of the B10.A, indicating additional non-H-2-linked control. In addition, the low responder C57BL/10 (H-2b) strain produced antibody levels as high as or higher than those of the congenic high responder B10.A (H-2a) strain when both strains were repeatedly immunized, indicating complexity even in the H-2-linked control of the response to this small monomeric protein. Polypeptide fragments of nuclease were also studied as immunogens. The antibody response to one fragment (residues 99-149) was found to follow the same pattern among five strains tested as that to whole nuclease. However, in this case the C57BL/10 was found to be a nonresponder rather than a low responder, failing to develop a response despite repeated immunizations. In contrast, the C57BL/10 showed a low but significant response to another fragment (residues 1-126) of nuclease. These results suggest that the apparent H-2-linked control of the response to whole nuclease is a reflection of the ability to recognize a determinant(s) in the region from residues 99 to 149, and that the eventual response of the C57BL/10 strain after hyperimmunization reflects the recognition of other determinants. If these observations reflect the common recognition of a determinant on native nuclease and on a random-conformation fragment, they have implications about the conformational specificity of the receptors, or the flexibility of the determinants, involved in H-2-linked Ir-gene control. In addition, evidence is presented for a possible second H-2-linked gene (or genes) controlling the response to other determinants of nuclease expressed on the polypeptide fragments.

Animals↗

PubMed1978

Genetic control of the immune response to staphylococcal nuclease. VIII. Mapping of genes for antibodies to different antigenic regions of nuclease.

Antibodies to staphylococcal nuclease have been fractionated into two populations on the basis of their ability to bind to the cyanogen bromide cleavage product of nuclease comprising the C-terminal portion of the molecule from the 99th to the 149th amino acid. The two populations of antibodies, anti-nuclease (1-99)n and anti-nuclease (99-149)N, have been prepared from a variety of strains, and analyzed using anti-idiotypic antisera raised against whole anti-nuclease antibodies from strains A/J, SJL, BALB/c, and B10.A(2R). Anti-nuclease (1-99)n, antibodies had the same pattern of reactivity with the anti-idiotypic antisera as did unfractionated antibodies, whereas a different pattern was found for anti-nuclease (99-149)n preparations. On the basis of these studies, five anti-nuclease idiotypes, designated NASE markers, have been identified and defined on the basis of their antigenic specificity and strain distribution. With these additional markers, it has been possible to provide more detailed maps of variable (V) region genes in the strains BALB/c, CB.20, and the recombinant BAB.14. A recombinational event between V region genes during the development of the BAB.14 strain is suggested by the positioning of these NASE markers.

Animals↗

PubMed1976

Genetic control of the immune response to nuclease. II. Detection of idiotypic determinants by the inhibition of antibody-mediated nuclease inactivation.

The humoral response of mice to staphylococcal nuclease has previously been shown to be controlled genetically by H-2-linked Ir gene(s). In order to examine the possible contributions of variable region immunoglobulin genes to this genetic control, we have developed a system for the detection of idiotypic determinants on anti-nuclease immunoglobulin molecules. Antisera to nuclease were raised in two high responder strains, A/J and SJL. The corresponding antibodies were purified by affinity chromotography on Sepharose-nuclease columns, and were used to immunize groups of Lewis rats. An assay system was developed to assess the inhibition of antibody-mediated inactivation of nuclease activity by the rat antisera thus produced. Despite the presence of many species-specific anti-mouse immunoglobulin antibodies in these sera, inhibition of antibody-mediated enzyme inactivation was found to be specific for anti-nuclease antibodies of the immunizing strain. The inhibition could not be removed by extensive absorption with normal serum proteins from the antibody-producing strain, and was shown to require antibodies directed toward binding sites of the anti-nuclease antibodies. This inhibition thus defines idiotypic determinants of anti-nuclease antibodies.

Absorption↗

PubMed1979

A kinetic study of the folding of nuclease B, a possible precursor of staphylococcal nuclease A.

Nuclease B, which contains an additional flexible amino acid sequence of 19 amino acid residues bound to the NH2-terminus of nuclease A, an extracellular nuclease of Staphylococcus aureus, has been investigated in order to determine the influence of the extra residues on the refolding of the nuclease A portion from the acid denaturated state by monitoring the change in tryptophan fluorescence using a stopped-flow technique. It was found that the kinetic parameters of this refolding is similar within experimental error for nuclease A and nuclease B for the entire course (up to 40 s) studied. Therefore, the extra residues do not appear to have any detectable effect on the dynamic events involved in the refolding process. Thus, the folding of the nuclease A portion of nuclease B appears to be thermodynamically and kinetically independent of the 19 residues at the amino-terminus.

Amino Acid Sequence↗

PubMed1999

On the advantage of being a dimer, a case study using the dimeric Serratia nuclease and the monomeric nuclease from Anabaena sp. strain PCC 7120.

The extracellular endonucleases from Serratia marcescens and Anabaena sp. are members of a family of nonspecific endonucleases. In contrast to the monomeric Anabaena nuclease, the Serratia nuclease is a dimer of two identical subunits. To find out whether the two active sites of the Serratia nuclease function independently of each other and what the advantage of being a dimer for this enzyme might be, we produced (i) dimers in which the two subunits were cross-linked, (ii) heterodimers consisting of a wild type and an inactive mutant subunit which were also cross-linked, and (iii) monomeric variants which are unable to dimerize. The monomeric H184R variant and the cross-linked S140C variant exhibit the same activity as the wild type enzyme, while the cross-linked heterodimer with one inactive subunit shows only half of the activity of the wild type enzyme, demonstrating functional independence of the two subunits of the Serratia nuclease. On the other hand at low enzyme and substrate concentrations dimeric forms of the Serratia nuclease are relatively more active than monomeric forms or the monomeric Anabaena nuclease in cleaving polynucleotides, not, however, oligonucleotides, which is correlated with the ability of dimeric forms of the Serratia nuclease to form large enzyme-substrate networks with high molecular weight DNA and to cleave polynucleotides in a processive manner. We conclude that in the natural habitat of Serratia marcescens where the supply of nutrients may become growth limiting the dimeric nuclease can fulfil its nutritive function more efficiently than a monomeric enzyme.

Anabaena↗

PubMed1995

[Serratia marcescens nucleases. I. Comparison of native and recombinant nucleases using electrospray mass spectrometry].

Isoforms of the natural and recombinant nucleases of Serratia marcescens were characterized by their molecular mass, which was determined by electrospray mass spectrometry. The natural nuclease was isolated from the S. marcescens B10M1 culture, whereas the recombinant nuclease was obtained from Escherichia coli MT102 cells carrying plasmid p403-SD2 with the nuclease gene nuc. The primary structure for each of the isoforms isolated from the nuclease preparations was determined by comparing its molecular mass with that of known amino acid sequence, which was determined from the nucleotide sequence of the nuc gene. Both preparations included identical nuclease variants with N-terminal amino acid residues removed. The number of isoforms in the natural nuclease was, however, significantly greater than in the recombinant nuclease. The structures of some of the isoforms were confirmed by N-terminal analysis.

Chromatography, DEAE-Cellulose↗

PubMed1978

Genetic control of the immune response to staphylococcal nuclease. VII. Role of non-H2-linked genes in the control of the anti-nuclease antibody response.

The role of non-H-2-linked genes in the control of the antibody response to staphylococcal nuclease has been investigated. 3 wk after immunization with nuclease in complete Freund's adjuvant, strain A/J (H-2 a) mice produced significantly higher titers of antibody than strain B10.A (H-2(a)) mice, whereas mice of strains A.BY (H-2(b)) and B10 (H-2(b)) produced barely detectable titers. With hyperimmunization, A/J and A.BY mice reached the same peak levels for antibody titers, both severalfold higher than those reached by B10.A and B10 mice. Analysis of the specificity of antibodies by assessment of binding to two fragments of nuclease showed similarities between strains of the same H-2 haplotype. These results suggest that although H-2-1inked genes determined initial responsiveness at 3 wk and the relative proportions of antibodies directed toward different antigenic determinants on the nuclease molecule, non-H-2-linked genes determined the overall magnitude of the hyperimmuneresponse. Measurement of the affinity of the antibodies to the nuclease fragment (1-126) showed that strains B10 and B10.A produced antibodies with 7- to 10-fold higher affinity than comparable antibodies from strains A.BY and A/J. In a backcross of (B10.A x A/J) x B10.A, the level of antibody segregated independently of the Ig-1(e) C(H) allotype and the A/J anti-nuclease idiotypes. Thus, a gene(s) linked to neither H-2 nor heavy chain structural genes appears to control the aggregate response to antigenic determinants on the nuclease molecule independent of subspecificities of these antibodies or their idiotype.

Animals↗

PubMed1984

Identification of a heat-labile cellular nuclease in Staphylococcus aureus with properties similar to the extracellular nuclease (EC 3.1.4.7).

Besides the well-known heat-stable extracellular staphylococcal nuclease (EC 3.1.4.7) and cell surface bound nuclease, one more nuclease, which is heat-labile, has been identified and purified on phosphorylated cellulose column and characterized. Analyses by Sephadex G-75 gel chromatography indicates that the heat-labile cellular nuclease has molecular weight of about 16,000 similar to those of extracellular and cell-surface bound nucleases. Like the heat-stable nucleases, the heat-labile enzyme acts on both DNA and RNA, is more active on heat-denatured DNA, requires Ca2+ ions for activity and maximum catalytic activity is observed at pH 9.8-10 and at 45 degrees C. The results suggest that the three enzymes have properties strikingly similar to one another and therefore may be related structurally.

Electrophoresis, Polyacrylamide Gel↗

PubMed1998

Mycoplasma nucleases able to induce internucleosomal DNA degradation in cultured cells possess many characteristics of eukaryotic apoptotic nucleases.

It was previously shown (Paddenberg et al (1996) Eur J Cell Biol 69, 105 - 119) that cells of established lines like NIH3T3 fibroblasts and the human pancreatic adenocarcinoma PaTu 8902 line only degrade their chromatin at internucleosomal sites after an apoptotic stimulus when infected with Mycoplasma hyorhinis. In order to distinguish mycoplasma nucleases (Mr 47 - 54 kDa) from already described eukaryotic apoptotic enzymes, the mycoplasma nucleases were partially purified from serum-free culture supernatants and further characterized. Here we demonstrate directly that the enriched mycoplasma nucleases were able to fragment the DNA of nuclease-negative substrate nuclei at internucleosomal sites. The DNA degradation was accompanied by morphological changes typical of apoptosis like chromatin condensation and margination followed by shrinkage of the nuclei. The biochemical characterization revealed that the mycoplasma nucleases had a neutral to weakly basic pH-optimum. They required both calcium and magnesium in the mM range for maximal activation and were inhibited by zinc chloride, EGTA and EDTA. In two dimensional zymograms they migrated as three spots with isoelectic points between 8.1 and 9.5. They were not inhibited by monomeric actin. Our data also demonstrate that nuclear extracts prepared from nuclei isolated from Mycoplasma hyorhinis infected cells contained the mycoplasma nuclease activities leading to their internucleosomal DNA-degradation after incubation in the presence of calcium and magnesium.

Apoptosis↗