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PubMed1980

Enzyme-catalyzed formation of semisynthetic staphylococcal nuclease using a new synthetic fragment, [48-glycine]synthetic-(6-49).

While trypsin can catalyze resynthesis of the peptide bond between fragments in the noncovalent complex of nuclease-T-(6-48) and nuclease-T-(49-149), this reaction leads to excision of Lys 49 and formation of inactive [des Lys 49]-nuclease-(6-149). To provide a method for making active active covalent semisynthetic nuclease, we chemically synthesized the fragment of residues 6 to 49 in which lysine 48 was replaced by glycine. This peptide was made using the recently described solid phase support, 4-(oxymethyl)phenylacetamidomethyl-polystyrene. The resultant crude polypeptide exhibited 30-50% of native nuclease-T enzymatic activity when added to native nuclease-T-(50-149). When the non-covalent complex formed by native nuclease-T-(50-149) and a 10-fold molar excess of [Gly 48]synthetic-(6-49) was equilibrated with trypsin in 90% glycerol, an increase in enzymatic activity from 8 to 32% (versus nuclease) was observed. Simultaneously, approximately 20% conversion of nuclease-T-(50-149) to nuclease-molecular weight material was observed by gel electrophoretic analysis. These data indicate that a covalent semisynthetic species is formed with activity about equal to that of native nuclease. The results confirm the importance of loop integrity on catalytic site organization. The Gly-48-containing fragment system defined above can allow preparation of semisynthetic nuclease sequence analogs.

Micrococcal Nuclease↗
PubMed1987

Secretion and processing of staphylococcal nuclease by Bacillus subtilis.

We have studied the secretion and processing of Staphylococcus aureus nuclease in Bacillus subtilis. We show that the initial species of nuclease found in the cell supernatants during short-term radioactive labeling (pulse-chase) had a molecular weight of approximately 18,800 and comigrated in a sodium dodecyl sulfate-polyacrylamide gel with staphylococcal nuclease B. This nuclease B form was processed to the mature nuclease A extracellularly by a phenylmethylsulfonyl fluoride-sensitive protease. The nuclease B-processing site is a consensus signal peptidase site, and the processing of nuclease B was coupled to secretion as judged by pulse-chase experiments. The nuclease A was shown by microsequencing of the N terminus to be 2 amino acid residues shorter than the nuclease A described for S. aureus Foggi. The nuclease B form was still the first species found in the culture supernatant after removal of the N-terminal 26 amino acids of the native 60-amino-acid signal peptide. However, removal of the N-terminal 72 amino acids abolishes secretion of any nuclease form and leads to the intracellular accumulation of nuclease.

Bacillus subtilis↗
PubMed1999

Protein structure and gene cloning of Syncephalastrum racemosum nuclease.

The complete amino acid sequence of the fungus Syncephalastrum racemosum (Sr-) nuclease has been delineated on the basis of protein sequencing of the intact protein and its protease-digested peptides. The resulting 250-residue sequence shows a carbohydrate side chain attached at Asn134 and two half-cystine residues (Cys242 and Cys247) cross-linked to form a small disulphide loop. On the basis of the sequence of Sr-nuclease, a computer search in the sequence database yielded 60% and 48% positional identities with the sequences of Cunninghamella echinulata nuclease C1 and yeast mitochondria nuclease respectively, and very little similarity to those of several known mammalian DNases I. Sequence alignment of the three similar nucleases reveals that the single small disulphide loop is unchanged but the carbohydrate attachment in Sr-nuclease is absent from the other two nucleases. Alignment also shows a highly conserved region harbouring Sr-nuclease His85, which is assigned as one of the essential residues in the active site. The cDNA encoding Sr-nuclease was amplified by using reverse transcriptase-mediated PCR with degenerate primers based on its amino acid sequence. Subsequently, specific primers were synthesized for use in the 3' and 5' rapid amplification of cDNA ends (RACE). Direct sequencing of the RACE products led to the deduction of a 1.1 kb cDNA sequence for Sr-nuclease. The cDNA contains an open reading frame of 320 amino acid residues including a 70-residue putative signal peptide and the 250-residue mature protein. Finally, the recombinant Sr-nuclease was expressed in Escherichia coli strain BL21(DE3) in which the recombinant protein, after solubilization with detergent and renaturation, showed both DNase and RNase activities. The assignment of His85 to the active site was further supported by evidence that the mutant protein Sr-nuclease (H85A), in which His85 was replaced by Ala, was not able to degrade DNA or RNA.

Amino Acid Sequence↗
PubMed1992

cDNA cloning, sequencing, expression and possible domain structure of human APEX nuclease homologous to Escherichia coli exonuclease III.

cDNA encoding the human homologue of mouse APEX nuclease was isolated from a human bone-marrow cDNA library by screening with cDNA for mouse APEX nuclease. The mouse enzyme has been shown to possess four enzymatic activities, i.e., apurinic/apyrimidinic endonuclease, 3'-5' exonuclease, DNA 3'-phosphatase and DNA 3' repair diesterase activities. The cDNA for human APEX nuclease was 1420 nucleotides long, consisting of a 5' terminal untranslated region of 205 nucleotide long, a coding region of 954 nucleotide long encoding 318 amino acid residues, a 3' terminal untranslated region of 261 nucleotide long, and a poly(A) tail. Determination of the N-terminal amino acid sequence of APEX nuclease purified from HeLa cells showed that the mature enzyme lacks the N-terminal methionine. The amino acid sequence of human APEX nuclease has 94% sequence identity with that of mouse APEX nuclease, and shows significant homologies to those of Escherichia coli exonuclease III and Streptococcus pneumoniae ExoA protein. The coding sequence of human APEX nuclease was cloned into the pUC18 SmaI site in the control frame of the lacZ promoter. The construct was introduced into BW2001 (xth-11, nfo-2) strain and BW9109 (delta xth) strain cells of E. coli. The transformed cells expressed a 36.4 kDa polypeptide (the 317 amino acid sequence of APEX nuclease headed by the N-terminal decapeptide derived from the part of pUC18 sequence), and were less sensitive to methylmethanesulfonate and tert-butyl-hydroperoxide than the parent cells. The N-terminal regions of the constructed protein and APEX nuclease were cleaved frequently during the extraction and purification processes of protein to produce the 31, 33 and 35 kDa C-terminal fragments showing priming activities for DNA polymerase on acid-depurinated DNA and bleomycin-damaged DNA. Formation of such enzymatically active fragments of APEX nuclease may be a cause of heterogeneity of purified preparations of mammalian AP endonucleases. Based on analyses of the deduced amino acid sequence and the active fragments of APEX nuclease, it is suggested that the enzyme is organized into two domains, a 6 kDa N-terminal domain having nuclear location signals and 29 kDa C-terminal, catalytic domain.

Amino Acid Sequence↗
PubMed1997

Nuclease activity of T7 RNA polymerase and the heterogeneity of transcription elongation complexes.

We have discovered that T7 RNA polymerase, purified to apparent homogeneity from overexpressing Escherichia coli cells, possesses a DNase and an RNase activity. Mutations in the active center of T7 RNA polymerase abolished or greatly decreased the nuclease activity. This nuclease activity is specific for single-stranded DNA and RNA oligonucleotides and does not manifest on double-stranded DNAs. Under the conditions of promoter-driven transcription on double-stranded DNA, no nuclease activity was observed. The nuclease attacks DNA oligonucleotides in mono- or dinucleotide steps. The nuclease is a 3' to 5' exonuclease leaving a 3'-OH end, and it degrades DNA oligonucleotides to a minimum size of 3 to 5 nucleotides. It is completely dependent on Mg2+. The T7 RNA polymerase-nuclease is inhibited by T7 lysozyme and heparin, although not completely. In the presence of rNTPs, the nuclease activity is suppressed but an unusual 3'-end-initiated polymerase activity is unmasked. RNA from isolated pre-elongation and elongation complexes arrested by a psoralen roadblock or naturally paused at the 3'-end of an oligonucleotide template exhibited evidence of nuclease activity. The nuclease activity of T7 RNA polymerase is unrelated to pyrophosphorolysis. We propose that the nuclease of T7 RNA polymerase acts only in arrested or paused elongation complexes, and that in combination with the unusual 3'-end polymerizing activity, causes heterogeneity in elongation complexes. Additionally, during normal transcription elongation, the kinetic balance between nuclease and polymerase is shifted in favor of polymerase.

Bacteriophage T7↗
PubMed2000

SURVEY AND SUMMARY: holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories.

Holliday junction resolvases (HJRs) are key enzymes of DNA recombination. A detailed computer analysis of the structural and evolutionary relationships of HJRs and related nucleases suggests that the HJR function has evolved independently from at least four distinct structural folds, namely RNase H, endonuclease, endonuclease VII-colicin E and RusA. The endonuclease fold, whose structural prototypes are the phage lambda exonuclease, the very short patch repair nuclease (Vsr) and type II restriction enzymes, is shown to encompass by far a greater diversity of nucleases than previously suspected. This fold unifies archaeal HJRs, repair nucleases such as RecB and Vsr, restriction enzymes and a variety of predicted nucleases whose specific activities remain to be determined. Within the RNase H fold a new family of predicted HJRs, which is nearly ubiquitous in bacteria, was discovered, in addition to the previously characterized RuvC family. The proteins of this family, typified by Escherichia coli YqgF, are likely to function as an alternative to RuvC in most bacteria, but could be the principal HJRs in low-GC Gram-positive bacteria and AQUIFEX: Endonuclease VII of phage T4 is shown to serve as a structural template for many nucleases, including MCR:A and other type II restriction enzymes. Together with colicin E7, endonuclease VII defines a distinct metal-dependent nuclease fold. As a result of this analysis, the principal HJRs are now known or confidently predicted for all bacteria and archaea whose genomes have been completely sequenced, with many species encoding multiple potential HJRs. Horizontal gene transfer, lineage-specific gene loss and gene family expansion, and non-orthologous gene displacement seem to have been major forces in the evolution of HJRs and related nucleases. A remarkable case of displacement is seen in the Lyme disease spirochete Borrelia burgdorferi, which does not possess any of the typical HJRs, but instead encodes, in its chromosome and each of the linear plasmids, members of the lambda exonuclease family predicted to function as HJRs. The diversity of HJRs and related nucleases in bacteria and archaea contrasts with their near absence in eukaryotes. The few detected eukaryotic representatives of the endonuclease fold and the RNase H fold have probably been acquired from bacteria via horizontal gene transfer. The identity of the principal HJR(s) involved in recombination in eukaryotes remains uncertain; this function could be performed by topoisomerase IB or by a novel, so far undetected, class of enzymes. Likely HJRs and related nucleases were identified in the genomes of numerous bacterial and eukaryotic DNA viruses. Gene flow between viral and cellular genomes has probably played a major role in the evolution of this class of enzymes. This analysis resulted in the prediction of numerous previously unnoticed nucleases, some of which are likely to be new restriction enzymes.

Amino Acid Sequence↗
PubMed1999

The NucE and NucD lysis proteins are not essential for secretion of the Serratia marcescens extracellular nuclease.

The nuclease of Serratia marcescens is an extracellular protein encoded by the nucA gene. Pre-nuclease carries a typical 21-amino-acid N-terminal signal sequence that interacts with the Sec machinery to allow the translocation of nuclease to the periplasm. In Escherichia coli the nuclease remains in the periplasm; however, S. marcescens has the capacity to secrete nuclease extracellularly. The nucC operon carrying the nucEDC genes of S. marcescens has been identified previously. NucC is a transcriptional activator necessary for expression of nuclease as well as the extracellular bacteriocin 28b. NucE resembles and can act as a bacteriophage holin, whereas NucD has homology to bacteriophage lysozyme-like proteins. When present on a multicopy plasmid, the nucC operon, and specifically the nucED genes, appeared to allow extracellular secretion of nuclease from E. coli. Here experiments are reported which demonstrate that, when the nucC operon was placed in the E. coli chromosome in single copy, nuclease secretion was lost and nuclease remained periplasmic. The converse experiment, deletion of the nucE and nucD genes from the chromosome of S. marcescens, likewise had no effect on nuclease secretion by S. marcescens. It is concluded therefore that NucD and NucE are not necessary for nuclease secretion.

Bacterial Proteins↗
PubMed1993

Properties of some nuclear nucleases of rat thymocytes and their changes in radiation-induced apoptosis.

Three nuclease activities have been found and characterized in rat thymocyte nuclear extracts. A Mn(2+)-dependent nuclease is loosely bound to nuclear components and can be extracted with 0.35 M NaCl. The enzyme is activated by Mn2+ but not by Mg2+, Ca2+, or both. Its molecular mass is 36-40 kDa when measured by gel filtration and 37 kDa by SDS/PAGE. An acidic nuclease is independent of divalent ions, produces DNA strand breaks with 5'-OH ends, its molecular mass is about 37 kDa. Two fractions of Ca2+/Mn(2+)-dependent nuclease, differing in binding to CM-Sepharose but identical in other respects, are active in the presence of Mn2+ but can be additionally activated by Ca2+. They are inactive in the presence of Mg2+ or Ca2+ but cleave DNA in Ca2+/Mg(2+)-containing medium. The molecular mass of the enzyme is 22 kDa as determined by both gel filtration and electrophoresis. The dependence of nuclease activities on pH, ions, and sulfhydryl reagents is described. Cycloheximide injection to both control and irradiated animals strongly inhibits the activities of Ca2+/Mn(2+)-dependent nuclease from thymocyte nuclei separated by chromatography on CM-Sepharose and does not change the activities of Mn(2+)-dependent and acidic nucleases. Nuclease activity in thymocyte nuclei from irradiated rats is increased in Ca2+/Mg(2+)-containing and Ca2+/Mn(2+)-containing media whereas there is no change in the activity of acidic nuclease. Ca2+/Mn(2+)-dependent nuclease is extracted from thymocyte nuclei of irradiated rats with 0.35 M NaCl but from control nuclei only with 0.5 M NaCl. Possible reasons of labilization of Ca2+/Mn(2+)-dependent-nuclease binding to the nuclear structures in dying thymocytes are discussed.

Animals↗
PubMed1990

Purification and characterization of a nuclease (3'-nucleotidase) from a Penicillium sp.

A nuclease (3'-nucleotidase) similar to P1 nuclease from Penicillium citrinum was purified from a commercial digestive from a Penicillium sp. The activity of the nuclease (PA) was separated to three fractions by diethylaminoethyl-Toyopearl 650M column chromatography, in total yield of 10%. The apparent molecular weight of these three nucleases, PA1, PA2 and PA3 was 35000, 33000, and 32000, respectively. All of them were homogeneous so far as checked by sodium dodecyl sulfate slab gel electrophoresis. The three nucleases differed in carbohydrate content, but their amino acid composition was practically the same, and very similar to that of P1 nuclease. The molecular weight of nuclease PA3, the major component of nuclease PA, was approximately 27000 after digestion by endoglycosidase F. The N-terminal and C-terminal amino acid sequences of nuclease PA3 were determined by Edman degradation and carboxypeptidase(s) digestion, respectively. The nuclease PA3 was inactivated in the presence of 10 mM ethylenediamine tetraacetic acid (EDTA) and 65% of its native enzyme activity restored by the addition of 20 mM ZnCl2. The pH-dependent photooxidative inactivation of nuclease PA3 was accelerated by removal of Zn ion by EDTA or trishydroxymethyl aminomethane, indicating the possible chelation of Zn2+ with some histidine residues.

Amino Acid Sequence↗
PubMed1996

Coupling between trans/cis proline isomerization and protein stability in staphylococcal nuclease.

The nucleases A produced by two strains of Staphylococcus aureus, which have different stabilities, differ only in the identity of the single amino acid at residue 124. The nuclease from the Foggi strain of S. aureus (by convention nuclease WT), which contains His124, is 1.9 kcal.mol-1 less stable (at pH 5.5 and 20 degrees C) than the nuclease from the V8 strain (by convention nuclease H124L), which contains Leu124. In addition, the population of the trans conformer at the Lys116-Pro117 peptide bond, as observed by NMR spectroscopy, is different for the two variants: about 15% for nuclease WT and 9% for nuclease H124L. In order to improve our understanding of the origin of these differences, we compared the properties of WT and H124L with those of the H124A and H124I variants. We discovered a correlation between effects of different residues at this position on protein stability and on stabilization of the cis configuration of the Lys116-Pro117 peptide bond. In terms of free energy, approximately 17% of the increase in protein stability manifests itself as stabilization of the cis configuration at Lys116-Pro117. This result implies that the differences in stability arise mainly from structural differences between the cis configurational isomers at Pro117 of the different variants at residue 124. We solved the X-ray structure of the cis form of the most stable variant, H124L, and compared it with the published high-resolution X-ray structure of the cis form of the most stable variant, WT (Hynes TR, Fox RO, 1991, Proteins Struct Funct Genet 10:92-105). The two structures are identical within experimental error, except for the side chain at residue 124, which is exposed in the models of both variants. Thus, the increased stability and changes in the trans/cis equilibrium of the Lys116-Pro117 peptide bond observed in H124L relative to WT are due to subtle structural changes that are not observed by current structure determination technique. Residue 124 is located in a helix. However, the stability changes are too large and follow the wrong order of stability to be explained simply by differences in helical propensity. A second site of conformational heterogeneity in native nuclease is found at the His46-Pro47 peptide bond, which is approximately 80% trans in both WT and H124L. Because proline to glycine substitutions at either residue 47 or 117 remove the structural heterogeneity at that position and increase protein stability, we determined the X-ray structures of H124L + P117G and H124L + P47G + P117G and the kinetic parameters of H124L, H124L + P47G, H124L + P117G, and H124L + P47G + P117G. The individual P117G and P47G mutations cause decreases in nuclease activity, with kcat affected more than Km, and their effects are additive. The P117G mutation in nuclease H124L leads to the same local conformational rearrangement described for the P117G mutant of WT (Hynes TR, Hodel A, Fox RO, 1994, Biochemistry 33:5021-5030). In both P117G mutants, the loop formed by residues 112-117 is located closer to the adjacent loop formed by residues 77-85, and residues 115-118 adopt a type I' beta-turn conformation with the Lys116-Gly117 peptide bond in the trans configuration, as compared with the parent protein in which these residues have a typeVIa beta-turn conformation with the Lys116-Pro117 peptide bond in the cis configuration. Addition of the P47G mutation appears not to cause any additional structural changes. However, the electron density for part of the loop containing this peptide bond was not strong enough to be interpreted.

Chemical Phenomena↗
PubMed1985

The ompA signal peptide directed secretion of Staphylococcal nuclease A by Escherichia coli.

The hybrid pre-enzyme formed by fusion of the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, to Staphylococcal nuclease A, a protein secreted by Staphylococcus aureus, is translocated across the cytoplasmic membrane of E. coli with concomitant cleavage of the signal peptide. A DNA fragment containing the coding sequence for the ompA signal peptide was initially ligated to a DNA fragment containing the coding sequence for nuclease A, with a linker sequence of 33 nucleotides separating the coding sequences. When this fused gene was induced, an enzymatically active nuclease was secreted into the periplasmic space; sequential Edman degradation of this protein revealed that the ompA signal peptide was removed at its normal cleavage site resulting in a modified version of the nuclease having 11 extra amino acid residues attached to the amino terminus of nuclease A. The 33 nucleotides between the coding sequences for the ompA signal peptide and the structural gene for nuclease A were subsequently deleted by synthetic oligonucleotide-directed site-specific mutagenesis. The nuclease produced by this hybrid gene was secreted into the periplasmic space and by sequential Edman degradation was identical to nuclease A. Thus, the ompA signal peptide is able to direct the secretion of fused staphylococcal nuclease A, and signal peptide processing occurs at the normal cleavage site. When the hybrid gene is expressed under the control of the lpp promoter, nuclease A is produced to the extent of 10% of the total cellular protein.

Amino Acid Sequence↗
PubMed1982

The involvement of nuclear nucleases in rat thymocyte DNA degradation after gamma-irradiation.

Possible mechanisms of internucleosomal DNA fragmentation in thymocytes of irradiated rats were studied. It was shown that thymocyte nuclei contain at least two nucleases that cleave DNA between nucleosomes--a Ca2+/Mg2+-dependent nuclease and an acidic one which does not depend on bivalent ions. 2 and 3 h after irradiation at a dose of 10 Gy the initial rate of DNA cleavage by Ca2+/Mg2+-dependent nuclease in isolated nuclei increased three and seven times, respectively, but the kinetics of DNA digestion by acidic nuclease did not change. The experiments with cycloheximide indicated that Ca2+/Mg2+-dependent endonuclease turns over at a high rate. The activity of the cytoplasmic acidic and Mg2+-dependent nucleases was shown to increase (by 40 and 50%, respectively) 3 h after irradiation. The effect is caused by the de novo synthesis of the nucleases. At the same time the activity of nuclear nucleases did not essentially change. The chromatin isolated from rat thymocytes 3 h after irradiation did not differ in its sensitivity to some exogenic nucleases (DNAase I, micrococcal nuclease and nuclease from Serratia marcescens) from the control. Thus, Ca2+/Mg2+-dependent endonuclease seems to be responsible for the postirradiation internucleosomal DNA fragmentation in dying thymocytes.

Animals↗
PubMed2002

Development of nonradioactive microtiter plate assays for nuclease activity.

We have developed two microtiter plate assays for the detection of DNA cleavage by nucleases, using 3'-biotinylated oligonucleotide substrates. In the covalently linked oligonucleotide nuclease assay (CLONA), the biotinylated substrates are phosphorylated at the 5' end to facilitate their covalent immobilization on CovaLink NH plates. The cleavage of the covalently immobilized substrate by nucleases results in biotin release. The uncleaved substrate molecules are detected with an enzyme-avidin conjugate. The affinity-linked oligonucleotide nuclease assay (ALONA) makes use of substrates with a digoxigenin on the 5' end of the 3'-biotinylated DNA strand. The substrate binds specifically to the wells of streptavidin-coated microtiter plates, in which the nuclease reaction takes place. Uncleaved substrate retains the digoxigenin label, which is detected with an enzyme-labeled anti-digoxigenin antibody. We assessed the efficiency of these two assays by measuring S1 nuclease and DNase I activities, and the inhibitory effect of EDTA and aurintricarboxylic acid on the reaction. Both methods are more convenient than the standard radioactive nuclease assay and are suitable for high-throughput screening of potential nuclease inhibitors, nucleases, and catalytic antibodies. The ALONA assay was found to be more sensitive than the CLONA assay, with a performance similar to that of the standard nuclease assay.

Aurintricarboxylic Acid↗
PubMed2002

The crystal structure of the nuclease domain of colicin E7 suggests a mechanism for binding to double-stranded DNA by the H-N-H endonucleases.

The bacterial toxin ColE7 contains an H-N-H endonuclease domain (nuclease ColE7) that digests cellular DNA or RNA non-specifically in target cells, leading to cell death. In the host cell, protein Im7 forms a complex with ColE7 to inhibit its nuclease activity. Here, we present the crystal structure of the unbound nuclease ColE7 at a resolution of 2.1A. Structural comparison between the unbound and bound nuclease ColE7 in complex with Im7, suggests that Im7 is not an allosteric inhibitor that induces backbone conformational changes in nuclease ColE7, but rather one that inhibits by blocking the substrate-binding site. There were two nuclease ColE7 molecules in the P1 unit cell in crystals and they appeared as a dimer related to each other by a non-crystallographic dyad symmetry. Gel-filtration and cross-linking experiments confirmed that nuclease ColE7 indeed formed dimers in solution and that the dimeric conformation was more favored in the presence of double-stranded DNA. Structural comparison of nuclease ColE7 with the His-Cys box homing endonuclease I-PpoI further demonstrated that H-N-H motifs in dimeric nuclease ColE7 were oriented in a manner very similar to that of the betabetaalpha-fold of the active sites found in dimeric I-PpoI. A mechanism for the binding of double-stranded DNA by dimeric H-N-H nuclease ColE7 is suggested.

Amino Acid Motifs↗
PubMed1989

Single-strand-preferring nuclease activity in wheat leaves is increased in senescence and is negatively photoregulated.

Single-strand-preferring nucleases (EC 3.1.30.1) selectively cleave internucleotide bonds in single-stranded regions of predominantly duplex DNA and DNA.RNA hybrids and extensively degrade denatured DNA and RNA. The functions of single-strand-preferring nuclease in plants are unknown. We have monitored this nuclease activity in flag leaves of wheat (Triticum aestivum L. cv. Chinese Spring) undergoing natural senescence and in primary leaves of wheat seedlings undergoing dark-induced senescence. In falg leaves, nuclease activity remained at basal levels during the first 2 weeks after anthesis, while chlorophyll content increased to a maximum. Nuclease activity then rose in concert with a decline in chlorophyll, reaching a 16-fold elevation at 5 weeks post-anthesis, when 53% of the chlorophyll had been lost. When 8-day-old wheat seedlings were induced to senesce by placing them in darkness, nuclease activity rose without apparent lag, reaching a 13-fold elevation in 7 days, when 61% of the chlorophyll had been lost. The increase in nuclease activity was reversible upon reexposure of seedlings to light, a decline beginning without apparent lag. Reversibility was complete for plants that had been held in darkness for 5 days, with activity returning to the control level in 2 days. These senescence-related changes in nuclease activity, measured in conventional assays, were consistent with concomitant analysis by activity staining of sodium dodecyl sulfate/polyacrylamide gels. We conclude that an increase in single-strand-preferring nuclease activity is closely associated with wheat leaf senescence and that nuclease activity is subject to negative photoregulation.

Journal Article↗
PubMed1999

Purification, characterization, and role of nucleases and serine proteases in Streptomyces differentiation. Analogies with the biochemical processes described in late steps of eukaryotic apoptosis.

Two exocellular nucleases with molecular masses of 18 and 34 kDa, which are nutritionally regulated and reach their maximum activity during aerial mycelium formation and sporulation, have been detected in Streptomyces antibioticus. Their function appears to be DNA degradation in the substrate mycelium, and in agreement with this proposed role the two nucleases cooperate efficiently with a periplasmic nuclease previously described in Streptomyces antibioticus to completely hydrolyze DNA. The nucleases cut DNA nonspecifically, leaving 5'-phosphate mononucleotides as the predominant products. Both proteins require Mg2+, and the additional presence of Ca2+ notably stimulates their activities. The two nucleases are inhibited by Zn2+ and aurin tricarboxylic acid. The 18-kDa nuclease from Streptomyces is reminiscent of NUC-18, a thymocyte nuclease proposed to have a key role in glucocorticoid-stimulated apoptosis. The 18-kDa nuclease was shown, by amino-terminal protein sequencing, to be a member of the cyclophilin family and also to possess peptidylprolyl cis-trans-isomerase activity. NUC-18 has also been shown to be a cyclophilin, and "native" cyclophilins are capable of DNA degradation. The S. antibioticus 18-kDa nuclease is produced by a proteolytic processing from a less active protein precursor. The protease responsible has been identified as a serine protease that is inhibited by Nalpha-p-tosyl-L-lysine chloromethyl ketone and leupeptin. Inhibition of both of the nucleases or the protease impairs aerial mycelium development in S. antibioticus. The biochemical features of cellular DNA degradation during Streptomyces development show significant analogies with the late steps of apoptosis of eukaryotic cells.

Amino Acid Sequence↗
PubMed1993

Membrane-associated nuclease activities in mycoplasmas.

Membrane-associated nucleases of various mycoplasmal species were investigated by using two nuclease assays. A lambda DNA assay was developed to measure nuclease activity associated with whole-cell suspensions, activity released from intact cells, and activity associated with detergent-disrupted cells. In most species, nuclease activities were entirely membrane associated, and disruption by a detergent had a stimulatory effect on these activities. All mycoplasmal species contained nuclease activity, but Mycoplasma capricolum was unusual because its activity was dependent upon magnesium and was inhibited by calcium. We developed a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system that produced reproducible nuclease patterns, and this system was used to determine the apparent molecular weights of the nuclease proteins. An examination of 20 mycoplasmal species failed to identify common bands in their nuclease patterns. An examination of 11 Mycoplasma pulmonis strains, however, indicated that nuclease patterns on polyacrylamide gels may provide a means for categorizing strains within a species. Our results suggest that nucleases are important constituents of mycoplasmal membranes and may be involved in the acquisition of host nucleic acids required for growth.

Animals↗
PubMed2001

In vivo evidence for two active nuclease motifs in the double-strand break repair enzyme RexAB of Lactococcus lactis.

In bacteria, double-strand DNA break (DSB) repair involves an exonuclease/helicase (exo/hel) and a short regulatory DNA sequence (Chi) that attenuates exonuclease activity and stimulates DNA repair. Despite their key role in cell survival, these DSB repair components show surprisingly little conservation. The best-studied exo/hel, RecBCD of Escherichia coli, is composed of three subunits. In contrast, RexAB of Lactococcus lactis and exo/hel enzymes of other low-guanine-plus-cytosine branch gram-positive bacteria contain two subunits. We report that RexAB functions via a novel mechanism compared to that of the RecBCD model. Two potential nuclease motifs are present in RexAB compared with a single nuclease in RecBCD. Site-specific mutagenesis of the RexA nuclease motif abolished all nuclease activity. In contrast, the RexB nuclease motif mutants displayed strongly reduced nuclease activity but maintained Chi recognition and had a Chi-stimulated hyperrecombination phenotype. The distinct phenotypes resulting from RexA or RexB nuclease inactivation lead us to suggest that each of the identified active nuclease sites in RexAB is involved in the degradation of one DNA strand. In RecBCD, the single RecB nuclease degrades both DNA strands and is presumably positioned by RecD. The presence of two nucleases would suggest that this RecD function is dispensable in RexAB.

Amino Acid Motifs↗