Identification and characterization of glucocorticoid-regulated nuclease(s) in lymphoid cells undergoing apoptosis

Apoptosis is a physiological process by which selected cells are deleted from a population in response to specific regulatory signals. A hallmark of apoptosis is the internucleosomal degradation of DNA prior to cell death. We are studying glucocorticoid-induced lymphocytolysis as a model system for apoptosis within the immune system. In rat thymocytes, the internucleosomal DNA cleavage which occurs following glucocorticoid treatment is both time- and dose-dependent, and is blocked by the glucocorticoid antagonist RU 486, indicating that this effect is mediated by the glucocorticoid receptor. Similar experiments using glucocorticoid-responsive (wt) and glucocorticoid-resistant (nt⁻) S49.1 lymphoma cell lines confirm that internucleosomal DNA degradation and cell death are glucocorticoid receptor-mediated events and thus reflect the direct effects of glucocorticoids on lymphocytes. In an effort to identify the nuclease(s) responsible for the DNA degradation, we have developed two assays to detect nucleases whose activity is altered by glucocorticoid treatment. The first assay involves electrophoresing extracts of nuclear protein from control and glucocorticoid-treated lymphoid cells into SDS-polyacrylamide gels containing [³²P]DNA within the gel matrix. This assay is used to estimate the molecular mass of the nuclease, based on the observed in situ nuclease activity. The second assay uses HeLa nuclei as a substrate to detect internucleosomal cleavage activity present in nuclear extracts of control and glucocorticoid-treated lymphoid cells. Using these assays we have identified a novel Ca²⁺, Mg²⁺-dependent nuclease with an apparent molecular weight of 18 kDa in both S49 wt cells and rat thymocytes treated with glucocorticoids. Furthermore, nuclear extracts of glucocorticoid-treated, but not control, rat thymocytes and S49 wt cells were capable of cleaving HeLa chromatin at internucleosomal sites. In an effort to determine the identity of the nuclease capable of internucleosomal cleavage of DNA, nuclear extracts from dex-treated rat thymocytes were fractionated by gel filtration chromatography under non-denaturing conditions, and the fractions were analyzed using the [³²P]DNA SDS-PAGE and HeLa nuclei assays. When analyzed under native conditions, the 18 kDa nuclease described previously appears to exist as a 25 kDa protein which may be part of a high molecular weight complex. Interestingly, only the 25 kDa form of the protein was associated with internucleosomal DNA cleavage activity where as the high molecular weight form of the enzyme was devoid of this activity.     

Apoptotic DNA degradation: evidence for novel enzymes

Apoptosis is characterized by multiple morphological and biochemical changes. One biochemical change that has been primarily associated with apoptosis is the cleavage of chromatin in the internucleosomal regions. We have taken two independent approaches to investigating the enzyme(s) responsible for such cleavage. First, using SDS-PAGE gels with (32)P-labelled DNA incorporated into the matrix, we identified a nuclease activity (termed NUC18) from apoptotic thymocytes. This enzyme has been purified to homogeneity and the activity of the pure protein is dependent on Ca(2+) and Mg(2+) while inhibited by Zn(2+) and aurintricarboxylic acid. This protein is found in the nucleus of apoptotic and nonapoptotic cells but is maintained in nondying cells in a large-molecular-weight inactive complex. NUC18 has a denatured molecular weight of 18 Kd but elutes from gel filtration columns with a native molecular weight of approximately 25 Kd. Although an exhaustive search has not been performed, NUC18 has been identified in several cell lines and tissues. Our second approach is designed specifically to detect internucleosomal cleavage of DNA, an obvious requirement for an apoptotic nuclease. By examining the degradation of HeLa chromatin, we have identified a low-molecular-weight of approximately 23 Kd native molecular weight) internucleosomal cleavage enzyme active in nuclear extracts from glucocorticoid-treated thymocytes. This activity is also dependent upon Ca(2+)and Mg(2+) and is inhibited by Zn(2+) as well as aurintricarboxylic acid. It is present in a variety of cell lines and tissues and is maintained in control cells in a latent state prior to apoptosis. In addition to similarities in physical properties, the two enzymes appear to be immunologically related to one another by virtue of their ability to interact with the same antibody. Overall, using independent approaches, we have identified two nucleases with similar biochemical properties whose activity correlates with apoptosis. The current work suggests that these are novel and perhaps closely related enzymes.     

Utilization of an in vitro assay to evaluate chromatin degradation by candidate apoptotic nucleases

Apoptosis is commonly associated with the catabolism of the genome in the dying cell. The chromatin degradation occurs in essentially two forms: (1) internucleosomal DNA cleavage to generate oligonucleosomal-length fragments (180-200 bp and multiples thereof), and (2) cleavage of higher order chromatin structures to generate approximately 30-50 Kb fragments. To investigate this component of apoptosis and identify the nuclease(s) responsible, we have developed and utilized an in vitro assay that recapitulates the genomic destruction seen during apoptosis in vivo and allows the simultaneous analysis of both forms of DNA degradation from the same sample. Using this assay we evaluated the digestion patterns of several candidate apoptotic nucleases: DNase I, DNase II, and cyclophilin (NUC18) as well as the bacterial enzyme micrococcal nuclease (not thought to be involved in apoptosis). Chromatin degraded by DNase I formed a smear of DNA on conventional static-field agarose gels and approximately amp;30 - 50 Kb DNA fragments on pulsed field gels. In contrast, DNase II, at a physiologically relevant pH, had no effect on the integrity of HeLa chromatin in either analysis. Similar to DNase I, cyclophilin C produced only approximately 30-50 Kb DNA fragments but did not generate internucleosomal fragments. In contrast, micrococcal nuclease generated both oligonucleosomal and approximately 30-50 Kb DNA fragments. Nuclear extracts from glucocorticoid-treated apoptotic thymocytes generated oligonucleosomal DNA fragments and the larger approximately 30-50 Kb DNA fragments, fully recapitulating both types of apoptotic DNA degradation. Previously, differential sensitivity of nucleases to inhibition by Zn2+ was used to argue that two distinct enzymes mediate approximately 30-50 Kb DNA cleavage and internucleosomal DNA degradation. While, the nuclease activity present in thymocyte nuclear extracts was differentially sensitive to inhibition by Zn2+ during short term incubations it was not during prolonged digestions, suggesting that differences in DNA detection are likely to account for previous results. Together our studies show that none of the nucleases commonly associated with apoptosis could fully recapitulate the DNA degradation seen in vivo.     

Characterization of apoptosis-like endonuclease activity in avian thymocytes.

In the current study the internucleosomal DNA cleavage activity associated with apoptosis was investigated in avian thymocytes. Thymocyte nuclear proteins from glucocorticoid-treated chickens were incubated with chicken red blood cell (cRBC) nuclei, and DNA degradation was analyzed by agarose gel electrophoresis and fluorescence-activated flow cytometry. The thymocyte nuclear extract contained an endonuclease activity that degraded cRBC chromatin at internucleosomal sites as detected by agarose gel electrophoresis. Flow cytometry analysis of cRBC nuclei that were treated with thymocyte nuclear proteins demonstrated a loss of cellular DNA as a function of the amount of added nuclease activity. Furthermore, it was demonstrated that the thymocyte nuclear extract contained a nuclease activity that was capable of degrading radiolabelled naked 32P-DNA into acid soluble DNA fragments. All three assay methods demonstrate that the thymocyte nuclease activity can be inhibited by EDTA, zinc ions and the nuclease inhibitor aurintricarboxylic acid. Based on the analysis of cofactor requirement of this nuclease activity and its susceptibility to inhibitors, the endonuclease activity present in avian apoptotic thymocytes appears to be identical to the mammalian counterpart.     

Sequence and expression of NUC1, the gene encoding the mitochondrial nuclease in Saccharomyces cerevisiae.

The DNA sequence and studies on the expression of the NUC1 gene from Saccharomyces cerevisiae are presented. The NUC1 locus is located in the distal portion of the left arm of Chromosome X and encodes the major nuclease found in mitochondria. The inferred amino acid sequence of NUC1 predicts that the nuclease is basic, rich in prolines, of average hydrophobicity, and has a molecular weight for the primary translation product of 37,209 daltons. NUC1 is very poorly expressed, consistent with the codon usage bias determined from the DNA sequence and our previous determination of the number of enzyme molecules per cell. Mapping of the 5' terminus of the NUC1 mRNA reveals that the mRNA has a long 400 base untranslated leader in which are found three open reading frames, each initiated by an AUG. The possibility that these upstream open reading frames contribute to the poor expression of the NUC1 gene is discussed.     

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.     

Similar actions of glucocorticoids and calcium on the regulation of apoptosis in S49 cells

Glucocorticoid-induced lymphocyte cell death is a programmed process which is thought to involve the calcium-dependent degradation of DNA into multiples of 180 basepairs, characteristic of internucleosomal degradation. We have used the glucocorticoid-sensitive mouse lymphoma cell line S49.1 [wild-type (wt)] and the glucocorticoid-resistant cell line S49.22r (nt-) to evaluate the role of both glucocorticoid receptors and calcium in the regulation of internucleosomal DNA degradation and expression of calcium-dependent deoxyribonuclease activity. DNA was isolated from untreated (control) and dexamethasone (dex)-treated viable cells and analyzed for internucleosomal DNA degradation by agarose gel electrophoresis, followed by ethidium bromide staining. Glucocorticoid treatment resulted in substantial internucleosomal DNA degradation in wt cells, but not in nt- cells. This effect was inhibited by coincubation of cells with dex and the glucocorticoid receptor antagonist RU486. In contrast to the glucocorticoid response, administration of either of two calcium ionophores, ionomycin or A23187, produced internucleosomal degradation of DNA in both wt and nt- cells, although the latter were less sensitive to ionophore treatment. Interestingly, A23187 treatment also resulted in a loss of cell viability in HeLa S3 cells, a cell line that does not exhibit glucocorticoid-induced apoptosis. No internucleosomal DNA degradation was detected in HeLa S3 cells killed by A23187. To determine whether similar nucleases are associated with this internucleosomal DNA degradation resulting from both glucocorticoid and calcium ionophore treatment, 0.3 M NaCl nuclear protein extracts were prepared from control and treated cells and analyzed for protein composition or nuclease activity. To assay for nuclease activity, nuclear extracts were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels impregnated with [32P]DNA. Nuclease activity was detected by removal of sodium dodecyl sulfate from the gel, activation with calcium, and subsequent visualization of the loss of [32P]DNA by autoradiography. Dex treatment of wt cells resulted in the appearance of several proteins within the mol wt range of 12-18 kDa, only one of which (16-18 kDa) exhibited calcium-dependent nuclease activity. The appearance of these proteins in nuclear extracts was inhibited by coincubation of glucocorticoid-treated cells with RU 486. Glucocorticoid treatment did not result in the appearance of nuclease activity in nuclear extracts from nt- cells. Interestingly, A23187 or ionomycin treatment resulted in an increase in activity of the 16- to 18-kDa nuclease in both wt and nt- cells. These findings indicate that both glucocorticoid receptors and calcium may share common features in the regulation of apoptosis in lymphoid cells.     

Development of an apoptosis endonuclease assay.

A biochemical hallmark of cells undergoing programmed cell death, or apopotosis, is the endonucleolytic cleavage of genomic DNA at internucleosomal sites. To study further the nuclease involved in this process, an assay system was developed to measure internucleosomal DNA degradation. Micrococcal nuclease (MNase), a bacterial enzyme that cleaves chromatin at internucleosomal intervals, was used to validate the assay procedure. Thymocyte nuclear proteins obtained from glucocorticoid-treated chickens, a source of internucleosomal DNA-degrading activity, were incubated with chicken red blood cell nuclei, and genomic DNA was subsequently extracted and analyzed by agarose gel electrophoresis. Generation of internucleosomal DNA degradation products by the thymocyte protein extract required ATP and was both time and protein concentration dependent. This nuclease activity could be inhibited by EDTA, EGTA, alkylating agents, or heat denaturation. Addition of purified proteinases, RNases, or other types of nucleases to the assay failed to generate discrete internucleosomal lengths of DNA, thus confirming the nuclease specificity of this assay. On the basis of these data, we believe that this assay system will be instrumental in isolating and characterizing the nuclease(s) associated with apoptosis.     

Identification of a glucocorticoid-induced nuclease in thymocytes. A potential "lysis gene" product

Glucocorticoids initiate a cytolytic process in lymphoid cells that is characteristic of programmed cell death. In vivo treatment of adrenalectomized rats with glucocorticoids results in the rapid degradation of the thymocyte genome at internucleosomal sites. This DNA degradation occurs prior to cell death, and considerable evidence indicates that this nucleolytic event is central to the initiation of lymphocytolysis. To further characterize this process, we have searched for the gene products in thymocytes which may be responsible for steroid-induced DNA degradation. Adrenalectomized rats were treated in vivo with dexamethasone or a vehicle control; nuclear thymocyte proteins were extracted with 0.6 M NaCl and analyzed for protein content or nuclease activity on sodium dodecyl sulfatepolyacrylamide gels containing calf thymus DNA. Glucocorticoid treatment resulted in the induction of two major protein families, a 30-32-kDa protein doublet and a series of 3-4 proteins of 12-19 kDa, both of which express prominent DNase activity. Induction of the lower molecular weight nucleases increased with time after steroid treatment and paralleled the time course of glucocorticoid-mediated DNA degradation. Nuclease induction was blocked by the glucocorticoid antagonist RU 486, indicating a steroid receptor-mediated process. When nuclei from glucocorticoid-resistant cells were incubated with nuclear extracts from glucocorticoid-treated rats, the DNA was cleaved at internucleosomal sites, whereas extracts from vehicle-treated animals were virtually inactive. Based on these findings we propose that glucocorticoids, acting via a receptor-mediated pathway, induce a nucleolytic "lysis gene" product(s) responsible for lymphocytolysis.     

Transformation in Bacillus subtilis: involvement of the 17-kilodalton DNA-entry nuclease and the competence-specific 18-kilodalton protein.

A protein complex, consisting of a 17-kilodalton (kDa) nuclease and an 18-kDa protein, is believed to be involved in the binding and entry of donor DNA during transformation of Bacillus subtilis (H. Smith, K. Wiersman, S. Bron, and G. Venema, J. Bacteriol. 156:101-108, 1983). In this paper, the nucleotide sequences of the genes encoding both the nuclease and the 18-kDa protein are presented. The genes are encoded by a 904-base-pair PstI-HindIII fragment. The open reading frames encoding both proteins are partly overlapping. A B. subtilis mutant was constructed by insertion of a Cmr marker into the gene encoding the nuclease. This mutant lacked the competence-specific nuclease activity and the 18-kDa protein but retained 5% residual transformation. The total DNA association of the mutant was higher than that of the wild-type cells, and DNA entry was reduced to 30% of the wild-type level. These results suggest that an alternative pathway exists for the internalization of transforming DNA. A mutant, exclusively deficient for the 18-kDa protein, previously suggested to be involved in the binding of transforming DNA, was constructed by insertion of a kanamycin resistance gene into the coding sequence of the gene. Since the mutant showed wild-type DNA-binding activity, the 18-kDa protein is probably not involved in the binding of donor DNA to competent cells. The transforming activity of the mutant was reduced to 25% of the wild-type level, indicating that the 18-kDa protein has a function in the transformation process. In vitro experiments showed that the 18-kDa protein is capable of inhibiting the activity of the competence-specific nuclease. Its possible role in transformation is discussed.     

An endogenous calcium-dependent, caspase-independent intranuclear degradation pathway in thymocyte nuclei: antagonism by physiological concentrations of K(+) ions

Calcium ions have been implicated in apoptosis for many years, however the precise role of this ion in the cell death process remains incomplete. We have extensively examined the role of Ca(2+) on nuclear degradation in vitro using highly purified nuclei isolated from non-apoptotic rat thymocytes. We show that these nuclei are devoid of CAD (caspase-activated DNase), and DNA degradation occurs independent of caspase activity. Serine proteases rather than caspase-3 appear necessary for this Ca(2+) -dependent DNA degradation in nuclei. We analyzed nuclei treated with various concentrations of Ca(2+) in the presence of both a physiological (140 mM) and apoptotic (40 mM) concentration of KCl. Our results show that a 5-fold increase in Ca(2+) is required to induce DNA degradation at the physiological KCl concentration compared to the lower, apoptotic concentration of the cation. Ca(2+) -induced internucleosomal DNA degradation was also accompanied by the release of histones, however the apoptotic-specific phosphorylation of histone H2B does not occur in these isolated nuclei. Interestingly, physiological concentrations of K(+) inhibit both Ca(2+) -dependent DNA degradation and histone release suggesting that a reduction of intracellular K(+) is necessary for this apoptosis-associated nuclear degradation in cells. Together, these data define an inherent caspase-independent catabolic pathway in thymocyte nuclei that is sensitive to physiological concentrations of intracellular cations.     

LDFF, the large molecular weight DNA fragmentation factor, is responsible for the large molecular weight DNA degradation during apoptosis in Xenopus egg extracts

DNA degradation is a biochemical hallmark in apoptosis. It has been demonstrated in many cell types that there are two stages of DNA fragmentation during the apoptotic execution. In the early stage, chromatin DNA is cut into large molecular weight DNA fragments, although the responsible nuclease(s) has not been recognized. In the late stage, the chromatin DNA is cleaved further into short oligonucleosomal fragments by a well-characterized nuclease in apoptosis,the caspase-activated DNase (CAD/DFF40). In this study, we demonstrate that large molecular weight DNA fragmentation also occurs in Xenopus egg extracts in apoptosis. We show that the large molecular weight DNA fragmentation factor (LDFF) is not the Xenopus CAD homolog XCAD. LDFF is activated by caspase-3. The large molecular weight DNA fragmentation activity of LDFF is Mg^2 -dependent and Ca^2 -independent, can occur in both acidic and neutral pH conditions and can tolerate 45℃ treatment. These results indicate that LDFF in Xenopus egg extracts might be a new DNase (or DNases) responsible for the large DNA fragmentation.     

Glucocorticoid action on the immune system

Glucocorticoids have profound effects on immune function that are mediated, in part, by steroid-induced cell death. Our studies have been aimed at identifying the mechanism of this lymphocytolytic process using the rat thymocyte as a model system. Administration of glucocorticoids in vivo resulted in internucleosomal cleavage of the lymphocyte genome that was detectable within 2 h of treatment and increased with time after hormone administration. Six h after steroid treatment greater than 50% of the genome was degraded, yet cell viability remained greater than 90% indicating that this event preceded cell death. Furthermore, this process appeared to be mediated by the glucocorticoid receptor since the antagonist RU 486 blocked glucocorticoid-mediated DNA degradation. To further characterize this lymphocytolysis we have analyzed glucocorticoid-treated thymocytes for nucleases. Two families of nuclear proteins have been identified, a 30-32 kDa doublet and a series of 3-4 proteins that are 12-19 kDa, both of which are induced by glucocorticoid treatment (137 +/- 6% and 342 +/- 24%, respectively) and have prominent nuclease activity. These nucleases can also be induced in vitro indicating that glucocorticoids act directly on thymocytes to mediate this response. Moreover, this nuclease induction, like glucocorticoid-mediated DNA degradation, could be blocked by RU 486. Based on these findings we propose a working model of glucocorticoid-mediated lymphocytolysis in which these steroids, acting via a receptor mediated process, induce the expression of a lysis gene product (nuclease) which degrades the genome and results in cell death.     

Glucocorticoid-induced lymphocytolysis is not mediated by an induced endonuclease

The mechanism of glucocorticoid-induced internucleosomal DNA cleavage and cytolysis of lymphatic cells is not known. Recent data (Compton, M.M., and Cidlowski, J.A. (1987) J. Biol. Chem. 262, 8288-8292) suggested that in vivo treatment of rat thymocytes with glucocorticoids induces a nucleolytic "lysis gene" product(s) responsible for lymphocytolysis. In this paper, the possibility that lymphocytolysis may result from glucocorticoid-induced nuclease(s) was examined. Using the rat thymocytes as a model system, we have shown by electrophoretic, enzymatic, and amino acid sequence analysis that the putative glucocorticoid-induced nucleases identified recently by Compton and Cidlowski are in fact H1, H1(0), and core histones, and their gross appearance is not the result of new histone protein synthesis, but a result of the release of histone-containing nucleosomes during chromatin breakdown. Evidence presented here shows that the putative induced nuclease activity is an artifact of the assay system employed. Because our data do not support induction of a glucocorticoid-induced nuclease(s), we examined the possibility that DNA cleavage might be induced by activation of a constitutive endogenous endonuclease. We have shown that it is possible to produce characteristic internucleosomal DNA cleavage of rat thymocytes, merely by incubating intact nuclei from untreated adrenalectomized rat thymocytes with Ca2+ and Mg2+ for a short period of time. However, in glucocorticoid-sensitive human CEM-C7 lymphocytes activation of internucleosomal DNA cleavage was independent of calcium uptake. We conclude that glucocorticoid induction of internucleosomal DNA fragmentation does not necessarily require expression of a new nuclease(s), but is the result of the activation of a constitutive endogenous endonuclease(s). Also, our data suggest that the mechanism which controls activation of internucleosomal DNA cleavage in rat thymocytes differs from that which operates in CEM-C7 lymphocytes.     

S(T)PXX motifs promote the interaction between the extended N-terminal tails of histone H2B with "linker" DNA.

The assembly of hybrid core particles onto long chicken DNA with histone H2B in the chicken histone octamer replaced with either wheat histone H2B(2) or sea urchin sperm histone H2B(1) or H2B(2) is described. All these histone H2B variants have N-terminal extensions of between 18 and 20 amino acids, although only those from sea urchin sperm have S(T)PXX motifs present. Whereas chicken histone octamers protected 167 base pairs (bp) (representing two full turns) of DNA against micrococcal nuclease digestion (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813), all the hybrid histone octamers protected an additional 17-bp DNA against nuclease digestion. This protection was more marked in the case of hybrid octamers containing sea urchin sperm histone H2B variants and similar to that described previously (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813) for hybrid histone octamers containing wheat histone H2A variants all of which also have S(T)PXX motifs present. Continued micrococcal nuclease digestion reduced the length of DNA associated with the core particle via 172-, 162-, and 152-bp intermediates until the 146-bp core particle was obtained. These DNA lengths were approximately 5 bp or half a helical turn longer than those reported previously for stripped chicken chromatin and for core particles containing histone octamers reconstituted using "normal" length histone H2B variants. This protection pattern was also found in stripped sea urchin sperm chromatin, demonstrating that the assembly/digestion methodology reflects the in vivo situation. The interaction between the N-terminal histone H2B extension and DNA of the "linker" region was confirmed by demonstrating that stripped sea urchin sperm chromatin precipitated between 120 and 500 mM NaCl in a manner analogous to unstripped chromatin whereas stripped chicken chromatin did not. Tryptic digestion to remove all the histone tails abolished this precipitation as well as the protection of DNA outside of the 167-bp core particle against nuclease digestion.     

Early-stage apoptosis is associated with DNA-damage-independent ATM phosphorylation and chromatin decondensation in NIH3T3 fibroblasts

Chromatin condensation and degradation of DNA into internucleosomal DNA fragments are key hallmarks of apoptosis. The phosphorylation of protein kinase ataxia telangiectasia mutated (ATM) and histone H2A.X was recently shown to occur concurrently with apoptotic DNA fragmentation. We have used immunofluorescence microscopy, Western blot analysis and alkali comet assays to show that phosphorylation of ATM in NIH3T3 fibroblasts occurs prior to apoptotic DNA fragmentation, nuclease degradation and phosphorylation of histone H2A.X in cells treated with low levels of either staurosporine (STS) or tumor necrosis factor-alpha mixed with cycloheximide (TNF-alpha/CHX). In extension to previous findings, ATM phosphorylation was associated with chromatin decondensation, i.e., by loss of dense foci of constitutive heterochromatin. These results suggest that chromatin is decondensed and that ATM is activated independently of DNA damage signaling pathways during the very early stages of apoptosis.     

Glucocorticoid-induced thymocyte apoptosis: protease-dependent activation of cell shrinkage and DNA degradation

Glucocorticoids are well known to stimulate apoptosis in immature thymocytes. Apoptosis in this and other cells is characterized by cell shrinkage, DNA fragmentation and activation of a class of proteases named caspases. We have utilized the flow cytometer to evaluate the coordinate regulation of cell shrinkage and DNA fragmentation in glucocorticoid-treated rat thymocytes and explore the role of caspases upstream of both changes. The results indicate that the activation of apoptosis by glucocorticoids in a cell population is an asynchronous event with only a percentage of the cells displaying apoptotic characteristics at any given time. Both cell shrinkage and chromatin degradation are tightly coupled with similar proportions of the cells displaying each characteristic. The coordinate appearance of these characteristics may suggest a similar mechanism of regulation. Incubation of thymocytes with the general caspase inhibitor Z-VAD-FMK completely blocked both cell shrinkage and DNA fragmentation in spontaneous and glucocorticoid-induced thymocyte apoptosis, implicating an early upstream role for proteases in the activation of thymocyte apoptosis.     

Purification and characterization of a Bacillus subtilis 168 nuclease, YokF, involved in chromosomal DNA degradation and cell death caused by thermal shock treatments

We purified and characterized a 39-kDa Bacillus subtilis 168 nuclease that has been suggested in this laboratory to be involved in chromosomal DNA degradation induced by lethal heat and cold shock treatments in vivo. The nuclease activity was inhibited in vitro by aurintricalboxylic acid but not by Zn(2+). By the mutant analysis, we identified the 39-kDa nuclease as a product of yokF gene. The yokF gene contained a putative lipoprotein signal peptide motif. After in vivo exposure to lethal heat and cold stresses, the chromosomal DNA fragmentation was reduced in the yokF mutant, which demonstrated about a 2-10-fold higher survival rate than the wild type. The yokF mutant was found to be more sensitive to mitomycin C than the wild type. The transformation efficiency of the yokF mutant was about 10 times higher than that of the wild type. It is suggested that when B. subtilis cells are exposed to a stressful thermal shock resulting in membrane perturbation, YokF nuclease consequently dislocates into the cytoplasm and then attacks DNA.     

The assembly of an H2A2,H2B2,H3,H4 hexamer onto DNA under conditions of physiological ionic strength

A novel nucleohistone particle is generated in high yield when a complex of DNA with the four core histones formed under conditions that are close to physiological (0.15 M NaCl, pH 8) is treated with micrococcal nuclease. The particle was found to contain 102 base pairs of DNA in association with six molecules of histones in the ratio 2H2A:2H2B:1H3:1H4 after relatively brief nuclease treatment. Prolonged nuclease digestion resulted in a reduction in the DNA length to a sharply defined 92-base pair fragment that was resistant to further degradation. Apparently normal nucleosome core particles containing two molecules each of the four core histones in association with 145 base pairs of DNA and a particle containing one molecule each of histones H2A and H2B in association with approximately 40 base pairs of DNA were also generated during nuclease treatment of the histone-DNA complexes formed under physiological ionic strength conditions. Kinetic studies have shown that the hexamer particle is not a subnucleosomal fragment produced by the degradation of nucleosome core particles. Furthermore, the hexamer particle was not found among the products of nuclease digestion when histones and DNA were previously assembled in 0.6 M NaCl. The high sedimentation coefficient of the hexameric complex (8 S) suggests that the DNA component of the particle has a folded conformation.     

Identification, genetic analysis and characterization of a sugar-non-specific nuclease from the cyanobacterium Anabaena sp. PCC 7120

A nuclease that could be recovered from the supernatant of cultures, as well as from cell-free extracts, of the cyanobacterium Anabaena sp. PCC 7120 was identified as a 29 kDa polypeptide by its ability to degrade DNA after electrophoresis in DNA-containing SDS-polyacrylamide gels. Some clones of a gene library of strain PCC 7120 established in Escherichia coli were found to produce the 29 kDa nuclease. The nucA gene encoding this nuclease was subcloned and sequenced. The deduced polypeptide, NucA, had a molecular weight of 29,650, presented a presumptive signal peptide in its N-terminal region and showed homology to the products of the nuc gene from Serratia marcescens and the NUC1 gene from Saccharomyces cerevisiae. The NucA protein from Anabaena itself, or from the cloned nucA gene expressed in E. coli, catalysed the degradation of both RNA and DNA, had the potential to act as an endonuclease, and functioned best in the presence of Mn2+ or Mg2+. An Anabaena nucA insertional mutant was generated which failed to produce the 29 kDa nuclease.