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Originally published In Press as doi:10.1074/jbc.M001087200 on May 11, 2000

J. Biol. Chem., Vol. 275, Issue 28, 21302-21308, July 14, 2000
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A Role of the Ca2+/Mg2+-dependent Endonuclease in Apoptosis and Its Inhibition by Poly(ADP-ribose) Polymerase*

Alexander G. YakovlevDagger , Geping WangDagger , Bogdan A. StoicaDagger , Hamid A. BoularesDagger , Alexander Y. SpoondeDagger , Koichiro Yoshihara§, and Mark E. SmulsonDagger

From the Dagger  Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, D. C. 20007 and the § Department of Biochemistry, Nara Medical University, Kashihara, Japan

Received for publication, February 8, 2000, and in revised form, April 17, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Apoptosis is characterized by various cell morphological and biochemical features, one of which is the internucleosomal degradation of genomic DNA. The role of the human chromatin-bound Ca2+- and Mg2+-dependent endonuclease (CME) DNAS1L3 and its inhibition by poly(ADP-ribosyl)ation in the DNA degradation that accompanies apoptosis was investigated. The nuclear localization of this endonuclease is the unique feature that distinguishes it from other suggested apoptotic nucleases. Purified recombinant DNAS1L3 was shown to cleave nuclear DNA into both high molecular weight and oligonucleosomal fragments in vitro. Furthermore, exposure of mouse skin fibroblasts expressing DNAS1L3 to inducers of apoptosis resulted in oligonucleosomal DNA fragmentation, an effect not observed in cells not expressing this CME, as well as in a decrease in cell viability greater than that apparent in the control cells. Recombinant DNAS1L3 was modified by recombinant human poly(ADP-ribose) polymerase (PARP) in vitro, resulting in a loss of nuclease activity. The DNAS1L3 protein also underwent poly(ADP-ribosyl)ation in transfected mouse skin fibroblasts in response to inducers of apoptosis. The cleavage and inactivation of PARP by a caspase-3-like enzyme late in apoptosis were associated with a decrease in the extent of DNAS1L3 poly(ADP-ribosyl)ation, which likely releases DNAS1L3 from inhibition and allows it to catalyze the degradation of genomic DNA.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Apoptosis, or programmed cell death, is an evolutionarily conserved process that is important in normal development, physiological homeostasis, and certain pathological conditions. It is mediated by a variety of intracellular enzymes, among which are endonucleases that catalyze the internucleosomal fragmentation of DNA, which is one of the hallmarks of apoptotic death (1, 2). Candidates for such endonucleases include the caspase-activated enzymes DFF40 (or CAD) (3-8) and NUC70 (9), divalent cation-dependent neutral (1, 2) or acidic (10, 11) endonucleases, leukemia-associated endo-exonucleases (12), and Ca2+- and Mg2+-dependent endonucleases (CMEs)1 (13-18).

CMEs introduce double strand breaks and single strand nicks into DNA, generating fragments with 5'-phosphate and 3'-hydroxyl termini, a mode of DNA fragmentation consistent with the products of chromatin degradation in apoptotic cells (19-21). CME activity is increased by a variety of stimuli that induce apoptosis (17, 21), and treatments that prevent apoptosis also prevent the induction of CME activity. A role for CMEs in apoptosis has also been supported by studies demonstrating the inhibition of DNA fragmentation by Ca2+ chelators or Zn2+ (13-15, 22).

One of earliest nuclear events in apoptosis is the poly(ADP-ribosyl)ation of various proteins by poly(ADP-ribose) polymerase (PARP), an enzyme that is activated by the presence of DNA strand breaks (23, 24). PARP catalyzes the modification of histones, topoisomerases I and II, SV40 large T antigen, DNA polymerase alpha , proliferating cell nuclear antigen, and various other DNA-binding proteins (25, 26). Indeed, we have previously shown that an early and transient burst of poly(ADP-ribosyl)ation of nuclear proteins, prior to the commitment to cell death, is required for apoptosis (23). Our observations suggested that subsequent cleavage of PARP by a caspase-3-like protease releases certain nuclear proteins from poly(ADP-ribosyl)ation-induced inhibition and thereby allows them to mediate DNA fragmentation and cell death (23). The activity of chromatin-bound CMEs purified from rat liver or thymus has been shown to be inhibited by poly(ADP-ribosyl)ation (27-31), however, until this time, a particular nuclease of this type had not been identified from human cells.

We previously identified the human homolog, DNAS1L3, of bovine and rat chromatin-bound CMEs. The nuclear localization of DNAS1L3 is the unique feature of this enzyme that distinguishes it from other suggested apoptotic nucleases (32). Our data indicated that this nuclease cleaves DNA into both oligonucleosomal and high molecular weight fragments (32). We now provide additional evidence that DNAS1L3 is regulated by PARP and that it may be responsible for apoptotic DNA degradation. The results also demonstrate that the activation of DNAS1L3 increases rates of cell death during apoptosis.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Mouse fibroblasts, immortalized by a standard 3T3 protocol, were kindly provided by Z. Q. Wang (International Agency for Research on Cancer, Lyon, France). Cells were grown under a humidified atmosphere of 5% CO2 in air at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 mg/ml). They were maintained in the logarithmic phase of growth by passage every 2-3 days.

DNAS1L3 cDNA was aligned to the coding region for six histidine residues followed by the FLAG epitope and cloned in pcDNA3.1 mammalian expression vector (Invitrogen). Addition of the His-FLAG tag was necessary for analysis of protein expression and also for purification of the recombinant DNAS1L3, because antibodies against the native enzyme were not available. Cells were transfected with the use of the Mirus TransIT-100 reagent (Panvera) and selected by culture for 2-3 weeks in the presence of G418 and then pooled. Staurosporin, tumor necrosis factor-alpha (TNF-alpha ), and antibodies to mouse Fas receptor were obtained from Sigma, Roche Molecular Biochemicals, and Kamiya Biomedical, respectively.

Production of Recombinant Proteins-- Recombinant human PARP was purified essentially as described (33). A pET21a(+) expression vector (Novagen) containing the coding region for DNAS1L3 was used for the production of recombinant nuclease; because the 20 NH2-terminal amino acids of both DNAS1L3 and its rat homolog (DNase gamma ) are thought to be removed after synthesis, resulting in enzyme activation, we used the coding sequence for the "processed" form of DNAS1L3. The DNAS1L3 expression plasmid was introduced into Escherichia coli strain BL21(DE3)pLysS. After induction of recombinant gene expression with 1 mM isopropyl-beta -D-thiogalactopyranoside, DNAS1L3 was isolated from bacterial inclusion bodies with the use of Ni-NTA agarose (Qiagen). The purified protein was refolded in enzyme storage buffer (25 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.3 M NaCl, 0.1% Triton X-100), concentrated, and stored at -80 °C. The purity of the DNAS1L3 preparation was estimated as >95% on the basis of Coomassie Blue staining of SDS-polyacrylamide gels (not shown).

Assay of DNAS1L3 Activity-- DNAS1L3 activity was measured by evaluation of the integrity of double stranded phage lambda  DNA during incubation with the recombinant nuclease with the use of a Pico Green double stranded DNA quantitation kit (Molecular Probes). The phage DNA (2 µg/ml) was first stained with the quantitation reagent (1:200 dilution in water) at room temperature for 30-60 min, and recombinant DNAS1L3 was diluted immediately before use to a final concentration of 10 µg/ml in a solution containing 25 mM Tris-HCl (pH 7.4), 150 mM KCl, and various combinations and concentrations of cations. Equal volumes (100 µl) of lambda  DNA and DNAS1L3 were mixed together in the wells of a 96-well microtiter plate, and the nuclease reaction was monitored continuously over 10 min at 37 °C with a CytoFluor 4000 fluorometer (PerSeptive Biosystems) by measuring the decrease in DNA fluorescence at excitation and emission wavelengths of 480 and 520 nm, respectively. Reaction mixtures without DNAS1L3 were included in each experiment to provide an indication of background changes in fluorescence. The emission from each well was plotted against time, and linear regression analysis of the initial slope of each curve yielded an activity value.

Analysis of Oligonucleosomal DNA Fragmentation-- Nuclei were isolated from rat cerebellum as described (34). In brief, minced tissue was homogenized with a Dounce homogenizer in a solution containing 15 mM Pipes-NaOH (pH 7.4), 80 mM KCl, 15 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, 0.5 mM spermidine, 0.2 mM spermine, 1 mM phenylmethylsulfonyl fluoride, and 250 mM sucrose. The homogenate was filtered through four layers of cheesecloth, after which an equal volume of homogenization buffer containing 2.3 M sucrose was added to the filtrate. The resulting mixture was layered over 10 ml of homogenization buffer containing 2.3 M sucrose in a Beckman SW28 centrifuge tube and then centrifuged at 22,000 rpm for 90 min at 4 °C. The resulting pellet was resuspended in homogenization buffer containing 50% glycerol at a concentration of 3 × 107 to 7 × 107 nuclei per milliliter and stored at -80 °C (35).

The reaction mixture for the DNA fragmentation assay contained 25 mM Tris-HCl (pH 7.4), 150 mM KCl, 5 × 105 nuclei, and various concentrations of MgCl2, CaCl2, and recombinant DNAS1L3 in a final volume of 60 µl. The reaction was performed at 37 °C for 15 or 60 min, after which DNA was isolated from the nuclei as described (36). In brief, nuclei were lysed in 300 µl of 7 M guanidine hydrochloride, and the lysates were mixed with 1 ml of Wizard Miniprep DNA purification resin (Promega) and drawn by vacuum through a Wizard Minicolumn (Promega). The column was washed with 3 ml of washing solution (90 mM NaCl, 9 mM Tris-HCl (pH 7.4), 2.25 mM EDTA, 55% ethanol) and dried by centrifugation in a microcentrifuge tube at 10,000 × g for 2 min. DNA was eluted from the column by adding 50 µl of 10 mM Tris-HCl (pH 8.4) and centrifuging in a new microcentrifuge tube. Residual RNA was removed from the eluate by addition of 1 µg of RNase A and incubation at 37 °C for 30 min. DNA was analyzed by electrophoresis through an agarose gel in the presence of ethidium bromide (0.5 µg/ml).

Genomic DNA of cultured fibroblasts was isolated and analyzed as described previously (36). In brief, cells collected from one 10-cm Petri dish were lysed in 1 ml of 7 M guanidine hydrochloride and mixed with 1 ml of Wizard Maxiprep Resin (Promega). The suspension was drawn by vacuum through a Wizard Midicolumn (Promega). Columns were washed, and DNA was eluted with 150 µl of 10 mM Tris-HCl (pH 8.4) and analyzed as described above.

Transverse Alternating-field Electrophoresis-- Isolated nuclei (2 × 106) were washed once with ice-cold phosphate-buffered saline, resuspended in 100 µl of lysis buffer (100 mM EDTA, 20 mM NaCl, 10 mM Tris-HCl (pH 8.0)), mixed with 150 µl of agarose solution (1% agarose in lysis buffer, maintained at 42 °C), and poured into a plug mold. After solidification, plugs were incubated twice for 24 h each time at 50 °C in five volumes of lysis buffer supplemented with proteinase K (1 mg/ml) and 1% sodium lauroyl sarcosinate. They were subsequently incubated for at least 24 h in 100 volumes of Tris-EDTA buffer (pH 7.4) at 4 °C, with at least two changes of buffer, and then stored at 4 °C until use. DNA was subjected to transverse alternating-field electrophoresis (TAFE) through 1% agarose in 1× TAFE buffer (20× TAFE buffer: 0.2 M Tris, 7.8 mM EDTA, and 0.5% glacial acetic acid) at 170 V for 30 min with 4-s pulses, followed by 150 V for 18 h with 35-s pulses. This protocol allowed for resolution of DNA molecules of up to 1000 kilobases (kb) in size. Lambda DNA ladders (50-1000 kb) were used as standards. DNA fragments were visualized by staining with ethidium bromide.

Assessment of Cell Viability-- Cell viability was measured by retention and de-esterification of calcein AM (Molecular Probes). Cells were washed once in Locke's buffer containing 154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 2.3 mM CaCl2, 1.2 mM MgCl2, 5.6 mM glucose, and 5 mM Hepes-NaOH (pH 7.4). After loading of cells with 5 µM calcein AM in Locke's buffer for 20 min, fluorescence was monitored with a CytoFluor 4000 fluorometer at excitation and emission wavelengths of 480 and 520 nm, respectively.

Reverse Transcription and Polymerase Chain Reaction-- Transcripts encoding the mouse homolog (LSDNase) of DNAS1L3 were detected by reverse transcription and polymerase chain reaction RT-PCR as described previously (36). In brief, total cellular RNA was isolated by acidic phenol extraction (37) and treated with DNase I. RT was performed with 10 µg of total RNA in a 20-µl reaction mixture, and one-tenth of the resulting cDNA was amplified by PCR. PCR primers were as follows (sense and antisense, respectively): mouse LSDNase, 5'-GAGACACAGACGTGTTTTCC-3' and 5'-GTCCACAAAGCACAATCCTG-3'; mouse beta -actin, 5'-GTTTGAGACCTTCAACACCCCAGCC-3' and 5'-ATGTCACGCACGATTTCCCTCTCAG-3'. Amplification was performed for 30 cycles of denaturation for 45 s at 94 °C, annealing for 30 s at 57 °C, and primer extension for 45 s at 72 °C. The amplification products were analyzed by electrophoresis through 2% agarose gels and staining with ethidium bromide. The identity of specific PCR products was confirmed by DNA sequencing.

Immunoblot Analysis-- Cells were harvested, washed once with ice-cold phosphate-buffered saline, and lysed on ice in a solution containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40, 0.25% sodium deoxycholate, leupeptin (5 µg/ml), and aprotinin (5 µg/ml). After removal of cell debris by centrifugation, the protein concentration of the cell lysate was determined with the Bio-Rad protein assay reagent. A portion of the lysate (30 µg of protein) was then fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) through a 4-20% gradient gel, and the separated proteins were transferred to a nitrocellulose filter. The filter was stained with Ponceau S to confirm equal loading and transfer of samples and was then probed with specific antibodies. Immune complexes were detected with appropriate secondary antibodies and chemiluminescence reagents (Pierce). Antibodies to RARP and poly(ADP-ribose) were obtained from Santa Cruz and Trevigen, respectively.

Assay of Caspase Activity-- Caspase-3-like activity was assayed as described (38). Portions of cytosolic extract (20 µg of protein in 100 µl of extraction buffer) were mixed in the wells of a 96-well microtiter plate with an equal volume of 40 µM fluorescent tetrapeptide substrate (Ac-DEVD-AMC; Bachem) in the same buffer. Accumulation of free aminomethylcoumarin (AMC), which was produced as a result of cleavage of the aspartate-AMC bond, was monitored continuously over 10 min with a CytoFluor 4000 fluorometer at excitation and emission wavelengths of 360 and 460 nm, respectively. The emission from each well was plotted against time, and linear regression analysis of the initial slope of each curve yielded the activity value.

Poly(ADP-ribosyl)ation in Vitro-- Purified recombinant DNAS1L3 and PARP were incubated for 30 min at room temperature in a reaction mixture (30 µl) containing 25 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 0.5 mM dithiothreitol, 5% glycerol, 40 pmol of [32P]NAD (800 Ci/mmol), and 0.1 µg of high molecular weight or activated rat genomic DNA. The reaction was terminated by the addition of an equal volume of SDS sample buffer and heating at 95 °C for 10 min, after which samples were analyzed by SDS-PAGE through a 12.5% gel. Gels were dried and subjected to autoradiography.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cation Dependence of DNA Cleavage by DNAS1L3-- The results of our previous study (32) suggested that DNAS1L3 mediates Ca2+- and Mg2+-dependent fragmentation of DNA both in vitro and in vivo. We have now examined the cation dependence of DNAS1L3 directly with the use of the purified recombinant enzyme. The purified protein required both Ca2+ and Mg2+ for maximal activity; the optimal concentrations of these cations were 2.5-5 mM (Fig. 1A) and 5 mM (Fig. 1B), respectively, values similar to those determined for CME activity in rat liver nuclei (32). The activity of DNAS1L3 was also supported by Mn2+ (Fig. 1C), with 20 mM Mn2+ yielding ~80% of the nuclease activity apparent in the presence of optimal concentrations of Ca2+ and Mg2+; this concentration of Mn2+ is ~10 times that shown to be optimal for the rat homolog (DNase gamma ) of DNAS1L3 (39). The activity of DNAS1L3 in the presence of Ca2+ and Mg2+, like that of DNase gamma  (39), was inhibited by Zn2+, with a median effective concentration of ~45 µM (Fig. 1D).


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  		Fig. 1.   Cation dependence of DNAS1L3 activity. The nuclease activity of recombinant DNAS1L3 was measured at 37 °C in the presence of either 5 mM MgCl2 and the indicated concentrations of CaCl2 (A); 2.5 mM CaCl2 and the indicated concentrations of MgCl2 (B); the indicated concentrations of MnCl2 (in the absence of Mg2+ and Ca2+) (C); or 2.5 mM CaCl2, 5 mM MgCl2, and the indicated concentrations of ZnSO4 (D). Data are expressed as a percentage of the nuclease activity apparent under optimal conditions (2.5 mM CaCl2 and 5 mM MgCl2) and are means of triplicates from an experiment that was repeated three times with similar results.

Characterization of the Nature of DNA Strand Cleavage by Recombinant DNAS1L3-- To examine whether DNAS1L3 preferentially introduces single stranded nicks or double stranded breaks into DNA substrates, we incubated various amounts of the recombinant enzyme with supercoiled and linear forms of pCR2.1 plasmid DNA (Invitrogen) in the presence of optimal concentrations of Ca2+ and Mg2+. Agarose gel electrophoresis of the reaction products revealed that DNAS1L3 cleaved both forms of DNA in a dose-dependent manner (Fig. 2). However, at low concentrations (2-4 µg/ml), the recombinant enzyme preferentially introduced single strand nicks rather than double strand breaks, as reflected by the relative abundance of relaxed and linear forms of DNA generated from the supercoiled plasmid (Fig. 2A).


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  		Fig. 2.   Analysis of mode of DNA strand cleavage catalyzed by recombinant DNAS1L3. Circular (A) or linear (B) pCR2.1 DNA (1 µg) was incubated for 1 h at 37 °C in the presence of 5 mM MgCl2, 2.5 mM CaCl2, and the indicated concentrations of purified recombinant DNAS1L3 in a final volume of 15 µl. The DNA was then analyzed by electrophoresis through a 1.5% agarose gel and ethidium bromide staining. S, L, and R indicate supercoiled, linear, and relaxed forms of the plasmid, respectively. M demonstrates the 1-kb DNA ladder molecular weight DNA standard.

Cleavage of Nuclear DNA by Recombinant DNAS1L3-- We previously analyzed Ca2+- and Mg2+-dependent cleavage of DNA in nuclei isolated from rat liver and cerebellum (32). Although liver nuclei exhibited such DNA cleavage, cerebellar nuclei did not. Consistent with the notion that DNase gamma  is responsible for the observed DNA degradation in the liver nuclei, transcripts encoding this enzyme were detected in rat liver but not in cerebellum. We investigated this conclusion further in the present study by incubating rat cerebellar nuclei with recombinant human DNAS1L3 in the absence or presence of Ca2+ and Mg2+ and then analyzing the generation of high molecular weight and oligonucleosomal DNA fragments. TAFE revealed the presence of small amounts of >1000-kb DNA fragments in cerebellar nuclei incubated without DNAS1L3 in the absence or presence of Ca2+ and Mg2+ (Fig. 3A). Although Mg2+ alone did not induce the production of high molecular weight DNA fragments in nuclei incubated in the presence of recombinant DNAS1L3, nuclei incubated with DNAS1L3 in the presence of 5 mM Mg2+ and 0.1 mM Ca2+ exhibited a marked increase in the amount of >1000-kb DNA fragments. A further increase in the Ca2+ concentration to 2.5 mM resulted in processing of the >1000-kb DNA molecules into ~50-kb fragments (Fig. 3A). Electrophoresis through 1.5% agarose gels revealed that DNAS1L3 cleaved DNA in cerebellar nuclei into oligonucleosomal fragments in a manner that was dependent on both enzyme dose as well as Ca2+ and Mg2+ (Fig. 3B). No such internucleosomal DNA fragmentation was apparent in the absence of added DNAS1L3. These results thus provide direct evidence that DNAS1L3 catalyzes the cleavage of DNA into both high molecular weight and oligonucleosomal fragments.


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  		Fig. 3.   Effect of recombinant DNAS1L3 on Ca2+- and Mg2+-dependent DNA fragmentation in isolated rat cerebellar nuclei. The DNA cleavage activity of the indicated amounts of DNAS1L3 was analyzed by incubation with rat cerebellar nuclei for 15 min (A) or 1 h (B) at 37 °C in the presence of the indicated concentrations of Mg2+ and Ca2+. The integrity of the DNA was then evaluated by TAFE (A) or conventional agarose gel electrophoresis (B). The leftmost lane in B contains molecular size standards.

Effect of DNAS1L3 Expression on DNA Fragmentation during Apoptosis-- We next examined the potential role of DNAS1L3 in DNA cleavage during apoptosis and its effect on cell viability in mouse skin fibroblasts stably transfected with DNAS1L3 cDNA (32). The expression vector encoded DNAS1L3 fused at its COOH terminus with six histidine residues to facilitate protein purification with Ni-NTA agarose. The presence of this COOH-terminal tag did not inhibit DNAS1L3 activity, given that the recombinant nuclease expressed in and purified from bacteria for our in vitro experiments was similarly tagged. Mouse skin fibroblasts were chosen, because RT-PCR analysis did not detect endogenous transcripts encoding the mouse homolog of DNAS1L3 (data not shown) and because they do not exhibit internucleosomal DNA fragmentation during apoptosis induced by a variety of agents.

Cells transfected with the DNAS1L3 expression plasmid or with the empty vector (control) were treated with staurosporin or with TNF-alpha plus cycloheximide, after which DNA fragmentation and cell viability were analyzed. Both treatments induced internucleosomal DNA fragmentation in the DNAS1L3-expressing cells but not in control cells (Fig. 4A). Analysis of cell viability revealed that the apoptosis-inducing effects of these treatments were markedly potentiated in cells expressing DNAS1L3 (Fig. 4B). The incidence of cell death after culture of cells in the absence of serum or in the presence of other inducers of apoptosis, such as etoposide or antibodies to mouse Fas, was also greater for cells expressing DNAS1L3 than for control cells (Fig. 4C).


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  		Fig. 4.   Effects of DNAS1L3 expression on DNA fragmentation in (A) and viability of (B and C) immortalized mouse fibroblasts exposed to inducers of apoptosis. Cells transfected with a DNAS1L3 expression plasmid or with the empty vector (control) were incubated either for 12 h in the absence or presence of 1 µM staurosporin or of TNF-alpha (10 ng/ml) plus 1 µM cycloheximide (A and B), or for 24 h under conditions of serum deprivation or in the absence or presence of either 50 µM etoposide or antibodies to mouse Fas (100 ng/ml) plus 1 µM cycloheximide, as indicated. The integrity of genomic DNA was then examined by electrophoresis through a 2% agarose gel (A), and cell viability was analyzed either by measurement of calcein fluorescence (B) or by light microscopy (C). Data in B are expressed as a percentage of the value for control cells not exposed to inducers of apoptosis and are means ± S.D. of six wells from an experiment that was repeated four times with similar results.

Tissue Distribution of Transcripts Encoding the Mouse Homolog of DNAS1L3-- RT-PCR analysis revealed that mRNA encoding the mouse homolog (LSDNase) of DNAS1L3 is present in a variety of tissues, with the highest amounts apparent in spleen, liver, and testes (Fig. 5). Smaller amounts were detected in heart, lungs, skeletal muscle, and kidney, but LSDNase mRNA was not detected in mouse brain. Similar analysis of mouse embryos at various stages of development revealed that LSDNase mRNA was not detected in 7-days mouse embryos, and small amounts of this mRNA were detected at day 11, and higher amounts were detected at days 15 and 17 (Fig. 5).


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  		Fig. 5.   RT-PCR analysis of the abundance of transcripts encoding the mouse homolog (LSDNase) of DNAS1L3 in various mouse tissues and during mouse embryogenesis. Total RNA from the indicated mouse tissues and from mouse embryos on the indicated days of embryogenesis was subjected to RT-PCR with primers specific for LSDNase or for beta -actin. The PCR products were analyzed by electrophoresis through a 2% agarose gel.

Poly(ADP-ribosyl)ation and Inactivation of Recombinant DNAS1L3 by PARP in Vitro-- The bovine chromatin-bound CME and related enzymes are inhibited by poly(ADP-ribosyl)ation (24-26). We therefore investigated whether human DNAS1L3 is also susceptible to such modification by PARP in vitro. Incubation of purified recombinant DNAS1L3 with recombinant human PARP in the presence of a low concentration of [32P]NAD and high molecular weight DNA resulted in marked poly(ADP-ribosyl)ation of both proteins (Fig. 6A). This effect was blocked in the presence of the PARP inhibitor 3-aminobenzamide (3-AB). In the absence of DNAS1L3, automodification of PARP was not detected; however, when high molecular weight DNA in the reaction mixture was replaced with activated DNA, poly(ADP-ribosyl)ation of PARP was apparent even in the absence of DNAS1L3. These results showed that DNAS1L3 activates PARP by introducing breaks into DNA strands, and that PARP, in turn, catalyzes the post-translational modification of the nuclease. The effect of the increasing NAD concentration of the reaction mixture from 1.3 µM to 3 mM on the length of the ADP-ribose chains attached to DNAS1L3 was investigated by performing the reaction with nonradioactive NAD. Under these conditions, the positions of both DNAS1L3 and PARP were shifted toward the top of the gel, reflecting the presence of long chains of ADP-ribose attached to these proteins (Fig. 6A).


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  		Fig. 6.   Poly(ADP-ribosyl)ation and inhibition of recombinant DNAS1L3 by PARP in vitro. A, poly(ADP-ribosyl)ation of DNAS1L3 by PARP. The indicated combinations of recombinant human PARP (150 ng), 1.3 µM [32P]NAD (lanes 1-5) or 3 mM nonradioactive NAD (lane 6), recombinant DNAS1L3 (4.5 µg), and 3 mM 3-AB were incubated in a volume of 30 µl with 0.1 µg of high molecular weight (lanes 1-4 and 6) or activated (lane 5) DNA. The reaction products were then analyzed by SDS-PAGE and autoradiography. The positions of PARP, PARP fragments, and DNAS1L3 are indicated. B, effect of poly(ADP-ribosyl)ation on DNAS1L3 activity. The indicated combinations of recombinant human PARP (0.3 µg), 0.3 mM nonradioactive NAD, recombinant DNAS1L3 (3 µg), and 3 mM 3-AB were incubated for 30 min at room temperature in a volume of 30 µl with 0.2 µg of high molecular weight DNA. The reaction mixtures were then placed on ice, and portions (1 µl) were subsequently incubated for 30 min at 37 °C with 2 µg of phage lambda  DNA in a final volume of 20 µl containing 25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, and 2.5 mM CaCl2. DNA integrity was then analyzed by electrophoresis through a 1.5% agarose gel. The leftmost lane contains 1-kb DNA size markers.

The effect of poly(ADP-ribosyl)ation on DNAS1L3 activity was assessed qualitatively with a DNA degradation assay. Thus, although the unmodified enzyme effectively degraded bacteriophage lambda  DNA in the presence of optimal concentrations of Ca2+ and Mg2+, poly(ADP-ribosyl)ated DNAS1L3 showed no such activity (Fig. 6B). The inclusion of 3-AB in, or the omission of NAD or PARP from, the poly(ADP-ribosyl)ation reaction mixture prevented the inhibition of DNAS1L3.

Effect of Poly(ADP-ribosyl)ation on DNAS1L3 Activity during Apoptosis-- The effect of poly(ADP-ribosyl)ation on DNAS1L3 activity was also examined in mouse fibroblasts stably expressing histidine-tagged DNAS1L3. The generation of oligonucleosomal DNA fragments was first evident in DNAS1L3-expressing cells ~9 h after exposure to TNF-alpha and cycloheximide, whereas DNA fragmentation was not detected in control fibroblasts at any time point examined (Fig. 7A). DNA degradation in the DNAS1L3-expressing fibroblasts was accompanied by an increase in caspase-3-like activity; this activity was maximal 9 h after exposure to TNF-alpha and cycloheximide and decreased thereafter (Fig. 7B). Similar changes in caspase-3-like activity were observed in control cells treated with these agents (data not shown). Immunoblot analysis revealed that cleavage of PARP to yield an apoptosis-specific 89-kDa fragment was first apparent in the DNAS1L3-expressing (Fig. 7C) or control (data not shown) cells 3 h after exposure to TNF-alpha and cycloheximide; after 12 h of incubation, almost all of the 113-kDa PARP protein had been cleaved and inactivated, presumably as a result of the increase in caspase-3-like activity.


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  		Fig. 7.   Effect of poly(ADP-ribosyl)ation on DNAS1L3-dependent DNA fragmentation during apoptosis in mouse fibroblasts. Cells transfected with a DNAS1L3 expression plasmid were incubated for various times in the presence of TNF-alpha (10 ng/ml) and 1 µM cycloheximide, after which internucleosomal DNA fragmentation was analyzed by electrophoresis through a 2% agarose gel (A); caspase-3-like activity was assayed fluorometrically (data are expressed as a percentage of the value for time zero and are from a representative experiment) (B); cleavage of PARP (C) and poly(ADP-ribosyl)ation of nuclear proteins (D) were monitored by immunoblot analysis with antibodies to PARP and to PAR, respectively; and the recombinant histidine-tagged DNAS1L3 protein was purified from the transfected cells and subjected to immunoblot analysis with antibodies to PAR (E). Data for control (C) cells transfected with the empty vector are also shown in A and E.

Immunoblot analysis with antibodies to poly(ADP-ribose) (PAR) revealed that the extent of poly(ADP-ribosyl)ation of nuclear proteins increased during exposure of DNAS1L3-expressing cells to TNF-alpha and cycloheximide (Fig. 7D); this effect was maximal at 6 h, a time at which cells remained viable, and had declined markedly by 12 h, concomitant with the increases in caspase-3-like activity, cleavage of PARP, and DNA fragmentation. In mock-transfected fibroblasts similar changes in PAR synthesis have been observed during a course of apoptosis (data not shown).

Poly(ADP-ribosyl)ation of nuclear proteins in response to DNA damage is transient and restricted to those proteins associated with PARP adjacent to DNA strand breaks (40). Although the bovine homolog of human DNAS1L3 was identified as a chromatin-bound enzyme (31, 32), our immunocytochemical data suggest that DNAS1L3 is localized mostly to the perinuclear region (not shown). Detection of poly(ADP-ribosyl)ation of DNAS1L3, a reaction that occurs in the nucleus, might therefore be expected to be difficult in intact cells, because only a small proportion of this protein normally enters the cell nucleus. To investigate whether DNAS1L3 undergoes poly(ADP-ribosyl)ation during apoptosis, we therefore isolated the histidine-tagged nuclease from extracts of DNAS1L3-expressing mouse fibroblasts with the use of Ni-NTA magnetic agarose beads (Qiagen). The purified protein was then subjected to immunoblot analysis with antibodies to PAR. The extent of poly(ADP-ribosyl)ation of DNAS1L3 was low under normal culture conditions, was markedly increased 6 h after exposure of cells to TNF-alpha and cycloheximide, and had returned to control values after 12 h (Fig. 7E).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Early studies showing that liver nuclei contain endonuclease activity responsible for specific degradation of DNA implicated CMEs in internucleosomal DNA fragmentation (41, 42), and subsequent studies established a link between intracellular Ca2+ and induction of apoptosis (2, 43, 44). Previous observations also indicated that chromatin-bound CMEs from various tissues are poly(ADP-ribosyl)ated and thereby inhibited by PARP (27-32). PARP has been shown to be important in various cellular models of apoptosis, although the precise molecular mechanisms involved remain poorly understood (23-25, 45). PARP activation has been proposed to result in cell death by depletion of cellular NAD and ATP (46, 47). On the other hand, PARP is rapidly cleaved and inactivated by caspases (48), and this cleavage is thought to be a key apoptotic event (49, 50).

We have recently shown that human DNAS1L3 is necessary for Ca2+- and Mg2+-dependent internucleosomal DNA cleavage in specific cell types (32). With the use of recombinant DNAS1L3, we have now confirmed that DNAS1L3 requires both Ca2+ and Mg2+ for maximal activity, with optimal concentrations of 2.5-5 mM for Ca2+ and 5 mM for Mg2+, consistent with the values we previously obtained for DNA fragmentation in isolated rat liver nuclei (32). We also showed that, like other DNase I-related nucleases (39, 51), human DNAS1L3 is activated by Mn2+ and inhibited by Zn2+. Kinetic analysis of plasmid DNA cleavage by recombinant DNAS1L3 has revealed that the nuclease preferentially introduces nicks into one strand of double stranded DNA rather than catalyzing the cleavage of both strands; however, at higher concentrations, the enzyme also mediates double strand scission.

We previously showed that rat liver nuclei, but not nuclei isolated from rat cerebellum, exhibit fragmentation of DNA into high molecular weight and oligonucleosomal fragments on incubation in the presence of Ca2+ and Mg2+ (32). Furthermore, we detected DNase gamma  mRNA in rat liver but not in rat cerebellum (32). We thus hypothesized that DNase gamma  is responsible for both types of DNA cleavage stimulated by Ca2+ and Mg2+ in rat liver nuclei. These results indicate that preferential appearance of oligonucleosomal or high molecular weight DNA fragments may depend on levels of CME activity.

Mouse fibroblasts expressing human DNAS1L3, but not control cells, exhibited extensive internucleosomal DNA fragmentation in response to inducers of apoptosis. Although internucleosomal DNA fragmentation is a biochemical marker of apoptosis, its precise role in cell death is unclear. Expression of DNAS1L3 in mouse fibroblasts markedly increased the incidence of apoptosis induced by various treatments, consistent with the results of previous studies (13-15, 22), suggesting that Ca2+- and Mg2+-dependent DNA degradation may contribute to the execution phase of apoptosis.

Activity of chromatin-bound CMEs has been previously detected in various mammalian tissues, including liver, spleen, lymph nodes, and placenta. However, conflicting data have been obtained with regard to the tissue distribution of the enzyme protein and mRNA (52, 53). We have now detected transcripts encoding the mouse homolog of DNAS1L3 in various tissues, with the highest amounts apparent in spleen, liver, and testes. The expression of the LSDNase gene also appeared to be developmentally regulated in mouse embryos. DNAS1L3 and its homologs may thus participate in apoptotic DNA fragmentation in various mammalian tissues.

The bovine CME is poly(ADP-ribosyl)ated and thereby inactivated by PARP (27, 29-31). We have now shown that PARP catalyzes the poly(ADP-ribosyl)ation of recombinant DNAS1L3 in vitro, resulting in inactivation of the nuclease. DNAS1L3 was able to activate PARP by introducing strand breaks into high molecular weight DNA. We also showed that DNAS1L3, like various other nuclear proteins, undergoes poly(ADP-ribosyl)ation during the early stages of apoptosis in transfected mouse fibroblasts. We have previously described a similar early and transient poly(ADP-ribosyl)ation of various nuclear proteins in several cell types undergoing apoptosis (23, 24). At later stages of apoptosis, PARP is cleaved by a caspase-3-like protease and thereby inactivated, and DNA undergoes extensive internucleosomal fragmentation. The observation that PAR is removed from DNAS1L3 at this time, presumably resulting in activation of the nuclease, suggests that this enzyme may contribute to the degradation of DNA.

Our results thus suggest a model for apoptosis (Fig. 8) in which the activation of DNAS1L3 by an increase in the intracellular concentration of Ca2+ results in the introduction of strand breaks into genomic DNA and the consequent activation of PARP. Poly(ADP-ribosyl)ation of DNAS1L3 by PARP, in turn, results in inhibition of nuclease activity. Subsequent cleavage and inactivation of PARP by caspases prevents further poly(ADP-ribosyl)ation of nuclear proteins, thereby allowing the activity of PAR glycohydrolase to remove polymer from these proteins and thereby release DNAS1L3 from inhibition. The activated nuclease may then catalyze the internucleosomal DNA fragmentation characteristic of the later stages of apoptosis. Currently, experiments initiated utilizing caspase-3-deficient cells strengthen our hypothesis, because established peptide inhibitors of caspase 3 have only limited specificity.


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  		Fig. 8.   Model for the role of DNAS1L3 in DNA fragmentation during apoptosis. The apoptotic stimulus may result in initial DNA damage directly or through Ca2+-induced activation of DNAS1L3. See text for further details.

Automodification of PARP, followed by the subsequent removal of the polymer during a cycling mechanism of protein activity and binding to DNA strands breaks during DNA replication and repair, has been verified in several studies, including reports by Lindahl and coauthors (54, 55) as well as by our data obtained with PARP deletion mutants (56). We have also recently demonstrated that spontaneous apoptosis in human osteosarcoma cells is associated with a marked increase in poly(ADP-ribosyl)ation of other nuclear proteins such as p53. The results demonstrate that the initiation of the cell death program is associated with a marked increase in the intracellular abundance of p53 that undergoes extensive modification by PARP early during the apoptosis (57). Subsequent degradation of PAR, attached to p53, was apparent in this cell system after activation of caspase 3 and PARP cleavage. Significantly, the decrease in the polymer covalently bound to p53 coincided with the marked induction of the expression of p53-responsive pro-apoptotic genes, such as Bax and Fas (57).

The results of the present study demonstrate transient modification and inhibition of DNAS1L3 by PARP and thereby confirm that poly(ADP-ribosyl)ation has a role in regulation of activity of nuclear proteins. Taken together, these data provide insight into the effect of transient poly(ADP-ribosyl)ation as a general mechanism for reversibly rendering DNA-binding proteins as a result of the highly negative charge conferred by the polymer.

    FOOTNOTES

* This work was supported by National Cancer Institute Grants CA25344 and 1P01-CA74175, U.S. Air Force Office of Scientific Research Grant AFOSR-89-0053, and U.S. Army Medical Research and Development Command Contract DAMD17-90C-0053 (all to M. E. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Basic Science Bldg., Rm. 351, 3900 Reservoir Rd., NW, Washington, D. C. 20007. Tel.: 202-687-1718; Fax: 202-687-7186; E-mail: smulson@bc.georgetown.edu.

Published, JBC Papers in Press, May 11, 2000, DOI 10.1074/jbc.M001087200

    ABBREVIATIONS

The abbreviations used are: CME, Ca2+- and Mg2+-dependent endonuclease; PARP, poly(ADP-ribose) polymerase; TNF-alpha , tumor necrosis factor-alpha ; TAFE, transverse alternating-field electrophoresis; RT-PCR, reverse transcription and polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; AMC, aminomethylcoumarin; 3-AB, 3-aminobenzamide; PAR, poly(ADP-ribose); Pipes, 1,4-piperazinediethanesulfonic acid; kb, kilobase(s).

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TOP
ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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