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Cytoplasmic poly(ADP-ribose) polymerase during the HeLa cell cycle
ARCHIVES
OF
BIOCHEMISTRY
Cytoplasmic JERRY Department
AND
171,
Poly(ADP-Ribose)
H. ROBERTS2 of Biochemistry,
BIOPHYSICS
PATRICIA Schools
305-315
(197%
Polymerase Cycle1
during
STARK, CHANDRAKANT SMULSON
of Medicine Received
and Dentistry, 20007 April
Georgetown
the HeLa Cell P. GIRL
University,
AND
MARK
Washington,
D.C.
24, 1975
Poly(ADP-ribose) polymerase, an enzyme that has reportedly been confined to the nucleus of eukaryotic cells, has been found in the cytoplasm of HeLa cells. The enzyme activity is stimulated more than 30-fold by the addition of both DNA and histones. These two macromolecules are absolutely necessary for maximal activity and they act in a synergistic manner. The product of the reaction was characterized as poly(ADP-ribose) by its acid insolubility, its insensitivity to hydrolysis by DNase, RNase, spleen phosphodiesterase or Pronase and by release of 5’-AMP and 2’-(5”-phosphoribosyl)-5’-AMP by incubation with snake venom phosphodiesterase. A covalent attachment between histone PI and poly(ADP-ribose) has been established by using the cytoplasmic enzyme. The enzyme is primarily associated with ribosomes, both free ribosomes and those found in polysomes. Inhibition of protein synthesis in the intact cell reduced the level of activity in the cytoplasm. The enzyme can be removed from the ribosomes by centrifugation through sucrose gradients containing 0.6 M ammonium chloride. A relationship between this enzyme and DNA replication is suggested by the fact that the specific activity in the cytoplasm parallels the rate of DNA synthesis during the HeLa cell cycle.
The enzyme poly(ADP-ribose) polymerase catalyzes the formation of a homopolymer of ADP-ribose units linked by l’-2’ glycosidic bonds (for reviews, see Ref. (l3)). The substrate for the reaction is NAD, and the enzyme successively adds ADPribose units onto an initial ADP-ribose residue that has been reported to be covalently attached to various nuclear proteins (4-6). The enzyme is found tightly bound to chromatin and has been detected in a large number of eukaryotic cells but has not been detected in prokaryotic organisms (7). Partially purified poly(ADP-ribose) polymerase from rat liver nuclei has been shown to have an absolute requirement for both DNA and histones for elongation of ADP-ribose units (8, 9). ’ A preliminary report of this work was given at an International Seminar on Poly(ADP-Ribose) and ADP-Ribosylation of Protein, held September 10-12, 1974, Tomakomai, Japan. * Present address: Department of Biology, The Catholic University of America, Washington, D.C. 20064. 305 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
A number of proposals have been made suggesting a role for poly(ADP-ribose) polymerase in the regulation of eukaryotic DNA replication or repair (10-13). Work from various laboratories indicates that formation of poly(ADP-ribose) caused perturbations of chromatin structure which led to either activation (14) or restriction of chromatin function with respect to DNA synthesis depending upon the source of tissue used (10-12). We have recently reported that the formation of poly(ADP-ribose) in HeLa cell nuclei or chromatin releases template restriction for DNA synthesis; in modified nuclei we observed two to three times more binding sites for Escherichia coli DNA polymerase I than in nonmodified nuclei (15). This effect was inversely related to the extent of natural template restriction during asynchronous growth (15) as well as during various phases of the HeLa cell cycle (16). In addition, we and others have attempted to relate the specific activity of the nuclear enzyme as a function of the cell cycle to its
ROBERTS
306
possible biological role in the intact cell (13, 17, 18). Interpretation of these data has been difficult because of the complexity of this enzyme system in the intact nuclei. In such assays the nuclear material serves as: (i) A source of various polymerizing enzymes and DNA which are required for elongation of poly(ADP-ribose) (19), and (ii) various histones and nor&stone acceptors for the polymer that may contain varying levels of endogenous polymer which had been synthesized in vivo. Any one of these variables could account for differences in specific activity, and hence interpretation of cell cycle data is quite complicated. In an attempt to eliminate these variables, in the present study we investigated the biosynthesis of this enzyme on cytoplasmic ribosomes during the time sequences of the cell cycle. This activity allows a convenient in vitro assay that shows strict dependence for activity on both histones and DNA, in contrast to the nuclear or chromatin assay, and hence better allows us to investigate the relationship of this enzyme to events of the cell cycle. MATERIALS
AND
METHODS
Materials. Calf thymus DNA, whole histones, and histone fractions were obtained from Sigma Chemical Co.; ribonuclease Tl and T2 were obtained from Calbiochem; pancreatic RNase, DNase I, snake venom phosphodiesterase, and spleen phosphodiesterase were purchased from Worthington Biochemical Corp.; [3HlNAD (3.7 Ci/mmol) was purchased from New England Nuclear. Assays. The reaction mixture for the poly(ADPribose) polymerase assay contained in a volume of 0.5 ml: 50 pmol of Tris-HCl buffer, pH 7.4, 0.5 pmol of dithiothreitol, 1.0 pmol of MgC& and VHINAD, DNA and histones as indicated in each experiment. The assays were performed at room temperature and terminated after 30 min by the addition of 1.0 ml of 20% trichloroacetic acid containing 5 mM sodium pyrophosphate. Insoluble material was collected on Whatmann GF/C filter disks, and the radioactivity was determined in a Packard scintillation counter. The assay for nuclear poly(ADP-ribose) polymerase was performed as previously described (13). Protein was determined by the method of Lowry et al. (20). DNA was determined by the method of Burton (21). Growth of cells and preparation of extracts. HeLa S3 cells were maintained at 37°C in spinner flasks in
ET
AL
Eagle’s spinner medium (22) containing 10% fetal calf serum. Cells were harvested by centrifugation and washed with spinner salts before being broken. The following three methods have been used to disrupt the cells: (i) Dounce homogenization according to the method of Sporn et al. (23); (ii) utilization of the detergent NP-403 as described by Borun et al. (24); and (iii) the method of Attardi et al. (25). Nuclei were collected by centrifugation at 115Og for 5 min. Postmitochondrial supernatant fractions were collected by centrifugation at 16,OOOg for 20 min. Cell synchronization. HeLa cells at 3 x lo5 cells/ml were blocked at the Gl-S boundary by addition of methotrexate (230 pgiliter). After 16 h, the block was reversed by addition of a solution containing 10 pM thymidine (16). The rate of DNA synthesis was determined by measuring the amount of 13H1thymidine incorporated into trichloroacetic acid-insoluble material by 2-ml samples of cells in 15 min. At specific times during the cell cycle, samples were collected for subsequent determination of DNA content and poly(ADP-ribose) polymerase activity in the postmitochondrial extracts. Preparation of polysomes, monoribosomes and ribosomal subunits. Samples of postmitochondrial fractions were layered on top of 15-30% sucrose gradients (17 ml) which were formed over a 64% sucrose cushion. The gradients were centrifuged at 4°C in an SW 27 rotor for 2-5 h (for polysome patterns) or 18 h (for monoribosomes and ribosomal subunit patterns). The gradients were analyzed by using an ISCO density gradient fractionator and uv-absorbante monitor. Poly(ADP-ribose) polymerase was removed from ribosomes as follows: The ribosomal pellet was resuspended in 0.1 mM EDTA, 1.0 mM dithiothreitol and 0.25 M sucrose and then made 0.6 M with respect to NH&l. Samples of 0.5 ml were layered on top of a discontinuous gradient prepared by layering 2.0 ml of 0.5 M sucrose over 2.0 ml of 1.0 M sucrose, both containing dithiothreitol, EDTA and 0.6 M NH&l. The gradients were centrifuged at 4°C in a Type 40 rotor (Beckman) for 20 h at 28,000 rpm to reisolate the washed ribosomes. The supernatant fluid was dialyzed against 50 mM Tris-HCl buffer, pH 7.2, containing 1 mM dithiothreitol. ADP-ribosylation of purified [‘Wtistone Fl. HeLa cells were labeled with a 14C-amino acid mixture for three generations, nuclei prepared and histone Fl purified by the method of Johns (26). An aliquot of the labeled material was mixed with commercially available histone Fl carrier, characterized by electrophoresis according to the method of Weber and Osborn (271, and utilized as acceptor with the poly(ADP-ribose) polymerase separated from ribosomes (see above), as described in the legend to Fig. 5. 3 Abbreviation
used:
NP-40,
Nonidet
P-40.
CYTOPLASMIC
POLY(ADP-RIBOSE)
POLYMERASE
307
RESULTS
Cytoplasmic ase
Poly(ADP-Ribose)
Polymer-
The postnuclear extract prepared from asynchronous cultures of HeLa cells contained little poly(ADP-ribose) polymerase activity when assayed by the standard procedure (Table I) used to assay the nuclear enzyme. This finding agrees with other reports demonstrating that poly(ADP-rihose) polymerase is confined to the nuclei of eukaryote cells (l-3). However, if DNA and whole histones were included in the reaction, the incorporation of 13HlNAD into material insoluble in 20% trichloroacetic acid was stimulated 30-fold (Table I). The incorporation was inhibited by nicotinamide and by thymidine, which are competitive inhibitors of poly(ADP-ribose) polymerase (28). The data in Fig. 1 demonstrate that the amount of poly(ADP-ribose) formed displays concentration dependence on both histones and DNA; the formation of poly(ADP-ribose) is stimulated by DNA and histone synergistically. When both DNA and histone are present, the amount of poly(ADP-ribose) formed is almost three-fold greater than that expected by the sum of the single additions. When the optimal concentrations of DNA and histones were utilized, after a short lag period (2-3 min), incorporation of [3H]NAD into poly(ADP-ribose) was linear with respect to time for at least 40 min and was also proportional to the quantity of extract TABLE PoLYCADP-RIBOSE) FRACTION
I
SYNTHESIS
IN
OF HELA
Sample additions
Complete, zero time Complete, 30 min, 20°C Complete, 30 min, 20°C + nicotinamide (20 mM) Complete, 30 min, 20°C + thymidine (10 mM)
THE
POSTNUCLEAR
CELLS”
Control @pm)
+DNA, +Histone (dpm)
120 370 119
91 10,290 164
115
170
’ Cells were broken by the method of Sporn et al. (23) and the postnuclear fraction isolated. The reaction was performed as described in Materials and Methods and contained 230 pg of protein and, where indicated, 50 Kg of calf tbymus DNA and 50 pg of calf tbymua whole histones. The reaction contained 0.1 PCi of PH]NAD (3 mCi/pmol).
FIG. 1. Dependency on added DNA and histone for poly(ADP-ribose) synthesis in HeLa cytoplasmic extracts. The components of the reaction mixture have been given in Table I. The effect of DNA concentration on poly(ADP-ribose) formation was determined in the presence of 50 pg of calf thymus whole histones. When the histone concentration was varied, each assay tube contained 35 pg of calf thymus DNA. The reactions were carried out for 30 min at 20°C and stopped by addition of 1.0 ml of 20% trichloroacetic acid containing 5 mM sodium pyrophosphate.
used. The product of the reaction is poly(ADP-ribose) as shown by its sensitivity to snake venom phosphodiesterase, resulting in total degradation (shown below). Our studies indicate that under optimal conditions for the cytoplasmic and nuclear enzymes, the incorporation of 13HlNAD into poly(ADP-ribose) by the cytoplasmic extracts sometimes reached 50% of that observed in nuclei (Table II). Thus a significant cellular potential for poly(ADP-ribosylation) exists outside of the nucleus. Although both assays were performed with identical NAD concentrations, it should be noted that the nuclear system does not require exogenous DNA or histones. Since we and others have reported on the changes in specific activity of the nuclear
308
ROBERTS TABLE
II
PoLY(ADP-RIBOSE) POLYMERASE ACTIVITY FROM DIFFERENT CYTOPLASMIC FRACTIONS OF HELA CELLS Sample
Total activity* Cpm X W4
Experiment I, Dounce homogenization” A Postnuclear supernatant SlOOg pellet 16,000g pellet 16,OOQg supernatant B
16,OOOg supernatant 100,OOO.g supernatant 100,OOOg pellet
7.0 6.1 35.1
100 15 13 72
26.8 3.8 20.5
100 13 71
Dpm X lo-’ Experiment II, Nonidet P-40 Cell lysisc A Postnuclear supernatant 16,00&g pellet 16,OOOg supernatant B
16,OOQg supernatant 100,OOOg supernatant 100,OOQg pellet
Percent
Percent
5.20 1.99 2.80
100 36 53
1.87 0.22 0.99
100 12 53
a In Exneriment I. 3.4 x lo8 cells were fractionated according to the method of Attardi et al. (25). The polysomes were collected by centrifugation at 100,OOOg for 90 min. Fractions in TKM buffer (25) were assayed for poly(ADPribose) polymerase activity by the method described in Table I. The 0.5-ml reaction mixture contained 2 nmol of 13HINAD (0.05 &/nmol), 100 fig of calf thymus whole histones and 50 fig of calf thymus DNA. * Total activity is defined as histone- and DNA-dependent radioactivity korporated per 30 min, normalized to the total volume of the respective fractions. Using purified nuclei from each experiment, the amount of poly(ADPribose) found was 27.4 x 10’ cpm and 6.27 x lo6 dpm for Experiments I and II, respectively. However, addition of histones and DNA are not required in these assays in contrast to the data above. c In Experiment II, 7.5 x 10’ cells were lysed with Nonidet P-40 as described by Borun et al. (24). A postnuclear fraction was prepared by pelleting the nuclei at 116O.g for 5 min. The mitochondria were pelleted by centrifugation at 16,OOqS for 20 min and the polysomes were collected as above. The fractions were assayed for poly(ADP-ribose) polymerase activity as described above.
enzyme during the cell cycle (13, 14, 17, 18), such an analysis of the cytoplasmic enzyme was deemed important especially since the amount of acceptor could be stringently controlled in these assays, in contrast to the nuclear system. Cytoplasmic poly(ADP-Ribose) Polymerase Activity during the Cell Cycle HeLa cells were synchronized as described in Materials and Methods and the cell cycle monitored by 13Hlthymidine in-
ET AL.
corporation into DNA, DNA content and cell number (Fig. 2). Initial rate measurements of poly(ADP-ribose) formation were performed with postmitochondrial extracts under conditions where the incorporation of r3H]NAD into poly(ADP-ribose) was linear with respect to time and protein concentration of the extracts. The specific activity of the enzyme was highest in S phase and closely correlated with scheduled DNA synthesis. The activity declined to low levels in G2-M phase and also in early Gl phase. Specific activity of the enzyme again increased as cells progressed into a second S phase as indicated by hour 24 in Fig. 2. These data suggest the possibility that the enzyme is synthesized during S phase of the cell cycle; such coordinate activity with DNA replication would be consistent with our observations (15) that, in intact nuclei, ADP-ribosylation of nuclear proteins leads to a release of template restriction and an increase in primer sites for DNA polymerase. Localization of Poly(ADP-Ribose) Polymerase in the HeLa Cell Cytoplasm Fractionation of HeLa cells by differential centrifugation indicated that between 50 and 70% of the cytoplasmic poly(ADPribose) polymerase activity could be recovered in the postmitochondrial supernatant fraction. Similar results were obtained either when the cells were ruptured in an isotonic solution by Dounce homogenization as shown in Experiment I, Table II, or by using the detergent NP-40 to lyse the cells (Experiment II, Table II). The use of NP-40 results in minimal nuclear disruption in normal and adenovirus-infected HeLa cells (24); these latter cells have been reported to possessvery fragile nuclei. This experiment strongly indicates that the cytoplasmic form of this enzyme is not due to nuclear leakage. In addition, in our studies we could find no nuclear leakage of DNA to the cytoplasmic fractions. Approximately 60% of the recovered activity in the postmitochondrial fraction could be pelleted with the microsomes by centrifugation at 100,OOOgfor l-2 h (Table II, Experiments IB, IIB). The remaining postnuclear activity that pellets with the mito-
CYTOPLASMIC
POLYCADP-RIBOSE)
Hours
POLYMERASE
309
after Reverial
2. Cytoplasmic poly(ADP-ribose) polymerase and the HeLa cell cycle. A 1500-ml culture of HeLa cells was synchronized at the Gl-S border with methotrexate. At zero time the block was released with lo-” M thymidine, and samples were collected at various times. The incorporation of [3H]thymidine into DNA (O-O), the cell number (O-0) and DNA content per lo5 nuclei (A-A) are shown in the top panel. Postmitochondrial extracts were prepared and aliquots containing 140-180 pg of protein were assayed for poly(ADP-ribosel polymerase activity in the standard 0.5-ml assay mixture. The rate of poly(ADP-ribosel formation (disintegrations per minute per microgram of protein per minute) was used to obtain the data shown in the lower panel. FIG.
chondria has not been characterized; however, Attardi et al. (25) have pointed out that the rough endoplasmic reticulum sediments with the mitochondria in HeLa cells, and this fraction contains up to 20% of the total ribosomes in the cell. Microsomal-bound NADase has been described in Ehrlich ascites cells (32); however, HeLa cells have been reported to have minimal NADase activity (33). We assayed for NADase on microsomal fractions containing high activity For poly(ADP-ribose) polymerase utilizing the cyanide method (34) and could detect no activity for NADase in the absence or presence of DNA and histones. We therefore conclude that the microsomal-bound polymerase activity is due to an enzyme distinct from microsomal NADase. In order to characterize further the location of the enzyme, the polyribosomes were fractionated on 15-30% sucrose gradients. The data shown in Fig. 3 demonstrate that activity for poly(ADP-ribose) polymerase, showing absolute requirement for histone and DNA, was detected in all classes of ribosomes, activity being roughly proportional to the absorbance at
260 nm. Treatment of the polyribosomes with 10 mM EDTA before centrifugation completely disrupted the polysomes into monosomes and ribosomal subunits; enzyme activity remained bound to the monoribosomes and ribosomal subunits. In this context, it is of interest that Baril and colleagues (31) have reported activity for rat liver DNA polymerase I, which is usually found only in the nucleus, on cytoplasmic ribosomes. In order to test whether the activity associated with ribosomes was due to de novo synthesis of enzyme, the effects of protein inhibitors on cytoplasmic polymerase activity were examined. HeLa cells were incubated for 4.5 h with cycloheximide (0.1 mM) or puromycin (0.1 mM), and the postmitochondrial fractions were prepared. Total protein synthesis was inhibited by 95% by these antibiotics; activity of poly(ADPribose) polymerase in the postmitochondrial supernatant fractions of these cells was inhibited approximately 45%. We conclude that, while a portion of the activity in HeLa cell cytoplasm is due to de nouo synthesis of poly(ADP-ribose) polymerase, the enzyme also exists in another form
310
ROBERTS
I
I
FRACTION
NUMBER
FIG. 3. DNAand histone-dependent poly(ADPribose) polymerase activity on polysomes of HeLa cells. Approximately 4.5 x 10’ HeLa cells were lysed in TKM buffer as described by Attardi et al. (25). Polysomes were isolated on sucrose gradients as described in Materials and Methods. Fractions of 0.6 ml each were collected and assayed for poly(ADP-ribose) activity. Absorbance at 260 nm (O-O); L3H]NAD incorporated (0-O). The direction of sedimentation was from left to right. [3H]ADP-ribosylation, disintegrations per minute per A,,, unit (A-A), was also calculated.
that is tightly bound to ribosomes and ribosomal subunits. Activity for poly(ADP-ribose) polymerase can be released from ribosomes by slow sedimentation through discontinuous sucrose gradients containing 0.6 M NH&l. The solubilized enzyme fraction also required histones and DNA in a 2:l ratio for optimal activity (see Fig. 1). This solubilized enzyme was used in studies to be described below. Chain Length Determination 3H-Labeled poly(ADP-ribose) was generated with microsomal-bound enzyme and histones and subsequently purified according to the method of Yamada and Sugimura (19). The polymer was too small to be completely precipitated by 20% trichloroacetic acid, but the majority of the labeled product was excluded from Sephadex G-50,
ET AL.
eluting near the void volume of the column (Fig. 4). This property of the polymer was utilized to examine its susceptibility to enzyme digestion. The majority of the labeled product was eluted ahead of a [14C]NAD marker (Fig. 4A), but the trailing radioactivity indicated that a heterogeneous distribution of polymer sizes was present in the preparation. Preincubation of the product with snake venom phosphodiesterase reduced the size of the product (Fig. 4B) resulting in elution of the degradation products at the same position as NAD. The labeled poly(ADP-ribose) was resistant to spleen phosphodiesterase, pancreatic DNase and a mixture of pancreatic, Tl and T2 RNases (Figs. 4C-E). This behavior is exactly as expected for a polymer of poly(ADP-ribose) (36) and confirms that the cytoplasmic system possessesthe properties of a poly(ADP-ribose) polymerase. The digestion products of snake venom phosphodiesterase were examined on thin layer cellulose-polyethyleneimine plates, as described by Lehmann et al. (37). The ratio of the radioactivity of 5’-AMP and 2’(5”-phosphoribosyl)-5’-AMP (37) was used to calculate an average chain length (8) of nine residues for the products of the cytoplasmic enzyme system (Table III) under our assay conditions. Specificity of DNA and Histone for Acceptance of Poly(ADP-Ribose) The specificity requirements for DNA and histones are shown by the data in Table IV. Both sonicated and singlestranded DNA can substitute for native calf thymus DNA; at these concentrations poly(A) or poly(1) cannot substitute nor can 10~ molecular weight RNA (4-5s) (data not shown). The protein requirement is quite specific for histones and cannot be fulfilled by bovine serum albumin, by the enzyme lysozyme (molecular weight approximately equal to that of the small histones) or by poly(L-lysine). Elongation factor 2 accepts ADP-ribose from NAD as catalyzed by diphtheria toxin (38) but did not act as an acceptor of ADP-ribose from NAD in the presence or absence of histones and DNA with the cytoplasmic form of poly(ADP-ribose) polymerase. Under optimal histone and DNA concentrations,
POLY(ADP-RIBOSE)
CYTOPLASMIC
311
POLYMERASE 300
300
200 100
I-J 200 !z P ,’
I” 0 ; 300
300 100
200 2 d s loo s
200 100
0 0
5 10 I5 FR*CTION NUMBER
20
0
10 20 0 10 FRACTlON NUMBER
20
0
FIG. 4. Susceptibility of the poly(ADP-ribose) to enzymatic digestion. The product of the poly(ADP-ribose) polymerase reaction using cytoplasmic extract as a source of enzyme was purified from histones by the method of Yamada and Sugimura (19). (A), A 100-~1 sample of the tritiated product was mixed with a small quantity of [‘%]NAD and passed over a Sephadex G50 column (0.5 x 8 cm) equilibrated with 10 mM Tris-HCl, pH 7.5, with 1.0 M NaCl. Five-drop fractions were collected directly into counting vials for determination of radioactivity. (B-E), 100-~1 samples of the 3H-labeled product containing 5 mM MgCl* were preincubated with 50 pg of the appropriate enzyme for a total of 1 h at 37°C. After 30 min, an additional 50 pg of enzyme was added. The entire sample (105-110 ~1) was passed through the column, and the radioactivity was determined as before. The activity of the enzymes was verified in separate control experiments using radioactively labeled DNA or RNA under the same conditions of incubation.
TABLE CHAIN RIBOSE)
LENGTH SYNTHESIZED
Digestion
PoLY(ADP-
POLYMERASE~
product
5’-AMP b”
OF PoLY(ADP-
BY CYTOPLASMIC
RIBOSE) Ehlple
TABLE
III
DETERMINATION
2’-(5”-Phosphori-
Radioactivity kpm)
711 6243
Average chain length (1 + b/a)
9.1
bosyl)-5’-AMP n 3H-labeled poly(ADP-ribose) was generated on histones, purified, and digested with snake venom phosphodiesterase (Fig. 4). The digestion products were mixed with 0.1 pm01 each of 5’-AMP and ADPR as markers and spotted on a 20 x 20-cm PEI plate. The plate was developed in two dimensions with 0.9 N acetic acid/O.3 M LiCl (37). The positions of the markers were detected with a uv light source and the plates were cut into 2 x 2-cm squares. After eluting the squares with 0.3 ml of 1.6 M LiCI, the radioactivity in each square was determined.
our data indicate that, while most histones act to accept poly(ADP-ribose), certain selectivity is evident. Lysine-rich histone Fl as isolated by the method of Johns (26) shows very high acceptor activity, whereas histone Fl isolated by the method of DeNooij and Westonbrink (39) is less active. The order of specificity for stimulation by the histones (50 pg per assay) was: Whole = Fl(26) > F3 > Fl(39) > F2a > F2b. The
SPECIFICITY
OF THE
REQUIREMENTS POLYMERASE IN THE Components
IV DNA
AND
HISTONE
FOR PoLY(ADP-RIBOSE) CYTOPLASM OF HELA
in assay
Radioactivity counts per minute
Complete mixture Complete mixture + nicotinamide (20 InM) - native DNA + heat denatured DNA - native DNA + sonicated DNA - whole histones + histone Fl - whole histones + bovine serum albumin - whole histones + lysozyme - whole histones + poly+lysine
CELLS”
Percent
560 34
100 6
506
90
569 569 82
102 105 15
68 33
16 6
a The complete 0.5-ml reaction mixture contained 50 pm01 of Tris-HCI, pH 7.4, 1 ymol of M&l%, 0.5 pm01 of dithiothreitol, 1 pCi of L3HINAD (0.5 &ilrmol), 35 pg of calf thymus DNA and 100 pg of calf thymus whole histones. Where other substitutions were made in the protocol, the final concentrations of DNA and protein were 35 and 100 pg, respectively. The reaction was started by addition of 40 ~1 of a postnuclear extract prepared from cells lysed at 1 x lo8 cells/ml. The reaction was incubated 30 min at 20°C before stopping the reaction with 1.0 ml of 20% trichloroacetic acid containing 5 nm~ sodium pyrophosphate.
requirement for DNA in this system was quite selective: DNase II-treated (all hydrolysis was 25%) DNA > DNase I-treated
312
ROBERTS
DNA > micrococcal nuclease-treated DNA >> denatured DNA = sonicated DNA = native DNA > apurinic DNA. Covalent Attachment of Poly(ADP-Ribose) to Histone Fl Because there has been some question as to whether poly(ADP-ribose) is covalently attached to histones, it was of importance to demonstrate that the partially purified enzyme from ribosomes formed a covalent bond between chains of ADP-ribose units and the histone acceptor. Radioactive [14Clhistone Fl was isolated by the method of Johns from nuclei derived from cells incubated with radioactive amino acids (26). This radioactively la-
ET
AL.
beled preparation was homogeneous on polyacrylamide-gel electrophoresis (Fig. 5A). 13H1NAD was used as a substrate for the partially purified cytoplasmic enzyme washed off ribosomes by NH,Cl to generate 3H-labeled poly(ADP-ribose) chains on the [14Clhistone Fl. This incubation mixture was chromatographed on a Bio-Rex 70 cation-exchange column, as indicated by the data in Fig. 5B. It should be noted that the free r3H]NAD was eluted directly through this column. A peak of 3H-labeled poly(ADP-ribose) appears in fractions 1217 which is coincidential with the peak of [14Clhistone Fl. No association was observed when [3H]NAD and [14C]histone Fl were mixed in the absence of enzyme and
FIG. 5. [3H]ADP-ribosylation of purified [Wlhistone Fl. (A), Electropherogram obtained by running an aliquot of purified [‘4C]histone (Materials and Methods) and commercial histone Fl as carrier on a polyacrylamide gel containing sodium dodecyl sulfate. Migration was from left to right, toward the anode. The optical density profile of stained protein bands is indicated by the solid line. Radioactivity in each 1.5mm slice is indicated by the bar graph. (B), lL4C]histone Fl was used as acceptor for 3H-labeled poly(ADP-ribose) as catalyzed by the cytoplasmic enzyme under conditions given in the footnote to Table I. The reaction was terminated by addition of an equal volume of 14% guanidine hydrochloride and the mixture subjected to chromatography on a Bio-Rex 70 column (Materials and Methods). Beginning with fraction 11, the guanidine hydrochloride concentration was increased from 7 to 14%. Aliquots of each fraction were counted for radioactivity. Fractions 12-17 were pooled, dialyzed against water and lyophilized. (Cl, The pooled column fractions were subjected to electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. The gels were sliced, the radioactivity in each slice determined and the data corrected for ‘*C spillover into the tritium channel. The distribution of 3H and W radioactivity is shown. The arrows at the top of the profile show the positions to which the five main histone fractions migrate under these conditions of electrophoresis. The direction of migration was from left to right, toward the anode.
CYTOPLASMIC
POLYCADP-RIBOSE)
applied to the column. In addition, the inclusion of nicotinamide in the complete reaction mixture reduced the amount of 3H-labeled poly(ADP-ribose) that was eluted with [14C]histone Fl. To prove that the chain of poly(ADP-ribose) was covalently attached to histone Fl, fractions 1217 from the Bio-Rex column were pooled and, after dialysis, subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (Fig. 5C). Again, there was strict coincidence between radioactivity from poly(ADP-ribose) and histone Fl. Treatment of the reaction mixture with snake venom phosphodiesterase reduced the amount of 3H-labeled poly(ADP-ribose) that was bound to [14C]histone Fl by only 25%. This result suggests that the polymer was short and that the histone conferred some protection on the polymer as also suggested by the work of others (2). Hence, chains generated by the solubilized enzyme are apparently shorter than those generated by the microsomal-bound enzyme (Table III). This result would explain why the poly(ADP-ribosylated) histone corn&-rates with free histone during chromatography on ion-exchange columns and during electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. DISCUSSION
Our results show that cytoplasmic extracts of HeLa cells possess an enzyme that can form poly(ADP-ribose) from the substrate molecule NAD. Numerous reports have shown that poly(ADP-ribose) polymerase is located in nuclei tightly bound to chromatin (l-3). Most of the cytoplasmic activity for this enzyme was found tightly associated with various ribosomal particles and subunits (Fig. 3). A portion of this bound activity was shown to be due to biosynthesis of the enzyme. However, a more functional role on the ribosome might also be indicated. In contrast to chromatin-bound poly(ADP-ribose) polymerase, the cytoplasmic form requires exogenous DNA and histones, and a 30-fold increase in polymer formation is observed when the reaction is supplemented with the optimal concentrations of these molecules (Table I). The present report clarifies
POLYMERASE
313
the role of histones as acceptors in the poly(ADP-ribose) reaction; strong evidence is provided for a covalent attachment between poly(ADP-ribose) and histone Fl (Fig. 5). A variety of observations indicate that the cytoplasmic form of poly(ADP-ribose) polymerase is not merely reflective of nuclear leakage of the chromatin-bound form of the enzyme. For example, the amount of activity found for this enzyme in the cytoplasm was substantial; in some cases the total potential activity to generate poly(ADP-ribose) in the cytoplasm of cells approached 50% of the total potential in the nuclei of these cells whether nuclei were assayed directly (by using the endogenous DNA and histone acceptors) or whether exogenous DNA and histones were added to the nuclei. In addition, no significant leakage of DNA out the the nucleus was observed. Other workers have not detected poly(ADP-ribose) polymerase in the cytoplasm, presumably because additions of DNA and histones have not been made. Gill has reported poly(ADP-ribose) polymerase in soluble extracts from whole animal cells (including nuclei), but the enzyme was complexed with nucleohistone and required addition of neither DNA nor histone for activity (43). Secondly, a variety of mild cell fractionation procedures have all yielded identical specific activity for the cytoplasmic enzyme. These procedures range from vigorous Dounce homogenization in a hypotonic solution (23) to gentle lysis with the nonionic detergent NP-40 in which no homogenization is used (24). Finally, the activity of cytoplasmic poly(ADP-ribose) polymerase was shown to fluctuate during the phases of the cell cycle; the specific activity of the enzyme was especially low in those periods of the cycle (G2-M) where nuclei have particularly fragile membranes. If the activity in the cytoplasm were purely due to nuclear leakage one would have expected activity to be high instead of low in the cytoplasm during these phases of the cell cycle (Fig. 2). Our report of a cytoplasmic form of poly(ADP-ribose) polymerase is analogous
314
ROBERTS
to observations that other “nuclear enzymes” have been detected in the cytoplasms of eukaryotic cells. An enzyme system responsible for synthesizing a polyadenylate homopolymer onto the 3’-end of heterogeneous nuclear RNA has been found in the cytoplasm of virally infected mouse L-cells (45) in addition to its usual location in the nucleus. Also, DNA polymerases have been detected in the cytoplasm of a number of eukaryotic cells. Baril and colleagues (31) have shown that the small nuclear DNA polymerase (DNA polymerase I) of rat liver is also found on cytoplasmic ribosomes. In rat liver cells, DNA polymerase II is associated with microsomal membranes, and it is this enzyme that fluctuates in a parallel fashion with the level of DNA synthesis in uiuo (31). In synchronized HeLa cells the specific activity of this latter cytoplasmic enzyme is highest in S phase of the cell cycle, and activity in extracts parallels the rate of thymidine incorporation in the intact cell (44). It is interesting that the activity of cytoplasmic poly(ADP-ribose) polymerase behaves in a similar fashion and does parallel changes in DNA synthesis during the cell cycle (Fig. 2). We have recently reported that ADP-ribosylation of HeLa nuclear proteins results in increased accessibility of Escherichia coli DNA polymerase I for primer sites in nuclei (15). Synthesis of histones in the cell cytoplasm also parallels the rate of DNA synthesis, and these histones are then transported into the nucleus (42). Additional experiments will have to be performed to discern if a portion of the high-activity poly(ADP-ribose) polymerase observed in S phase of the cell cycle (Fig. 2) is also eventually transported into the nucleus with histones. Such a coupled mechanism might account for a relationship between this nuclear protein-modifying system bound to ribosomes and the need for transport of nuclear proteins from their site of synthesis to their site of function in the nucleus. ACKNOWLEDGMENTS We thank Dr. Oeschker, Dr. 0. Gabriel town University) and Dr. M. Cashel (NIH) cally reviewing this manuscript. This work tially supported by U.S. Public Health Grants No. CA13195 and CA11950.
(Georgefor critiwas parService
ET
AL
REFERENCES 1. HONJO, T., AND HAYAISHI, 0. (1973) in Current Topics in Cellular Regulation (Horecker, B. L., and Stadtman, E. R., eds.), Vol. 7, pp. 87127, Academic Press, New York. 2. SUGIMURA, T. (1973) in Progress in Nucleic Acid Research and Molecular Biology (Davidson, J. N., and Cohn, W. E., eds.), Vol. 13, pp. 127151, Academic Press, New York. 3. PoIy(ADP-Ribose): An International Symposium, Forgarty International Center Proceeding No. 26, U.S. Government Printing Office, Washington, D.C. 4. NISHIZUKA, Y., UEDA, K., YOSHIHARA, K., YAMAMURA, H., TAKEDA, M., AND HAYAISHI, 0. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, 781-785. 5. OTAKE, H., MIWA, M., FUJIMURA, S., AND SUGIMURA, T. (1969) J. Biochem. (Tokyo) 65, 145146. 6. BURZIO, L., AND KOIDE, S. S. (1972) FEBS Lett. 20, 29-32. 7. NISHIZUKA, Y., UEDA, K., AND HAYAISHI, 0. (1971) in Methods in Enzymology, eds. (McCormick, D. B., and Wright, L. D., eds.), Vol. 18B, pp. 230-233, Academic Press, New York. 8. YAMADA, M., MIWA, M., AND SUGIMURA, T. (1971) Arch. Biochem. Biophys. 146, 579-586. 9. YOSHIHARA, K. (1972) Biochem. Biophys. Res. Commun. 47, 119-125. 10. BURZIO, L., AND KOIDE, S. S. (1970) Biochem. Biophys. Res. Commun. 40, 1013-1020. 11. BURZIO, L., AND KOIDE, S. S. (1971) Biochem. Biophys. Res. Commun. 42, 1185-1190. 12. NAGAO, M., YAMADA, M., MIWA, M., AND SUGIMURA, T. (1972) Biochem. Biophys. Res. Commun. 48,219-225. 13. SMULSON, M., HENRIKSEN, O., AND RIDEAU, C. (1971) Biochem. Biophys. Res. Commun. 43, 1266-1273. 14. ROBERTS, J. H., STARK, P., AND SMULSON, M. (1973) Biochem. Biophys. Res. Commun. 52, 43-49. 15. ROBERTS, J. H., STARK, P., AND SMULSON, M. (1974) Proc. Nat. Acad. Sci. USA 71, 32133216. 16. SMULSON, M., STARK, P., GAZZOLI, M., ANDROBERTS, J. H. (1975) Exp. Cell Res. 90, 175-182. 17. SOLAO, P. B., AND SHALL, S. (1971). Exp. Cell Res. 69, 295-300. 18. MIWA, M., SUGIMURA, T., IRVI, N., AND TAKAYANA, S. (1973) Cancer Res. 33, 1306-1315. 19. YAMADA, M., AND SUGIMURA, T. (1973) Biochemistry 12, 3303-3308. 20. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (19505. Biol. Chem. 193, 265-275. 21. BURTON, K. (1956) Biochem. J. 62, 315-323. 22. SMULSON, M., AND THOMAS, J. (1969) J. Biol. Chem. 244, 5309-5312.
CYTOPLASMIC
POLY(ADP-RIBOSE)
23. SPORN, M. B., BERKOWITZ, D. M., GLINKI, R. P., ASH, A. B., AND STEVENS, C. L. (1969) Science 164, 1408-1410. 24. BORUN, T. W., SCHARFF, M. D., AND ROBBINS, E. (1967)Biochim. Biophys. Actu 149,302-304. 25. ATTARDI, B., CRAVIOTO, B., AND ATTARDI, G. (1969) J. Mol. Biol. 44, 47-70. 26. JOHNS, E. W. (1964) Biochem. J. 92, 55-59. 27. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-4412. 28. PREISS, J., SCHLAEGER, R., AND HILZ, H. (1971) FEBS Lett. 19, 244-246. 29. MIWA, M., AND SLIGIMURA, T. (1971) J. Biol. Chem. 246, 6362-6364. 30. UEDA, K., OKA, J., NARUNIZA, S., MIYAKANA, N., AND HAYAISHI, 0. (1972) Biochem. Biophys. Res. Commun. 46, 516-523. 31. BARIL, E. F., JENKINS, M. D., BROWN, 0. E., LASZLO, J., AND MORRIS, H. P. (1973) Cancer Res. 33, 1187-1195. 32. GREER, S., AND BODARSKY, 0. (1965) J. Biol. Chem. 240, 2574-2579. 33. HILZ, H., AND KITTER, M. (1971) Hoppe-Seyler’s 2. Physiol. Chem. 352, 1693-1704. 34. ZATMAN, L. J., KAPLAN, N. O., AND COLOWICK,
POLYMERASE
315
S. P. (1953) J. Biol. Chem. 200, 197-212. 35. SHAFRITZ, D. A. AND ANDERSON, W. F. (1960) J. Biol. Chem. 245, 5553-5559. 36. NISHIZUKA, Y., UEDA, K., NAKAZAWA, K., AND HAYAISHI, 0. (1967) J. Biol. Chem. 242,31643171. 37. LEHMANN, A. R., KIRK-BELL, S., SHALL, S., AND WHISH, W. J. D. (1974) Ezp. Cell Res. 83, 6372. 38. HONJO, T., NISHIZUKA, Y., HAYAISHI, O., AND KATA, I. (1968)J. Biol. Chem. 243,3553-3555. 39. DENOOIJ, E. H., AND WESTONBRINK, H. G. K. (1962) Biochim. Biophys. Actu 62, 608-609. 40. GURLEY, L. R., WALTERS, R. A., AND ENCER, M. D. (1970) Biochem. Biophys. Res. Commun. 40, 428-436. 41. UEDA, K., REEDER, R. H., HONJO, T. NISHIZUKA, Y., AND HAYAISHI, 0. (1968) Biochem. Biophys. Res. Commun. 31, 379-385. 42. ROBBINS, E., AND BORUN, T. W. (1967) Proc. Nat. Acad. Sci. USA 57, 409-416. 43. GILL, D. M. (1972) J. Biol. Chem. 247, 59645971. 44. CHIU, R., AND BARIL, E. (1974). Fed. Proc. 33, 1281.
OF
BIOCHEMISTRY
Cytoplasmic JERRY Department
AND
171,
Poly(ADP-Ribose)
H. ROBERTS2 of Biochemistry,
BIOPHYSICS
PATRICIA Schools
305-315
(197%
Polymerase Cycle1
during
STARK, CHANDRAKANT SMULSON
of Medicine Received
and Dentistry, 20007 April
Georgetown
the HeLa Cell P. GIRL
University,
AND
MARK
Washington,
D.C.
24, 1975
Poly(ADP-ribose) polymerase, an enzyme that has reportedly been confined to the nucleus of eukaryotic cells, has been found in the cytoplasm of HeLa cells. The enzyme activity is stimulated more than 30-fold by the addition of both DNA and histones. These two macromolecules are absolutely necessary for maximal activity and they act in a synergistic manner. The product of the reaction was characterized as poly(ADP-ribose) by its acid insolubility, its insensitivity to hydrolysis by DNase, RNase, spleen phosphodiesterase or Pronase and by release of 5’-AMP and 2’-(5”-phosphoribosyl)-5’-AMP by incubation with snake venom phosphodiesterase. A covalent attachment between histone PI and poly(ADP-ribose) has been established by using the cytoplasmic enzyme. The enzyme is primarily associated with ribosomes, both free ribosomes and those found in polysomes. Inhibition of protein synthesis in the intact cell reduced the level of activity in the cytoplasm. The enzyme can be removed from the ribosomes by centrifugation through sucrose gradients containing 0.6 M ammonium chloride. A relationship between this enzyme and DNA replication is suggested by the fact that the specific activity in the cytoplasm parallels the rate of DNA synthesis during the HeLa cell cycle.
The enzyme poly(ADP-ribose) polymerase catalyzes the formation of a homopolymer of ADP-ribose units linked by l’-2’ glycosidic bonds (for reviews, see Ref. (l3)). The substrate for the reaction is NAD, and the enzyme successively adds ADPribose units onto an initial ADP-ribose residue that has been reported to be covalently attached to various nuclear proteins (4-6). The enzyme is found tightly bound to chromatin and has been detected in a large number of eukaryotic cells but has not been detected in prokaryotic organisms (7). Partially purified poly(ADP-ribose) polymerase from rat liver nuclei has been shown to have an absolute requirement for both DNA and histones for elongation of ADP-ribose units (8, 9). ’ A preliminary report of this work was given at an International Seminar on Poly(ADP-Ribose) and ADP-Ribosylation of Protein, held September 10-12, 1974, Tomakomai, Japan. * Present address: Department of Biology, The Catholic University of America, Washington, D.C. 20064. 305 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
A number of proposals have been made suggesting a role for poly(ADP-ribose) polymerase in the regulation of eukaryotic DNA replication or repair (10-13). Work from various laboratories indicates that formation of poly(ADP-ribose) caused perturbations of chromatin structure which led to either activation (14) or restriction of chromatin function with respect to DNA synthesis depending upon the source of tissue used (10-12). We have recently reported that the formation of poly(ADP-ribose) in HeLa cell nuclei or chromatin releases template restriction for DNA synthesis; in modified nuclei we observed two to three times more binding sites for Escherichia coli DNA polymerase I than in nonmodified nuclei (15). This effect was inversely related to the extent of natural template restriction during asynchronous growth (15) as well as during various phases of the HeLa cell cycle (16). In addition, we and others have attempted to relate the specific activity of the nuclear enzyme as a function of the cell cycle to its
ROBERTS
306
possible biological role in the intact cell (13, 17, 18). Interpretation of these data has been difficult because of the complexity of this enzyme system in the intact nuclei. In such assays the nuclear material serves as: (i) A source of various polymerizing enzymes and DNA which are required for elongation of poly(ADP-ribose) (19), and (ii) various histones and nor&stone acceptors for the polymer that may contain varying levels of endogenous polymer which had been synthesized in vivo. Any one of these variables could account for differences in specific activity, and hence interpretation of cell cycle data is quite complicated. In an attempt to eliminate these variables, in the present study we investigated the biosynthesis of this enzyme on cytoplasmic ribosomes during the time sequences of the cell cycle. This activity allows a convenient in vitro assay that shows strict dependence for activity on both histones and DNA, in contrast to the nuclear or chromatin assay, and hence better allows us to investigate the relationship of this enzyme to events of the cell cycle. MATERIALS
AND
METHODS
Materials. Calf thymus DNA, whole histones, and histone fractions were obtained from Sigma Chemical Co.; ribonuclease Tl and T2 were obtained from Calbiochem; pancreatic RNase, DNase I, snake venom phosphodiesterase, and spleen phosphodiesterase were purchased from Worthington Biochemical Corp.; [3HlNAD (3.7 Ci/mmol) was purchased from New England Nuclear. Assays. The reaction mixture for the poly(ADPribose) polymerase assay contained in a volume of 0.5 ml: 50 pmol of Tris-HCl buffer, pH 7.4, 0.5 pmol of dithiothreitol, 1.0 pmol of MgC& and VHINAD, DNA and histones as indicated in each experiment. The assays were performed at room temperature and terminated after 30 min by the addition of 1.0 ml of 20% trichloroacetic acid containing 5 mM sodium pyrophosphate. Insoluble material was collected on Whatmann GF/C filter disks, and the radioactivity was determined in a Packard scintillation counter. The assay for nuclear poly(ADP-ribose) polymerase was performed as previously described (13). Protein was determined by the method of Lowry et al. (20). DNA was determined by the method of Burton (21). Growth of cells and preparation of extracts. HeLa S3 cells were maintained at 37°C in spinner flasks in
ET
AL
Eagle’s spinner medium (22) containing 10% fetal calf serum. Cells were harvested by centrifugation and washed with spinner salts before being broken. The following three methods have been used to disrupt the cells: (i) Dounce homogenization according to the method of Sporn et al. (23); (ii) utilization of the detergent NP-403 as described by Borun et al. (24); and (iii) the method of Attardi et al. (25). Nuclei were collected by centrifugation at 115Og for 5 min. Postmitochondrial supernatant fractions were collected by centrifugation at 16,OOOg for 20 min. Cell synchronization. HeLa cells at 3 x lo5 cells/ml were blocked at the Gl-S boundary by addition of methotrexate (230 pgiliter). After 16 h, the block was reversed by addition of a solution containing 10 pM thymidine (16). The rate of DNA synthesis was determined by measuring the amount of 13H1thymidine incorporated into trichloroacetic acid-insoluble material by 2-ml samples of cells in 15 min. At specific times during the cell cycle, samples were collected for subsequent determination of DNA content and poly(ADP-ribose) polymerase activity in the postmitochondrial extracts. Preparation of polysomes, monoribosomes and ribosomal subunits. Samples of postmitochondrial fractions were layered on top of 15-30% sucrose gradients (17 ml) which were formed over a 64% sucrose cushion. The gradients were centrifuged at 4°C in an SW 27 rotor for 2-5 h (for polysome patterns) or 18 h (for monoribosomes and ribosomal subunit patterns). The gradients were analyzed by using an ISCO density gradient fractionator and uv-absorbante monitor. Poly(ADP-ribose) polymerase was removed from ribosomes as follows: The ribosomal pellet was resuspended in 0.1 mM EDTA, 1.0 mM dithiothreitol and 0.25 M sucrose and then made 0.6 M with respect to NH&l. Samples of 0.5 ml were layered on top of a discontinuous gradient prepared by layering 2.0 ml of 0.5 M sucrose over 2.0 ml of 1.0 M sucrose, both containing dithiothreitol, EDTA and 0.6 M NH&l. The gradients were centrifuged at 4°C in a Type 40 rotor (Beckman) for 20 h at 28,000 rpm to reisolate the washed ribosomes. The supernatant fluid was dialyzed against 50 mM Tris-HCl buffer, pH 7.2, containing 1 mM dithiothreitol. ADP-ribosylation of purified [‘Wtistone Fl. HeLa cells were labeled with a 14C-amino acid mixture for three generations, nuclei prepared and histone Fl purified by the method of Johns (26). An aliquot of the labeled material was mixed with commercially available histone Fl carrier, characterized by electrophoresis according to the method of Weber and Osborn (271, and utilized as acceptor with the poly(ADP-ribose) polymerase separated from ribosomes (see above), as described in the legend to Fig. 5. 3 Abbreviation
used:
NP-40,
Nonidet
P-40.
CYTOPLASMIC
POLY(ADP-RIBOSE)
POLYMERASE
307
RESULTS
Cytoplasmic ase
Poly(ADP-Ribose)
Polymer-
The postnuclear extract prepared from asynchronous cultures of HeLa cells contained little poly(ADP-ribose) polymerase activity when assayed by the standard procedure (Table I) used to assay the nuclear enzyme. This finding agrees with other reports demonstrating that poly(ADP-rihose) polymerase is confined to the nuclei of eukaryote cells (l-3). However, if DNA and whole histones were included in the reaction, the incorporation of 13HlNAD into material insoluble in 20% trichloroacetic acid was stimulated 30-fold (Table I). The incorporation was inhibited by nicotinamide and by thymidine, which are competitive inhibitors of poly(ADP-ribose) polymerase (28). The data in Fig. 1 demonstrate that the amount of poly(ADP-ribose) formed displays concentration dependence on both histones and DNA; the formation of poly(ADP-ribose) is stimulated by DNA and histone synergistically. When both DNA and histone are present, the amount of poly(ADP-ribose) formed is almost three-fold greater than that expected by the sum of the single additions. When the optimal concentrations of DNA and histones were utilized, after a short lag period (2-3 min), incorporation of [3H]NAD into poly(ADP-ribose) was linear with respect to time for at least 40 min and was also proportional to the quantity of extract TABLE PoLYCADP-RIBOSE) FRACTION
I
SYNTHESIS
IN
OF HELA
Sample additions
Complete, zero time Complete, 30 min, 20°C Complete, 30 min, 20°C + nicotinamide (20 mM) Complete, 30 min, 20°C + thymidine (10 mM)
THE
POSTNUCLEAR
CELLS”
Control @pm)
+DNA, +Histone (dpm)
120 370 119
91 10,290 164
115
170
’ Cells were broken by the method of Sporn et al. (23) and the postnuclear fraction isolated. The reaction was performed as described in Materials and Methods and contained 230 pg of protein and, where indicated, 50 Kg of calf tbymus DNA and 50 pg of calf tbymua whole histones. The reaction contained 0.1 PCi of PH]NAD (3 mCi/pmol).
FIG. 1. Dependency on added DNA and histone for poly(ADP-ribose) synthesis in HeLa cytoplasmic extracts. The components of the reaction mixture have been given in Table I. The effect of DNA concentration on poly(ADP-ribose) formation was determined in the presence of 50 pg of calf thymus whole histones. When the histone concentration was varied, each assay tube contained 35 pg of calf thymus DNA. The reactions were carried out for 30 min at 20°C and stopped by addition of 1.0 ml of 20% trichloroacetic acid containing 5 mM sodium pyrophosphate.
used. The product of the reaction is poly(ADP-ribose) as shown by its sensitivity to snake venom phosphodiesterase, resulting in total degradation (shown below). Our studies indicate that under optimal conditions for the cytoplasmic and nuclear enzymes, the incorporation of 13HlNAD into poly(ADP-ribose) by the cytoplasmic extracts sometimes reached 50% of that observed in nuclei (Table II). Thus a significant cellular potential for poly(ADP-ribosylation) exists outside of the nucleus. Although both assays were performed with identical NAD concentrations, it should be noted that the nuclear system does not require exogenous DNA or histones. Since we and others have reported on the changes in specific activity of the nuclear
308
ROBERTS TABLE
II
PoLY(ADP-RIBOSE) POLYMERASE ACTIVITY FROM DIFFERENT CYTOPLASMIC FRACTIONS OF HELA CELLS Sample
Total activity* Cpm X W4
Experiment I, Dounce homogenization” A Postnuclear supernatant SlOOg pellet 16,000g pellet 16,OOQg supernatant B
16,OOOg supernatant 100,OOO.g supernatant 100,OOOg pellet
7.0 6.1 35.1
100 15 13 72
26.8 3.8 20.5
100 13 71
Dpm X lo-’ Experiment II, Nonidet P-40 Cell lysisc A Postnuclear supernatant 16,00&g pellet 16,OOOg supernatant B
16,OOQg supernatant 100,OOOg supernatant 100,OOQg pellet
Percent
Percent
5.20 1.99 2.80
100 36 53
1.87 0.22 0.99
100 12 53
a In Exneriment I. 3.4 x lo8 cells were fractionated according to the method of Attardi et al. (25). The polysomes were collected by centrifugation at 100,OOOg for 90 min. Fractions in TKM buffer (25) were assayed for poly(ADPribose) polymerase activity by the method described in Table I. The 0.5-ml reaction mixture contained 2 nmol of 13HINAD (0.05 &/nmol), 100 fig of calf thymus whole histones and 50 fig of calf thymus DNA. * Total activity is defined as histone- and DNA-dependent radioactivity korporated per 30 min, normalized to the total volume of the respective fractions. Using purified nuclei from each experiment, the amount of poly(ADPribose) found was 27.4 x 10’ cpm and 6.27 x lo6 dpm for Experiments I and II, respectively. However, addition of histones and DNA are not required in these assays in contrast to the data above. c In Experiment II, 7.5 x 10’ cells were lysed with Nonidet P-40 as described by Borun et al. (24). A postnuclear fraction was prepared by pelleting the nuclei at 116O.g for 5 min. The mitochondria were pelleted by centrifugation at 16,OOqS for 20 min and the polysomes were collected as above. The fractions were assayed for poly(ADP-ribose) polymerase activity as described above.
enzyme during the cell cycle (13, 14, 17, 18), such an analysis of the cytoplasmic enzyme was deemed important especially since the amount of acceptor could be stringently controlled in these assays, in contrast to the nuclear system. Cytoplasmic poly(ADP-Ribose) Polymerase Activity during the Cell Cycle HeLa cells were synchronized as described in Materials and Methods and the cell cycle monitored by 13Hlthymidine in-
ET AL.
corporation into DNA, DNA content and cell number (Fig. 2). Initial rate measurements of poly(ADP-ribose) formation were performed with postmitochondrial extracts under conditions where the incorporation of r3H]NAD into poly(ADP-ribose) was linear with respect to time and protein concentration of the extracts. The specific activity of the enzyme was highest in S phase and closely correlated with scheduled DNA synthesis. The activity declined to low levels in G2-M phase and also in early Gl phase. Specific activity of the enzyme again increased as cells progressed into a second S phase as indicated by hour 24 in Fig. 2. These data suggest the possibility that the enzyme is synthesized during S phase of the cell cycle; such coordinate activity with DNA replication would be consistent with our observations (15) that, in intact nuclei, ADP-ribosylation of nuclear proteins leads to a release of template restriction and an increase in primer sites for DNA polymerase. Localization of Poly(ADP-Ribose) Polymerase in the HeLa Cell Cytoplasm Fractionation of HeLa cells by differential centrifugation indicated that between 50 and 70% of the cytoplasmic poly(ADPribose) polymerase activity could be recovered in the postmitochondrial supernatant fraction. Similar results were obtained either when the cells were ruptured in an isotonic solution by Dounce homogenization as shown in Experiment I, Table II, or by using the detergent NP-40 to lyse the cells (Experiment II, Table II). The use of NP-40 results in minimal nuclear disruption in normal and adenovirus-infected HeLa cells (24); these latter cells have been reported to possessvery fragile nuclei. This experiment strongly indicates that the cytoplasmic form of this enzyme is not due to nuclear leakage. In addition, in our studies we could find no nuclear leakage of DNA to the cytoplasmic fractions. Approximately 60% of the recovered activity in the postmitochondrial fraction could be pelleted with the microsomes by centrifugation at 100,OOOgfor l-2 h (Table II, Experiments IB, IIB). The remaining postnuclear activity that pellets with the mito-
CYTOPLASMIC
POLYCADP-RIBOSE)
Hours
POLYMERASE
309
after Reverial
2. Cytoplasmic poly(ADP-ribose) polymerase and the HeLa cell cycle. A 1500-ml culture of HeLa cells was synchronized at the Gl-S border with methotrexate. At zero time the block was released with lo-” M thymidine, and samples were collected at various times. The incorporation of [3H]thymidine into DNA (O-O), the cell number (O-0) and DNA content per lo5 nuclei (A-A) are shown in the top panel. Postmitochondrial extracts were prepared and aliquots containing 140-180 pg of protein were assayed for poly(ADP-ribosel polymerase activity in the standard 0.5-ml assay mixture. The rate of poly(ADP-ribosel formation (disintegrations per minute per microgram of protein per minute) was used to obtain the data shown in the lower panel. FIG.
chondria has not been characterized; however, Attardi et al. (25) have pointed out that the rough endoplasmic reticulum sediments with the mitochondria in HeLa cells, and this fraction contains up to 20% of the total ribosomes in the cell. Microsomal-bound NADase has been described in Ehrlich ascites cells (32); however, HeLa cells have been reported to have minimal NADase activity (33). We assayed for NADase on microsomal fractions containing high activity For poly(ADP-ribose) polymerase utilizing the cyanide method (34) and could detect no activity for NADase in the absence or presence of DNA and histones. We therefore conclude that the microsomal-bound polymerase activity is due to an enzyme distinct from microsomal NADase. In order to characterize further the location of the enzyme, the polyribosomes were fractionated on 15-30% sucrose gradients. The data shown in Fig. 3 demonstrate that activity for poly(ADP-ribose) polymerase, showing absolute requirement for histone and DNA, was detected in all classes of ribosomes, activity being roughly proportional to the absorbance at
260 nm. Treatment of the polyribosomes with 10 mM EDTA before centrifugation completely disrupted the polysomes into monosomes and ribosomal subunits; enzyme activity remained bound to the monoribosomes and ribosomal subunits. In this context, it is of interest that Baril and colleagues (31) have reported activity for rat liver DNA polymerase I, which is usually found only in the nucleus, on cytoplasmic ribosomes. In order to test whether the activity associated with ribosomes was due to de novo synthesis of enzyme, the effects of protein inhibitors on cytoplasmic polymerase activity were examined. HeLa cells were incubated for 4.5 h with cycloheximide (0.1 mM) or puromycin (0.1 mM), and the postmitochondrial fractions were prepared. Total protein synthesis was inhibited by 95% by these antibiotics; activity of poly(ADPribose) polymerase in the postmitochondrial supernatant fractions of these cells was inhibited approximately 45%. We conclude that, while a portion of the activity in HeLa cell cytoplasm is due to de nouo synthesis of poly(ADP-ribose) polymerase, the enzyme also exists in another form
310
ROBERTS
I
I
FRACTION
NUMBER
FIG. 3. DNAand histone-dependent poly(ADPribose) polymerase activity on polysomes of HeLa cells. Approximately 4.5 x 10’ HeLa cells were lysed in TKM buffer as described by Attardi et al. (25). Polysomes were isolated on sucrose gradients as described in Materials and Methods. Fractions of 0.6 ml each were collected and assayed for poly(ADP-ribose) activity. Absorbance at 260 nm (O-O); L3H]NAD incorporated (0-O). The direction of sedimentation was from left to right. [3H]ADP-ribosylation, disintegrations per minute per A,,, unit (A-A), was also calculated.
that is tightly bound to ribosomes and ribosomal subunits. Activity for poly(ADP-ribose) polymerase can be released from ribosomes by slow sedimentation through discontinuous sucrose gradients containing 0.6 M NH&l. The solubilized enzyme fraction also required histones and DNA in a 2:l ratio for optimal activity (see Fig. 1). This solubilized enzyme was used in studies to be described below. Chain Length Determination 3H-Labeled poly(ADP-ribose) was generated with microsomal-bound enzyme and histones and subsequently purified according to the method of Yamada and Sugimura (19). The polymer was too small to be completely precipitated by 20% trichloroacetic acid, but the majority of the labeled product was excluded from Sephadex G-50,
ET AL.
eluting near the void volume of the column (Fig. 4). This property of the polymer was utilized to examine its susceptibility to enzyme digestion. The majority of the labeled product was eluted ahead of a [14C]NAD marker (Fig. 4A), but the trailing radioactivity indicated that a heterogeneous distribution of polymer sizes was present in the preparation. Preincubation of the product with snake venom phosphodiesterase reduced the size of the product (Fig. 4B) resulting in elution of the degradation products at the same position as NAD. The labeled poly(ADP-ribose) was resistant to spleen phosphodiesterase, pancreatic DNase and a mixture of pancreatic, Tl and T2 RNases (Figs. 4C-E). This behavior is exactly as expected for a polymer of poly(ADP-ribose) (36) and confirms that the cytoplasmic system possessesthe properties of a poly(ADP-ribose) polymerase. The digestion products of snake venom phosphodiesterase were examined on thin layer cellulose-polyethyleneimine plates, as described by Lehmann et al. (37). The ratio of the radioactivity of 5’-AMP and 2’(5”-phosphoribosyl)-5’-AMP (37) was used to calculate an average chain length (8) of nine residues for the products of the cytoplasmic enzyme system (Table III) under our assay conditions. Specificity of DNA and Histone for Acceptance of Poly(ADP-Ribose) The specificity requirements for DNA and histones are shown by the data in Table IV. Both sonicated and singlestranded DNA can substitute for native calf thymus DNA; at these concentrations poly(A) or poly(1) cannot substitute nor can 10~ molecular weight RNA (4-5s) (data not shown). The protein requirement is quite specific for histones and cannot be fulfilled by bovine serum albumin, by the enzyme lysozyme (molecular weight approximately equal to that of the small histones) or by poly(L-lysine). Elongation factor 2 accepts ADP-ribose from NAD as catalyzed by diphtheria toxin (38) but did not act as an acceptor of ADP-ribose from NAD in the presence or absence of histones and DNA with the cytoplasmic form of poly(ADP-ribose) polymerase. Under optimal histone and DNA concentrations,
POLY(ADP-RIBOSE)
CYTOPLASMIC
311
POLYMERASE 300
300
200 100
I-J 200 !z P ,’
I” 0 ; 300
300 100
200 2 d s loo s
200 100
0 0
5 10 I5 FR*CTION NUMBER
20
0
10 20 0 10 FRACTlON NUMBER
20
0
FIG. 4. Susceptibility of the poly(ADP-ribose) to enzymatic digestion. The product of the poly(ADP-ribose) polymerase reaction using cytoplasmic extract as a source of enzyme was purified from histones by the method of Yamada and Sugimura (19). (A), A 100-~1 sample of the tritiated product was mixed with a small quantity of [‘%]NAD and passed over a Sephadex G50 column (0.5 x 8 cm) equilibrated with 10 mM Tris-HCl, pH 7.5, with 1.0 M NaCl. Five-drop fractions were collected directly into counting vials for determination of radioactivity. (B-E), 100-~1 samples of the 3H-labeled product containing 5 mM MgCl* were preincubated with 50 pg of the appropriate enzyme for a total of 1 h at 37°C. After 30 min, an additional 50 pg of enzyme was added. The entire sample (105-110 ~1) was passed through the column, and the radioactivity was determined as before. The activity of the enzymes was verified in separate control experiments using radioactively labeled DNA or RNA under the same conditions of incubation.
TABLE CHAIN RIBOSE)
LENGTH SYNTHESIZED
Digestion
PoLY(ADP-
POLYMERASE~
product
5’-AMP b”
OF PoLY(ADP-
BY CYTOPLASMIC
RIBOSE) Ehlple
TABLE
III
DETERMINATION
2’-(5”-Phosphori-
Radioactivity kpm)
711 6243
Average chain length (1 + b/a)
9.1
bosyl)-5’-AMP n 3H-labeled poly(ADP-ribose) was generated on histones, purified, and digested with snake venom phosphodiesterase (Fig. 4). The digestion products were mixed with 0.1 pm01 each of 5’-AMP and ADPR as markers and spotted on a 20 x 20-cm PEI plate. The plate was developed in two dimensions with 0.9 N acetic acid/O.3 M LiCl (37). The positions of the markers were detected with a uv light source and the plates were cut into 2 x 2-cm squares. After eluting the squares with 0.3 ml of 1.6 M LiCI, the radioactivity in each square was determined.
our data indicate that, while most histones act to accept poly(ADP-ribose), certain selectivity is evident. Lysine-rich histone Fl as isolated by the method of Johns (26) shows very high acceptor activity, whereas histone Fl isolated by the method of DeNooij and Westonbrink (39) is less active. The order of specificity for stimulation by the histones (50 pg per assay) was: Whole = Fl(26) > F3 > Fl(39) > F2a > F2b. The
SPECIFICITY
OF THE
REQUIREMENTS POLYMERASE IN THE Components
IV DNA
AND
HISTONE
FOR PoLY(ADP-RIBOSE) CYTOPLASM OF HELA
in assay
Radioactivity counts per minute
Complete mixture Complete mixture + nicotinamide (20 InM) - native DNA + heat denatured DNA - native DNA + sonicated DNA - whole histones + histone Fl - whole histones + bovine serum albumin - whole histones + lysozyme - whole histones + poly+lysine
CELLS”
Percent
560 34
100 6
506
90
569 569 82
102 105 15
68 33
16 6
a The complete 0.5-ml reaction mixture contained 50 pm01 of Tris-HCI, pH 7.4, 1 ymol of M&l%, 0.5 pm01 of dithiothreitol, 1 pCi of L3HINAD (0.5 &ilrmol), 35 pg of calf thymus DNA and 100 pg of calf thymus whole histones. Where other substitutions were made in the protocol, the final concentrations of DNA and protein were 35 and 100 pg, respectively. The reaction was started by addition of 40 ~1 of a postnuclear extract prepared from cells lysed at 1 x lo8 cells/ml. The reaction was incubated 30 min at 20°C before stopping the reaction with 1.0 ml of 20% trichloroacetic acid containing 5 nm~ sodium pyrophosphate.
requirement for DNA in this system was quite selective: DNase II-treated (all hydrolysis was 25%) DNA > DNase I-treated
312
ROBERTS
DNA > micrococcal nuclease-treated DNA >> denatured DNA = sonicated DNA = native DNA > apurinic DNA. Covalent Attachment of Poly(ADP-Ribose) to Histone Fl Because there has been some question as to whether poly(ADP-ribose) is covalently attached to histones, it was of importance to demonstrate that the partially purified enzyme from ribosomes formed a covalent bond between chains of ADP-ribose units and the histone acceptor. Radioactive [14Clhistone Fl was isolated by the method of Johns from nuclei derived from cells incubated with radioactive amino acids (26). This radioactively la-
ET
AL.
beled preparation was homogeneous on polyacrylamide-gel electrophoresis (Fig. 5A). 13H1NAD was used as a substrate for the partially purified cytoplasmic enzyme washed off ribosomes by NH,Cl to generate 3H-labeled poly(ADP-ribose) chains on the [14Clhistone Fl. This incubation mixture was chromatographed on a Bio-Rex 70 cation-exchange column, as indicated by the data in Fig. 5B. It should be noted that the free r3H]NAD was eluted directly through this column. A peak of 3H-labeled poly(ADP-ribose) appears in fractions 1217 which is coincidential with the peak of [14Clhistone Fl. No association was observed when [3H]NAD and [14C]histone Fl were mixed in the absence of enzyme and
FIG. 5. [3H]ADP-ribosylation of purified [Wlhistone Fl. (A), Electropherogram obtained by running an aliquot of purified [‘4C]histone (Materials and Methods) and commercial histone Fl as carrier on a polyacrylamide gel containing sodium dodecyl sulfate. Migration was from left to right, toward the anode. The optical density profile of stained protein bands is indicated by the solid line. Radioactivity in each 1.5mm slice is indicated by the bar graph. (B), lL4C]histone Fl was used as acceptor for 3H-labeled poly(ADP-ribose) as catalyzed by the cytoplasmic enzyme under conditions given in the footnote to Table I. The reaction was terminated by addition of an equal volume of 14% guanidine hydrochloride and the mixture subjected to chromatography on a Bio-Rex 70 column (Materials and Methods). Beginning with fraction 11, the guanidine hydrochloride concentration was increased from 7 to 14%. Aliquots of each fraction were counted for radioactivity. Fractions 12-17 were pooled, dialyzed against water and lyophilized. (Cl, The pooled column fractions were subjected to electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. The gels were sliced, the radioactivity in each slice determined and the data corrected for ‘*C spillover into the tritium channel. The distribution of 3H and W radioactivity is shown. The arrows at the top of the profile show the positions to which the five main histone fractions migrate under these conditions of electrophoresis. The direction of migration was from left to right, toward the anode.
CYTOPLASMIC
POLYCADP-RIBOSE)
applied to the column. In addition, the inclusion of nicotinamide in the complete reaction mixture reduced the amount of 3H-labeled poly(ADP-ribose) that was eluted with [14C]histone Fl. To prove that the chain of poly(ADP-ribose) was covalently attached to histone Fl, fractions 1217 from the Bio-Rex column were pooled and, after dialysis, subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (Fig. 5C). Again, there was strict coincidence between radioactivity from poly(ADP-ribose) and histone Fl. Treatment of the reaction mixture with snake venom phosphodiesterase reduced the amount of 3H-labeled poly(ADP-ribose) that was bound to [14C]histone Fl by only 25%. This result suggests that the polymer was short and that the histone conferred some protection on the polymer as also suggested by the work of others (2). Hence, chains generated by the solubilized enzyme are apparently shorter than those generated by the microsomal-bound enzyme (Table III). This result would explain why the poly(ADP-ribosylated) histone corn&-rates with free histone during chromatography on ion-exchange columns and during electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. DISCUSSION
Our results show that cytoplasmic extracts of HeLa cells possess an enzyme that can form poly(ADP-ribose) from the substrate molecule NAD. Numerous reports have shown that poly(ADP-ribose) polymerase is located in nuclei tightly bound to chromatin (l-3). Most of the cytoplasmic activity for this enzyme was found tightly associated with various ribosomal particles and subunits (Fig. 3). A portion of this bound activity was shown to be due to biosynthesis of the enzyme. However, a more functional role on the ribosome might also be indicated. In contrast to chromatin-bound poly(ADP-ribose) polymerase, the cytoplasmic form requires exogenous DNA and histones, and a 30-fold increase in polymer formation is observed when the reaction is supplemented with the optimal concentrations of these molecules (Table I). The present report clarifies
POLYMERASE
313
the role of histones as acceptors in the poly(ADP-ribose) reaction; strong evidence is provided for a covalent attachment between poly(ADP-ribose) and histone Fl (Fig. 5). A variety of observations indicate that the cytoplasmic form of poly(ADP-ribose) polymerase is not merely reflective of nuclear leakage of the chromatin-bound form of the enzyme. For example, the amount of activity found for this enzyme in the cytoplasm was substantial; in some cases the total potential activity to generate poly(ADP-ribose) in the cytoplasm of cells approached 50% of the total potential in the nuclei of these cells whether nuclei were assayed directly (by using the endogenous DNA and histone acceptors) or whether exogenous DNA and histones were added to the nuclei. In addition, no significant leakage of DNA out the the nucleus was observed. Other workers have not detected poly(ADP-ribose) polymerase in the cytoplasm, presumably because additions of DNA and histones have not been made. Gill has reported poly(ADP-ribose) polymerase in soluble extracts from whole animal cells (including nuclei), but the enzyme was complexed with nucleohistone and required addition of neither DNA nor histone for activity (43). Secondly, a variety of mild cell fractionation procedures have all yielded identical specific activity for the cytoplasmic enzyme. These procedures range from vigorous Dounce homogenization in a hypotonic solution (23) to gentle lysis with the nonionic detergent NP-40 in which no homogenization is used (24). Finally, the activity of cytoplasmic poly(ADP-ribose) polymerase was shown to fluctuate during the phases of the cell cycle; the specific activity of the enzyme was especially low in those periods of the cycle (G2-M) where nuclei have particularly fragile membranes. If the activity in the cytoplasm were purely due to nuclear leakage one would have expected activity to be high instead of low in the cytoplasm during these phases of the cell cycle (Fig. 2). Our report of a cytoplasmic form of poly(ADP-ribose) polymerase is analogous
314
ROBERTS
to observations that other “nuclear enzymes” have been detected in the cytoplasms of eukaryotic cells. An enzyme system responsible for synthesizing a polyadenylate homopolymer onto the 3’-end of heterogeneous nuclear RNA has been found in the cytoplasm of virally infected mouse L-cells (45) in addition to its usual location in the nucleus. Also, DNA polymerases have been detected in the cytoplasm of a number of eukaryotic cells. Baril and colleagues (31) have shown that the small nuclear DNA polymerase (DNA polymerase I) of rat liver is also found on cytoplasmic ribosomes. In rat liver cells, DNA polymerase II is associated with microsomal membranes, and it is this enzyme that fluctuates in a parallel fashion with the level of DNA synthesis in uiuo (31). In synchronized HeLa cells the specific activity of this latter cytoplasmic enzyme is highest in S phase of the cell cycle, and activity in extracts parallels the rate of thymidine incorporation in the intact cell (44). It is interesting that the activity of cytoplasmic poly(ADP-ribose) polymerase behaves in a similar fashion and does parallel changes in DNA synthesis during the cell cycle (Fig. 2). We have recently reported that ADP-ribosylation of HeLa nuclear proteins results in increased accessibility of Escherichia coli DNA polymerase I for primer sites in nuclei (15). Synthesis of histones in the cell cytoplasm also parallels the rate of DNA synthesis, and these histones are then transported into the nucleus (42). Additional experiments will have to be performed to discern if a portion of the high-activity poly(ADP-ribose) polymerase observed in S phase of the cell cycle (Fig. 2) is also eventually transported into the nucleus with histones. Such a coupled mechanism might account for a relationship between this nuclear protein-modifying system bound to ribosomes and the need for transport of nuclear proteins from their site of synthesis to their site of function in the nucleus. ACKNOWLEDGMENTS We thank Dr. Oeschker, Dr. 0. Gabriel town University) and Dr. M. Cashel (NIH) cally reviewing this manuscript. This work tially supported by U.S. Public Health Grants No. CA13195 and CA11950.
(Georgefor critiwas parService
ET
AL
REFERENCES 1. HONJO, T., AND HAYAISHI, 0. (1973) in Current Topics in Cellular Regulation (Horecker, B. L., and Stadtman, E. R., eds.), Vol. 7, pp. 87127, Academic Press, New York. 2. SUGIMURA, T. (1973) in Progress in Nucleic Acid Research and Molecular Biology (Davidson, J. N., and Cohn, W. E., eds.), Vol. 13, pp. 127151, Academic Press, New York. 3. PoIy(ADP-Ribose): An International Symposium, Forgarty International Center Proceeding No. 26, U.S. Government Printing Office, Washington, D.C. 4. NISHIZUKA, Y., UEDA, K., YOSHIHARA, K., YAMAMURA, H., TAKEDA, M., AND HAYAISHI, 0. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, 781-785. 5. OTAKE, H., MIWA, M., FUJIMURA, S., AND SUGIMURA, T. (1969) J. Biochem. (Tokyo) 65, 145146. 6. BURZIO, L., AND KOIDE, S. S. (1972) FEBS Lett. 20, 29-32. 7. NISHIZUKA, Y., UEDA, K., AND HAYAISHI, 0. (1971) in Methods in Enzymology, eds. (McCormick, D. B., and Wright, L. D., eds.), Vol. 18B, pp. 230-233, Academic Press, New York. 8. YAMADA, M., MIWA, M., AND SUGIMURA, T. (1971) Arch. Biochem. Biophys. 146, 579-586. 9. YOSHIHARA, K. (1972) Biochem. Biophys. Res. Commun. 47, 119-125. 10. BURZIO, L., AND KOIDE, S. S. (1970) Biochem. Biophys. Res. Commun. 40, 1013-1020. 11. BURZIO, L., AND KOIDE, S. S. (1971) Biochem. Biophys. Res. Commun. 42, 1185-1190. 12. NAGAO, M., YAMADA, M., MIWA, M., AND SUGIMURA, T. (1972) Biochem. Biophys. Res. Commun. 48,219-225. 13. SMULSON, M., HENRIKSEN, O., AND RIDEAU, C. (1971) Biochem. Biophys. Res. Commun. 43, 1266-1273. 14. ROBERTS, J. H., STARK, P., AND SMULSON, M. (1973) Biochem. Biophys. Res. Commun. 52, 43-49. 15. ROBERTS, J. H., STARK, P., AND SMULSON, M. (1974) Proc. Nat. Acad. Sci. USA 71, 32133216. 16. SMULSON, M., STARK, P., GAZZOLI, M., ANDROBERTS, J. H. (1975) Exp. Cell Res. 90, 175-182. 17. SOLAO, P. B., AND SHALL, S. (1971). Exp. Cell Res. 69, 295-300. 18. MIWA, M., SUGIMURA, T., IRVI, N., AND TAKAYANA, S. (1973) Cancer Res. 33, 1306-1315. 19. YAMADA, M., AND SUGIMURA, T. (1973) Biochemistry 12, 3303-3308. 20. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (19505. Biol. Chem. 193, 265-275. 21. BURTON, K. (1956) Biochem. J. 62, 315-323. 22. SMULSON, M., AND THOMAS, J. (1969) J. Biol. Chem. 244, 5309-5312.
CYTOPLASMIC
POLY(ADP-RIBOSE)
23. SPORN, M. B., BERKOWITZ, D. M., GLINKI, R. P., ASH, A. B., AND STEVENS, C. L. (1969) Science 164, 1408-1410. 24. BORUN, T. W., SCHARFF, M. D., AND ROBBINS, E. (1967)Biochim. Biophys. Actu 149,302-304. 25. ATTARDI, B., CRAVIOTO, B., AND ATTARDI, G. (1969) J. Mol. Biol. 44, 47-70. 26. JOHNS, E. W. (1964) Biochem. J. 92, 55-59. 27. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-4412. 28. PREISS, J., SCHLAEGER, R., AND HILZ, H. (1971) FEBS Lett. 19, 244-246. 29. MIWA, M., AND SLIGIMURA, T. (1971) J. Biol. Chem. 246, 6362-6364. 30. UEDA, K., OKA, J., NARUNIZA, S., MIYAKANA, N., AND HAYAISHI, 0. (1972) Biochem. Biophys. Res. Commun. 46, 516-523. 31. BARIL, E. F., JENKINS, M. D., BROWN, 0. E., LASZLO, J., AND MORRIS, H. P. (1973) Cancer Res. 33, 1187-1195. 32. GREER, S., AND BODARSKY, 0. (1965) J. Biol. Chem. 240, 2574-2579. 33. HILZ, H., AND KITTER, M. (1971) Hoppe-Seyler’s 2. Physiol. Chem. 352, 1693-1704. 34. ZATMAN, L. J., KAPLAN, N. O., AND COLOWICK,
POLYMERASE
315
S. P. (1953) J. Biol. Chem. 200, 197-212. 35. SHAFRITZ, D. A. AND ANDERSON, W. F. (1960) J. Biol. Chem. 245, 5553-5559. 36. NISHIZUKA, Y., UEDA, K., NAKAZAWA, K., AND HAYAISHI, 0. (1967) J. Biol. Chem. 242,31643171. 37. LEHMANN, A. R., KIRK-BELL, S., SHALL, S., AND WHISH, W. J. D. (1974) Ezp. Cell Res. 83, 6372. 38. HONJO, T., NISHIZUKA, Y., HAYAISHI, O., AND KATA, I. (1968)J. Biol. Chem. 243,3553-3555. 39. DENOOIJ, E. H., AND WESTONBRINK, H. G. K. (1962) Biochim. Biophys. Actu 62, 608-609. 40. GURLEY, L. R., WALTERS, R. A., AND ENCER, M. D. (1970) Biochem. Biophys. Res. Commun. 40, 428-436. 41. UEDA, K., REEDER, R. H., HONJO, T. NISHIZUKA, Y., AND HAYAISHI, 0. (1968) Biochem. Biophys. Res. Commun. 31, 379-385. 42. ROBBINS, E., AND BORUN, T. W. (1967) Proc. Nat. Acad. Sci. USA 57, 409-416. 43. GILL, D. M. (1972) J. Biol. Chem. 247, 59645971. 44. CHIU, R., AND BARIL, E. (1974). Fed. Proc. 33, 1281.