Presence of poly (ADP-ribose) polymerase and poly (ADP-ribose) glycohydrolase in the dinoflagellate Crypthecodinium cohnii

Eur. J. Biochem. 139, 81 -86 (1984) c FEBS 1984

Presence of poly (ADP-ribose) polymerase and poly (ADP-ribose) glycohydrolase in the dinoflagellate Crypthecodinium cohnii

Edgar WERNER, Silvia SOHST, Felix GROPP, Dietrich SIMON, Herbert WAGNER, and Hans KROGER Robert Koch-Institut. Berlin

(Received July 31/Novcmber 4. 1983) - EJB 83 0838

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase have been detected in chromatin extracts from the dinoflagellate Crypthecodinium cohnii. Poly(ADP-ribose) glycohydrolase was detected by the liberation of ADP-ribose from poly(ADP-ribose). Poly(ADP-ribose) polymerase was proved by (a) demonstration of phosphoribosyl-AMP in the phosphodiesterase digest of the reaction product, (b) demonstratioil of ADP-ribose oligomers by fractionation of the reaction product on DEAE-Sephadex. The (ADP-ribose)-protein transfer is dependent on DNA; it is inhibited by nicotinamide, thymidine, theophylline and benzamide. The protein-(ADP- ribose bond is susceptible to 0.1 M NaOH (70 %) and 0.4 M NH,OH (33 %). Dinoflagellates, nucleated protists, are unique in that their chromatin lacks histones and shows a conformation like bacterial chromatin [Loeblich, A. R., I11 (1976) J. Protozool. 23, 13 - 281; poly(ADP-ribose) polymerase, however, has been found only in eucaryotes. Thus our results suggest that histones were not relevant to the establishment of poly(ADP-ribose) during evolution.

Poly(ADP-Rib) polymerase is a nuclear enzyme which transfers the ADP-Rib moiety of NAD to nuclear proteins forming the homopolymer poly(ADP-Rib). It catalyzes both the linkage of ADP-Rib to the protein and the linkage to the subsequent ADP-Rib residues [I]. The enzyme has been detected in a series of eucaryotic cells from unicellular organisms up to plants and mammals [I]. It has usually been demonstrated by incubating isolated nuclei with NAD and collecting the ADP-Rib residues bound to protein. The enzyme in vitro charges a series of proteins of the histone and non-histone group [2]. What is charged in tiivo, however, remains to be elucidated. The number of proteins seems to be reduced [2]. For histone H1 a poly(ADP-ribosy1)ation in vivo has been documented [3]. Another candidate favoured as an acceptor in vivo is the enzyme itself [4].

Poly(ADP-Rib) has a high turnover in vivo, half-life times of as short as 1 min have been reported [5]. The enzyme poly(ADP-Rib) glycohydrolase has been considered rcspon- sible for the degradation of poly(ADP-Rib). A chromatin- bound phosphodiesterase (from rat liver) which also degrades poly(ADP-Rib) seemed not to be relevant because of its lower specificity and affinity to the polymer [6].

There have been various proposals as to the role poly (ADP-Rib) plays in the cell [I] . In the past few years a spectrum of data lent support to the idea that it might be involved in DNA excision repair [7]. Proposals to explain the molecular mechanism were that poly(ADP-Rib) destabilizes the linkage of chromatin proteins to DNA due to its dense negative charge. In this way, access of the repair machinery to the damaged DNA would become possible [8].

Up to now, poly(ADP-Rib) polymerase has not been detected in procaryotes. Only mono(ADP-Rib) transferases have been extracted from bacteria [9,1]. So the poly(ADP- Rib) systcm does not seem to be present in bacteria. One may speculate that it had been introduced during evolution when the eucaryotic chromatin was established.

New aspects to this suspected interdcpendence between the chromatin structure and the Occurrence of poly(ADP-Rib) may emerge from studies on dinoflagellates. Dinoflagellates are peculiar with respect to the composition of their chromatin. Although located in a nucleus, it has a spatial arrangement as in procaryotes. The diameter of the chromatin fiber is 6.0 - 8.0 nm in contrast to 10 nm or more in eucaryotes. Histones and a nucleosomal subunit structure, as in eucaryotes, are absent [I0 - 121. The DNA, however, contains a portion of repeated sequences (60 %) typical for eucaryotes [13]. In addition the size of the large rRNAs is not grossly divergent from other algae [14] and the 5s rRNA is composed of sequences that point to a relatively high evolutionary position of the dinoflagellates [15]. Since there is little molecular data on dinoflagellates beyond that mentioned here. the results of this paper may also contribute to the evolutionary classification of dinoflagellates.

We show here that the dinoflagellate Crypthecodinium cohnii is equipped with a poly(ADP-Rib) system by demon- strating poly(ADP-Rib) polymerase and poly(ADP-Rib) glycohydrolase activity in citro. To demonstrate the polymerization, it was necessary to detach the euyme from the chromatin and to separate it from the predominant glycohydrolase and other degrading activities.

Abbreviations. ADP-hb, adenosine(5')diphospho(5')-[&~-ribose; (ADP-Rib),, oligomers and polymers of ADP-Rib; AMP(P-Rib), 2'- (5-phospho-~-~-ribosyl)-adenosine 5'-phosphate ; Mes, 4-morpholine- cthanesulfonic acid: CI,AcOH, trichloroacetic acid; SDS, sodium dodecyl sulfate.

Enzymes. Poly(ADP-Rib) polymerase (EC 2.4.99. -) ; poly(ADP- Rib) glycohydrolase (EC 3.2.1. -); phosphodiesterase I (EC 3.1.4.1); alkaline phosphatase (EC 3.1.3.1).

MATERIALS AND METHODS

Materials

[adeni~e-2,8-~H]NAD (3.4 Ci/mmol) was from NEN, [ud~nine-'~C]NAD (280 Ciimol) was from the Amersham International plc. ADP-Rib and AMP were from Calbiochem.

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NAD, ATP, thymidine, proteinase K and yeast RNA were from Boehringer, Mannheim. Benzamide and theophylline were from Sigma. 5-Methylnicotinamide was a gift of Eli Lilly Corp. Alkaline phosphatase (Escherichia coli), the purest preparation offered, and phosphodiesterase (Crotalus ada- manteus) were from Worthington. DEAE-cellulose (DE 52) was from Whatman, DEAE-Sephadex (A25) was from Phar- macia. Calf thymus DNA, bovine albumin, poly(ethy1ene- glycol) (M, ZOOOO), Dowex 1 x 2 and pronase were from Serva. Soluenc 350 was from Packard Instruments. All other substances were of analytical quality, except for the cultivation medium where lower qualities were used.

Growth conditions

Crypthecodinium cohnii strain GC was axenically grown in a modified AXM medium [16]. Following the MLH medium [17], 1.-histidine was reduced to 0.2 gjl, betaine.HC1 was raised to 1.5 g/l, AMP was omitted. The cells grew at 25 -27 "C under continuous illumination (Osram L-fluora) without shaking (1-1 penicillin flask).

Poly i ADP-Rib)

This was prepared from rat liver nuclei which had been incubated with [,H]NAD (17 Ci/mol). The method was essentially that described by Sugimura et al. [I81 including the chromatography on hydroxyapatite. The mean chain length of the preparation was 13 units, the A,so/A,,o ratio was 0.25 -0.29.

Protein

This was determined according to Bradford [19].

Isolation o j chromutin ; preparation and purification of the chromatin extract

Late-log-phasc dinoflagellates (10 flasks) were harvested by centrifugation at 450 x g , washed with 4 mM EDTA/Na, pH 7.0, containing 12 I:(, (w/v) sorbitol and suspended in 150 ml buffer A (50 mM Tris/HCl, pH 7.6, 10 mM EDTA, 0.5 mM dithioerythritol). The suspension, about 1 vol. cells plus 7 vol. buffer, was passed twice through the French press at 28MPa. The homogenate was layered on 10ml 30% (w/w) sucrose in buffer A and centrifuged 20min at 10000rev.jmin in an HB-4 rotor. The sediment was resuspended in 35 ml buffer A containing 0.2 % Triton X-100. After 10 min in ice it was centrifuged again, resuspended in buffer A and centrifuged through 10 ml20 % (w/w) sucrose in buffer A. The sediment, resuspended in buffer A, was used as 'chromatin'.

For the extraction of the enzymes the sediment was resuspended in 15 ml buffer A supplemented with 1.5 ml 5 M NaCI. The highly viscous suspension was cleared by Centrifugation (20 min at 18000 rev./min, SS 34). The supernatant ( I8 ml) was supplemented with another 1.8 ml 5 M NaCl and 0.5 M dithioerythritol to 2 mM and sonicated twice for 1 min under cooling in ethanoliice and centrifuged at high spced (45 min at 45000rev./min, Ti50). 1Opl 10% polymin P (adjusted to pH 7.9 with HC1) were added per ml supernatant and the precipitate was spun down. The supernatant was dialyzed against 3 vol. of 3.6 M (NH&S04 in buffer A overnight. The resulting precipitate was collected and washed twice with 2.7 M (NH4),S04 in buffer A and dissolved

in a small volume of buffer B (50 mM TrisjHCl, pH 7.8, 0.5 mM EDTA, 5 mM 2-mercaptoethanol). After dialysis for 2 h against buffer B containing 0.25 M NaC1, any particulate material was spun down. The supernatant was layered on a column (0.9 x 3 cm) of DEAE-cellulose which had been equilibrated with buffer B containing 0.3 M NaC1. The un- bound material was washed through with the same solution. The fractions which contained protein (color reaction) were combined (about 5 ml), placed in a dialysis tube and embedded in dry poly(ethyleneglyco1) in order to reduce the volume to 0.5 ml ('high-salt extract'). The solution was centrifuged either immediately or the next morning. For this centrifugation it was layered on a density gradient of 15 -41 % (v/v) glycerol in buffer B containing 0.3 M NaCl and 0.5 mM dithioerythritol and centrifuged in a VTi 65 rotor for 4 h at 64000 rev./min. After the run the tubes were pierced at the bottom and fractions of about 0.23 ml were collected. The respective enzyme tests were carried out immediately and the fractions kept at -70 "C. The fractions of the rapidly sedimenting peak (see Fig. 2) were poolcd later and served as the enzyme source to be generally used ('purified chromatin extract').

Enzyme assays

Poly(ADP-Rib) polymerase. The reaction mixture (1 50 pl) consisted of 50 mM TrisjHCl, pH 7.8, 5 mM CaCI,. 3 mM dithioerythritol, 0.14 mM [3H]NAD (8.5 Ci/mol), 10 pg calf thymus DNA and 8 - 12 pg protein in 20 pl buffer B containing 0.3 M NaCl and about 30 % (v/v) glycerol. After 20 rnin at 20 "C, the tubes were placed in ice and 40 p1 yeast RNA (10 mg/ml) in 0.2 % SDS and 75 $20 7; C1,AcOH were added. After another 20 min the samples were filtered on GF/C filter discs, washed with 20 ml C1,AcOH containing 5 mM sodium pyrophosphate and 5 ml 75 ethanol. The dried filters were soaked for 30 rnin at room temperature in 0.6 ml Soluene/HzO (9:l) which were finally completed with 5 ml toluene-based scintillation cocktail (Quickszint 501). The recovery of radioactivity was about 35 %.

Poly(ADP-Rib) glycohydrolase or poly(ADP-Rib) degradation. The assay was the same as for the poly(ADP- Rib)polymerase except that [,H]NAD was replaced by I0 pl [3H]poly(ADP-Rib) (1.2 units, 240 counts min pl- I ) .

Product analysis

Determination of the mean chain length of ( A D P- Rib) n. A twofold poly(ADP-Rib) polymerase reaction mixture containing [3H]NAD of higher specific activity (39 Ciimol) was stopped after incubation with 0.1 ml yeast RNA in 0.2 SDS, 25 pl30 mM NAD and 250 p120 % C1,AcOH. The precipitate was spun down, washed twice with 5 '4 CI,AcOH (with the aid of sonication) and twice with 75 "/, ethanol containing 0.2 M sodium acetate, pH 5.0. In the first ethanol wash 25 p1 30 mM NAD were included. The dried sediment was dissolved in 0.2 ml0.1 M NaOH plus 0.2 ml50 mM Tris/HCl, pH 7.6, and kept at 30 "C for 15 min. After adjusting the pH to 8 with 0.1 M HCI, 10 p10.2 M MgCl,, and 22 p1 phosphodiesterase (100 units/ml) were added. After 2 h at 37 "C, 11 pl pronase ( 1 0 mg/ml, predigested for 2 h at 37 "C) were added and the incubation continued for a further 15 min. The digestion was stopped with 35 pl100 % C1,AcOH. The precipitate was spun down and the supernatant was treated with ether. The pH was adjusted to 7 - 8 with Tris base; ADP-Rib, AMP and adenosine were added and the volume was doubled by adding HzO. The digest was

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layered on a column (1 x 3 cm) of Dowex 1 x 2 (C1- form) which had been previously washed with H20. The ion- exchanger was treated consecutively with 5 ml H20, 4 ml 3.5 mM HCl, and three 6-ml portions of 3.5 M HC1 containing 40 mM, 150 mM and 250 mM NaC1, respectively. Fractions of 0.5 ml each were collected, mixed with 5 ml Triton/toluene scintillation cocktail (Quickszint 402) and the radioactivity was counted. Adenosine was found in the flow-through, AMP was eluted by 40 mM NaCl, ADP-Rib by 1 SO mM NaCl and some oligomers by 250 mM NaCl. AMP(P-Rib) was eluted at the position of ADP-Rib. It was discriminated from ADP-Rib by its susceptibility to alkaline phosphatase.

Poly(ADP-Rib) degradation. An appropriate multiple of the poly(ADP-Rib) degradation reaction mixture was incubated and stopped with RNA and C1,AcOH as described above. The precipitate was removed, the supernatant was treated with ether and neutralized with Tris base. One part was processed directly on a Dowex 1 x 2 column, the other part was incubated with phosphatase (1 unitlml) for 30min at 37 "C, stopped with Cl,AcO€I, treated with ether and neutralized as above. The separation of the degradation products on Dowex 1 x 2 was performed as described before.

D EA E- Sephadex chroma t ograph y of ( A D P- R ib) , An eightfold poly(ADP-Rib) polymerase assay was in-

cubated with NAD of double specific activity (16 Ci/mol). When stopping the reaction the usual RNA was replaced by bovine serum albumin (0.8 mg). The other conditions of preparing the reaction product were as described for the product analysis. The final sediment was incubated in 25 mM TrislHCl, pH 7.8, 2 SDS and 1 mg/ml proteinase K for 16 h at 37°C. SDS was removed by precipitation with KCl. The digest was treated with phenol, the H 2 0 phase with ether, brought to 7 M urca, diluted with 2 vol. 7 M urca, com- plcmented with the markers adenosine, AMP, ATP and ADP-Rib and applied to a DEAE-Sephadex A25 column (0.9 x 12 cm) which had been equilibratcd with 7 M urea in 20 mM Tris/HCl, pH 7.6. The column was washed with the same buffer until adenosine and AMP were eluted. Finally, a linear salt gradient (2 x 60 ml, 0 -0.75 M NaCl in the equilib- ration buffer) was applied and fractions of 0.5 ml were collected.

Partition of (ADP- Rib), in the chloroformlwater system

An appropriate multiple of the poly(ADP-Rib)polymerase reaction mixture was incubated under standard conditions except that [3H]NAD was replaced by [14C]NAD (6.7 Ci/mol). The reaction product was isolated as described before. The dried sediment was redissolved in 10 mM Mes- Na', pH 6.2, and after adjusting the actual pH to 6.2 with NaOH, sarcosyl was added to 0.2 %. After incubation in NaOH and NH20H the samples were titrated back to pH 7.0 with 1 M HCl. The control sample was incubated in 0.1 M NaCl, the proteinase K sample was brought to 0.1 M NaCl after digestion. The phase partition was accomplished by adding an equal volume of chloroform/isoamyl alcohol (24:l) to the samples and whirling them. The H 2 0 phase was aspirated carefully, the chloroform-plus interphase was repeatedly extracted with H,O until the radioactivity in the H 2 0 extract was below 100 counts/min. Finally the chloroform was distilled away, the remaining particulate

material was dissolved in 0.6 ml Soluene/H,O (9 :1) and supplemented with 10 ml toluene scintillation cocktail for counting. The H 2 0 phases were supplemented with Tri- ton/tolucne scintillation cocktail. Their radioactivities were presented as the sum of all extracts. Protein-bound (ADP- Rib), counted in the Triton/toluene system gave about 80 % of the values counted in the Soluene/toluene system. Protein-free (ADP-Rib), , however, gave similar recoveries with both systems.

RESULTS

Detection of the polymerization reaction

Poly(ADP-Rib) polymerase has usually been demonstrated by incubation of isolated nuclei with NAD (see introduction). Since isolation of nuclei from Crypthecodinium cohnii is very inconvenient (our results), we replaced the nuclei by a chromatin-like material which is easily prepared (see Materials and Methods). When it was incubated with NAD a transfer of the ADP-Rib moiety to protein was observed. The rcaction appeared to be a mono(ADP-ribosyl)ation, because almost exclusively AMP was set free by digestion of the product with snake venom phosphodiesterase. AMP(P-Rib), which would have indicated (ADP-Rib) polymerization- amountcd to maximally 1 %. AMP is generated from chain

ends including mono(ADP-Rib), while AMP(P-Rib) is generated from links inside the chain. Mono(ADP-ribosy1)ation also occurred with a high-salt extract from the chromatin-like material. When, on the other hand, poly(ADP-Rib) (isolated from rat liver) was incubated with chromatin-like material or with the high-salt extract a rapid degradation of the polymer was observed: 60 - 70 % of the material rendered acid-soluble by chromatin was ADP-Rib (Fig. 1). It was identified as ADP- Rib by its position being identical with marker ADP-Rib and by its resistance to phosphatase. ADP-Rib is the specific product of the poly(ADP-Rib) glycohydrolase reaction. This enzyme splits the ribose-ribose linkage in poly(ADP-Rib) ; it does not, however, attack the protein-ADP-Rib linkage of the first protein-linked ADP-Rib residue of the chain [6]. Thus the formation of mono(ADP-Rib)-protein conjugates observed with the dinoflagellate extract was suspected to arise from the

looot Ado AMP ADP-Rib

"0 10 20 3c LO Fraction number

Fig. 1. Chromatography qf poly (ADP-Rib) residues rendered acid- soluble by incubation with chromatin from C. cohnii. (3H]Poly (ADP-Rib) (0.22 A260 unit) was incubated with chromatin (equi- valent to about 120 pg DNA) in a total volume of 1.2 ml and processed as described in Materials and Methods. Equal volumes of the hydrolysate were chromatographed either directly (0) or after a consecutive digestion with phosphatase (0) on Dowcx 1 x 2 by stepwise elution. A separate chromatography of the hydrolysate with a linear salt gradient up to 0.25 M NaCl confirmed that radioactivity was eluted only at the positions of AMP and ADP-Rib

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Table 1. Mean chain length of (ADP-Rib),,,formed bjJ fractiuns q f t h e density gradient centrlfugution of the chromatin extract The reaction products of the respective fractions in Fig. 2 were prepared and processcd as described in Materials and Methods. 'AMP(P-Rib)' indicates the radioactivity at the position of ADP-Rib which served as markcr for AMP(P-Rib) during the chromatography on Dowex 1 x 2 . The mean chain length is the quotient of the radioactivity in AMP + AMP(P-Rib) to that in AMP

Fraction AMP 'AMP(P-Rib)' Mean chain length ~

counts iiiln - ~

9 1657 2588 2.56 11 24x4 1547 1.6 13 1013 170 1.1 1s 3304 97 1.02 17 2489 342 1.09

I 0 ' '0 0 5 10 15 20

Fract ion number

Pig. 2. Glycerol density gradient centrifugation oj' the chromatin ertracf. 0.5 ml chromatin extract containing 2.5 mg protein were centrifuged in a VTi 65 rotor (Beckman). The poly(ADP-Rib) polymerase assay was conducted in the presence (0) and in the absence (0) of DNA. Thc degradation of poly(ADP-Rib) (W) is represented as the difference between total precipitable poly(ADP-Rib) and poly (ADP-Rib) remaining precipitable after incubation. Protein (A). For more details see Materials and Methods. The direction of sedimenta- tion is from right to left. the positions of alcohol dehydrogenase (ADH; hfr 148000) and ovalbumin (Ov; M , 4S000) were determined in a parallel tube

simultaneous synthesis and degradation of poly(ADP-Rib). Consequently a separation of both activities was attempted which would allow the detection of the polymerization.

Separation was accomplished by centrifuging the high-salt extract in a glycerol density gradient (Fig. 2 and Table 1). Polymerization was detected in part of the fractions showing (ADP-Rib)-transferring activity (fractions 9 - 13 of Fig. 2). Phosphodiesterase digestion analysis revealed a maximal mean chain length of 2.6 in fraction 9 dropping to 1.6 in fraction I 1 and to 1 , O - 1 . I in fractions 13, 15, 17. Reduction of the chain length progressing towards the top of the gradient might be explained by the course of the poly(ADP-Rib) degradation (Fig. 2). Fractions 7 - 11 showed the lowest level of degradation in the whole gradient whereas degradation increascd towards the top of thc gradient. The highest level of poly(ADP-Rib)-degrading activity was at the bottom of the gradient. It is considered to be poly(ADP-Rib) glycohydrolase

1250 -Ado AMP ADP-Rib ATP

I 1 1 I C -1000 -

3, 750 - - 0.75 E

2 500 -

250 -

0

.-

C 3 - - I

- - - I

0 25 50 75 100 125 Fract ion number

Fig. 3. DEA E-Sephadex A25 chromatography of the reactiori product ,formed by the poly (ADP-Rib) polj~nerase reaction mixrure. The reaction product freed from protein was chromatographed in 7 M urea as outlined in Materials and Methods. (0) Radioactivity, (--) NaCl concentration. The position of the markers is indicated by the arrows. The eniyme protein, the purified chromatin extract, was stored for several weeks at -70°C which led to a high portion of mono(ADP- Rib) (see text)

since ADP-Rib was the sole product liberated from poly(ADP- Rib) (not shown).

The bipartite appearance of the ADP-Rib incorporation was reproducible. All the following experiments were carried out with the pooled fractions of the peak from the bottom of the gradient. The reaction product of the fractions from the top of the gradient probably consisted of (ADP-Rib)-protein conjugates. The supporting facts were : the incorporation was dependent on DNA and it was inhibited by nicotinamide and thymidine; ADP-Rib was set free from thc protein by NH,OH (results not shown). The reason for the bipartite appearance is unknown.

The ratio of AMP(P-Rib)+AMP to AMP indicated the average distribution of the chains (Table 1). The actual distribution of the chains was identified when the reaction product digested by protease was chromatographed on DEAE-Sephadex A25, resolving the polymers according to their length. Radioactivity was eluted from the ion-exchanger in the range of 0.12 M NaCl up to about 0.5 M NaCl, forming peaks at 0.28 M and 0.43 M NaCl (Fig. 3). At 0.4 M NaCl. poly(AMP) stretches of 16 -20 units in length are eluted (not shown). Since the monomer of poly(ADP-Rib) contains two phosphate residues, an oligo(ADP-Rib) of 8 ~ 10 units may make up this peak. The high proportion of mono(ADP-Rib) residues in the chromatogram was surprising. It varied with the age of the enzyme preparation, being relatively low with freshly prepared enzyme and high with an enzyme kept at -70 "C for several weeks. We also observed that 10 -20 % of the radioactivity in the Cl,AcOH/ethanol-washed reaction product became C1,AcOH-soluble aftcr dissolution in the usual Mes buffer. An ensuing incubation for 60 min at 37 "C. however, released only minute additional radioactivity. On the other hand, that part of the reaction product which remained acid-precipitable after digestion of the protein, was found to have a considerably higher mean chain length than that of the total reaction product; 8 - 10 units in contrast to about 2 units were determined (not shown). This size fits well with the expected length or the peak at 0.43 M NaCI. Apparantly, the solubility of most of the freed ADP-Rib residues in acid led to an enrichment of longer ADP-Rib oligomcrs.

Properties of the polymerization reactiori

Wherever in the gradient fractions (Fig. 2) radioactivity was incorporated into acid-prccipitable material. the reac-

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Table 2. Stability of the ADP-Rib-protein linkage in alkali and hydroxylamirze Protein-(ADP-Rib), conjugates were prepared with the purified chromatin extract as described in Materials and Methods. The samples were incubated at 37 “C for 60 min in the conditions indicated. For the ensuing partition into the H 2 0 and CHCl3 phases see Materials and Methods Compound ADP-Rib incorporated

Table 3. Effect of inhibitors on /he (ADP-Rib)-protein transfer The compounds were included in the standard reaction mixture at a concentration of 0.1 5 mM. Incubation and processing of the assays were performed as described in Materials and Methods. Enzyme source was the purified chromatin extract

Treatment CHCI, phase H,O phase Nicotinamide ”/, inhibition 25

Thymidine 39 - -~

total acid-precipitable Theophylline 62 5-Methyl nicotinamide 31 Benzamide 48 counts/niin (”/, total)

NaCl (0.1 M) 6600 (89) 810 (11) 0 ( 0) Proteinase K 240 ( 3) 8140 (97) 2630 (32)

NaOH (0.1 M) 2290 (30) 5230 (70) 3660 (53) NH,OH 4800 (67) 2400 (33) 785 (47)

(1 00 Pg!ml)

(0.4 M, pH 7.4)

tion was dependent on DNA. Traces of incompletely removed endogenous DNA may have caused the weak incorporation measured in the lower numbered fractions. In the reaction mixture the Mg2+ generally used was replaced by Ca2+ since Ca2 + suppressed the poly(ADP-Rib) degradation more strongly than Mg*+ (not shown).

I t is a characteristic of the protein-poly(ADP-Rib) linkage to be sensitive to alkali and hydroxylamine. Generally it is split completely in 0.1 M NaOH and partially in hydroxylamine, the extent being dependent on the state of the cells used [20]. Liberation of the chains from the protein was demonstrated by utilizing the partition of protein and (ADP-Rib), between chloroform and water (Table 2). Protein-bound ADP-Rib (if not too long) would reside in the interphase, free ADP-Rib in the H 2 0 phase. Protease treatment resulted in the transfer of the radioactivity from the interphase into the H20 phase. The radioactivity in the H20 phase of the untreated sample may be identical with the radioactivity which became acid-soluble when the Cl,AcOH/ethanol-washed reaction product was dissolved in the pH-6.2 buffer (see above). After incubation (60 min at 37 “C) in 0.1 M NaOH and neutral hydroxylamine 70 and 30 % respectively of the total radioactivity was in the H 2 0 phases. The H,O phases were further examined for their content of C1,AcOH-insoluble rddioactivities. It amounted to 43 ”/; and 37 % respectively of the H 2 0 phases. After protease digestion it amounted to 32 %. Apparently the bond betwccn protein and ADP-Rib was sensitive to NaOH and NH20H. Notably, there was a preference for polymers, as documented by the enrichment of C1,AcOH-precipitable material in the H 2 0 phase. The composition of the phases, however, was not analyzed further, for example by DEAE-Sephadex chromatography. Thus it is uncertain whether incubation with 0.1 M NaOH shifted the oligomers completely into the H 2 0 phase. The incubation conditions were ascertained to be saturating. Any linkage of ADP-Rib to DNA conferring alkali stability was excluded by the failure of DNase to reduce the acid- insoluble radioactivity. RNase had no effect either (not shown).

Several substances inhibit the poly(ADP-Rib) polymerase reaction. Nicotinamide, thymidine, theophylline, benzamide and 5-methyl nicotinamide, being the most frequently used compounds, were tested in the dinoflagellate system (Table 3).

At concentrations equimolar to NAD the degree of inhibition was not as high as reported for mammalian systems [21,22].

DISCUSSION

The results presented in this communication provide evidence that thc dinoflagellate Crypthecodinium cohnii contains a poly(ADP-Rib) polymerase. The enzyme resembles other poly(ADP-Rib) polymerases in so far as its reaction is dependent on DNA and is sensitive to characteristic inhibitors, and the bonds it forms with the acceptor proteins are susceptible to alkali and hydroxylamine. The detection of the specifically degrading enzyme poly(ADP-Rib) glycohydrolase was helpful in detecting the poly(ADP-Rib) polymerase. The presence of both enzymes in C. cohnii led to the conclusion that dinoflagellates possess a poly(ADP-Rib) system as do other eucaryotes.

A considerable portion of monomers was incorporated by the protein preparation which catalyzed the formation of poly(ADP-Rib) (Fig. 3). The origin of the ADP-Rib monomers is not clear Poly(ADP-Rib) polymerase seems to be involved in their production since the incorporation of (ADP- Rib), into protein is dependent on DNA. Other DNA- dependent enzymes consuming NAD are not known. It is not satisfactory, however, to explain the bulk of the monomers as the remainders of a ‘frustrated’ polymerization reaction. The relatively long ADP-Rib chains (Fig. 3) should not survive the presumed radical degradation. We rather suppose that chemi- cally formed protcin-ADP-Rib adducts play the leading role Kun et al. [23] called attention to the nonenzymically formed linkage between thc free nitrogen of lysine in the protein and the aldehydic group in the terminal ribose of ADP-Rib. Free ADP-Rib might have been produced by the action of the poly(ADP-Rib) glycohydrolase. Possibly such ADP-Rib- protein adducts can help to explain why 10 ~ 20 7; of the ADP- Rib residues became acid-soluble when dissolving the washed reaction product in a pH-6.5 buffer, and why 0.1 M NaOH did not totally split ADP-Rib from the protein. However, this point is still not proved.

Early attempts to settle the evolutionary position of the dinoflagellates are reflected by the proposal to call them ‘mesocaryotes’ [24]. Thereby dinoflagellates were interpreted as being on the route from procaryotes to eucaryotes. Since then, howcver, molecular data have accumulated pointing to a plainly eucaryotic classification of the dinoflagellates. Cor- respondingly, new interpretations as to how the histone-less chromatin had evolved were put forward. It has been suggested that dinoflagellates branched from an eucaryotic ancestor and

86

that the absence of histones was secondary [15]. T h e manifest procaryotic arrangement of the chromatin would merely represent that arrangemcnt which a histonc-free DNA would adopt in the nuclear environment. This new tendency seems t o be supported by our finding of a poly(ADP-Ribjpolymerase in dinoflagellates.

O u r results show, on the other hand, that histones a rc not a prerequisite for the existence of the poly(ADP-Ribjpoly- merase even if they were factually acceptors. One is tempted t o consider that some property of the chromatin in itself made the poly(ADP-ribosy1)ation of chromosomal proteins necessary. For instance, processes on thc D N A such as DNA repair, would require a topical relaxation of the DNA-protein binding. Poly(ADP-Rib) would accomplish this task by conferring negativc charges onto the proteins. Potential targets would be all proteins with a high affinity t o D N A including histones, if prcsent.

If any evolutionary interdepencence of poly(ADP-Rib) and histones did not exist, one could still speculate on the interdependence between the establishment of the nucleus and poly(ADP-Rib). With regard to this speculation a n d t o the evolutionary classification of dinoflagellates (see above), one would likc to know definitely whether or not procaryotes do possess poly(ADP-Rib j polymerase. The search for poly (ADP-Rib) polymerase in procaryotes conducted in the early ycars of the poly(ADP-Rib) history produced a negative result [25]. Since then, howcver, archaebacteria have been character- ized as a new kingdom apart f rom procaryotes a n d eucaryotes, which s h w striking similarities to eucaryotes, e. g. regarding the R N A polymerase [XI. Since the older report did not include representatives of archaebacteria, an effort to search for poly(ADP-Rib) polymerase in archaebacteria would be worthwhile.

We thank Miss c'. Schulz for valuable assistance. Part of this work was supported by Deitt iche Forschungsgemeinschuft (SFB 29).

REFERENCES

1 Mandel, P , Okdznki. H & Niedergang, C (1982) Piogr Nutleic 4 c d Rcu Mi)/ BIOI 27. 1-51

2. Adamietz, P. (1982) in ADP-Ribo.s.ylation Reactions (Hayaishi, 0.

3. Adamietz, P., Bredehorst, R. & Hilz, H. (1978) Eur. J . Biocheni.

4. Ogata, N., Kawaichi, M., Ueda, K. & Hayaishi, 0. (1980) Biochem. Int. I , 229 -236.

5. Wielckens, K.. Schmidt, A,, Gcorge, E., Bredehorst, R. & Hilz, H. (1982) J . Bid. Chern. 257. 12872-12877.

6. Miwa, M. & Sugimura. T. (1982) in ADP-Ribosvlation Reactions (Hayaishi, 0. & Ueda, K. , cds) pp. 263 -277, Academic Press. New York.

7. Shall, S. (1982) in ADP-Rihosylation Reuctions (Hayaishi. 0. & Ueda, K., eds) pp. 477 - 520, Academic Press, New York.

8. Benjamin, R. C. & Gill, D. M. (1980) J . Biol. Cheni. 255.

9. Skorko, R. & Kur, J. (1981) Eur. J . Biochez. I l 6 , 317-322. 10. Loeblich, A. R., I11 (1976) J. Protozool. 23, 13 -28. 11. Rizzo, P. J. & Nooden, L.-D. (1974) Biochirn. Biophys. Acta, 349.

12. Bodansky, S., Mintz, L. B. & Holmes, D. S. (1979) Riochem.

13. Allen, R. J., Roberts, T. M.. Locblich. A. R. 111 & Klotz, L. C.

14. Werncr-Schlenzka, H.. Werner, E. & Kroger, H. (1978) Comp.

15. Hinnebusch, A. G., Klotz, L. C., Blanken. R. L. & Loeblich, A. R.

16. Provasoli, L. & Gold, K. (1962) Arch. Mikrobiol. 42. 196 -203. 17. Tuttle, R. C. & Loehlich. A. R. 111 (1975) Phycologin, 14, 1-8. 18. Sugimura, T., Yoshimura, N., Miwa, M., Nagai, €-I. & Nagao, M.

19. Bradford. M. (1976) Anal. Biochern. 72, 248-254. 20. Adamietz. P., Bredehorst, R., Oldckop. M. & Hilz, H. (1974)

21. Hilz, H. & Stone, P. (1976) Rea. Physiol. Biochem. 76. 1 -58. 22. Levi, V., Jacobson, E. L. &Jacobson, M. K. (1978) FEBS Lett. 88,

23. Kun, E., Chang. A. C. Y.. Sharmu, M. L., Fcrro, A. M. &Nitecki. D. (1976) Proc. Natl Acad. Sci. USA, 73, 3131 -3135.

24. Dodge, J . (1965) Exp. Med. Int. Congr. Ser. 91, 339. 25. Nishizuka, Y., Ueda, K., Nakazawa. K. & Hayaishi, 0. (1967) J.

26. Huet, J., Schnabel, R., Scnthac, A. & Zillig. W. (1983) EMBO J. .

& Ueda, K., eds) pp. 78-101, Acadcmic Press, New York.

91, 317-326.

10502 - 10508.

415 -427.

Biophys. Res. Cornmun. 88, 1329 - 1336.

(1975) Cell, 6 , 161 -169.

Biochem. Ph.vsio1. 61B, 587 -591.

111 (1981) J. Mo~. E ~ o l . 17, 334-347.

(1971) Arch. Biochem Biophys. 147, 660 -665.

FEBS Lett. 43, 318 -322.

144-146.

B i d . Chem. 242. 3164-3171.

2, 1291 - 1294.

E. Werner, S. Sohst. D. Simon, H. Wagner, and H. Kroger, Robert Koch-Institut, Nordufer 20, D-1000 Berlin (West)

F. Gropp. Max-Planck-Institut fur Biochemie, Am Klopferspit7 18a. 11-8033 Martinsried, Federal Republic of Germany

Documents

Presence of poly (ADP-ribose) polymerase and poly (ADP-ribose) glycohydrolase in the dinoflagellate Crypthecodinium cohnii