Vol. 259, No. 8, Issue of April 25, pp. 4770-4776 1984 Printed in &%A.

THE JOURNAL OF BIOLOGICAL CHEMlSTRY 0 1984 by The American Society of Biological Chemists, Inc.

Poly(ADP-Ribose) Synthetase SEPARATION AND IDENTIFICATION OF THREE PROTEOLYTIC FRAGMENTS AS THE SUBSTRATE- BINDING DOMAIN, THE DNA-BINDING DOMAIN, AND THE AUTOMODIFICATION DOMAIN*

(Received for publication, December 12,1983)

Isamu Kameshita, Zen'e Matsuda, Taketoshi Taniguchi, and Yutaka Shizuta From t h Department of Medical Chemistry, Kochi Medical School, Nankoku, Kochi 781-51, Japan

Poly(ADP-ribose) synthetase of M, = 120,000 is cleaved by limited proteolysis with a-chymotrypsin into two fragments of M. = 54,000 (54K) and M, = 66,000 (66K). When the native enzyme is modified with 3-(bromoacetyl)pyridine, both portions of the en- zyme are alkylated; however, alkylation of the 54K portions of the enzyme is protected by the addition of the substrate, NAD, or its analog, nicotinamide, sug- gesting that the substrate-binding site is localized in the 54K fragment. When the enzyme previously auto- modified with a low concentration of [adenine-U-''C] NAD is digested with a-chymotrypsin, the radioactiv- ity is detected exclusively in the 66K fragment. The 66K fragment thus labeled is further cleaved with papain into two fragments of M, = 46,000 and M, = 22,000. With these two fragments, the label is detected only in the 22K fragment, but not in the 46K fragment. The 46K fragment binds to a DNA-cellulose column with the same affinity as that of the native enzyme, while the 22K fragment and the 54K fragment have little affinity for the DNA ligand. These results indi- cate that poly(ADP-ribose) synthetase contains three separable domains, the first possessing the site for binding of the substrate, NAD, the second containing the site for binding of DNA, and the third acting as the site(s) for accepting poly(ADP-ribose).

Poly(ADP-ribose) synthetase, an enzyme localized in the nucleus of eukaryotic cells, catalyzes the polymerization of the ADP-ribose moiety of NAD to form poly(ADP-ribose) which is covalently bound to various nuclear proteins (1, 2). Although the physiological function of this enzyme reaction is not yet fully understood, it has been suggested that the reaction participates in DNA repair (3,4), DNA synthesis (5), RNA synthesis (6), or cell differentiation (7).

During the past few years, the enzyme has been purified to near homogeneity from calf thymus (%lo), rat liver ( l l ) , Ehrlich ascites tumor cells (12), and other sources (13-15). It has been demonstrated that the purified enzyme absolutely requires double-stranded DNA for activity and that the acti- vation occurs as the result of binding of the enzyme protein to sites of strand breaks on DNA (16, 17). While histone H1 and histone H2B are known to serve as the acceptors of poly(ADP-ribose) in vitro (18, 19) and in uiuo (20), several

* This work was supported in part by the Naito Foundation Re- search Grant, the Yamada Science Foundation Research Grant, and Grants-in-Aid for Scientific and Cancer Research from the Ministry of Education, Science, and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

lines of evidence have indicated that the major acceptor of the polymer is the enzyme itself rather than histones (21-23).

In a preceding paper (24), we demonstrated that poly(ADP- ribose) synthetase can be split into two fragments by mild digestion with papain, one retaining the ability to bind to DNA and the other containing the site(s) for poly(ADP- ribosy1)ation. Nevertheless, which fragments contain the site for binding of the substrate, NAD, and whether it is separable from the other two functional sites have remained to be answered. In order to elucidate the localization of the site for the binding of NAD in the enzyme molecule, we employed the technique of limited proteolysis by a-chymotrypsin in addi- tion to partial digestion by papain after chemical modification of the enzyme protein with 3-(bromoacety1)pyridine.

In this paper, evidence is presented to show that the enzyme consists of three different functional fragments, the first one of MI = 54,000 possessing the site for the substrate binding, the second one of MI = 46,000 retaining the site for DNA binding, and the third one of MI = 22,000 containing the site(s) for accepting poly(ADP-ribose).

EXPERIMENTAL PROCEDURES

[odenine-U-"C]NAD (286 mCi/mmol) from the Radiochemical Materials-Commercial sources of materials used were as follows:

Centre, Amersham; sodium b~ro[~H]hydride (347 mCi/mmol) from New England Nuclear; NAD, phenylmethylsulfonyl fluoride, calf thymus DNA, calf thymus histone H1, a-chymotrypsin (Type 11), bovine serum albumin, ovalbumin, and lysozyme from Sigma; 8- galactosidase and phosphorylase a from Boehringer Mannheim; pa- pain and snake venom phosphodiesterase from Worthington Bio- chemical Corp.; polyethyleneimine cellulose plates from Macherey- Nagel; Tonein TP from Otsuka Assay Laboratories. All other chem- icals were from Nakarai Chemicals and were of analytical grade. Ep- 475, a specific inhibitor of papain (25), was a generous gift from Dr. T. Murachi, Department of Clinical Science, Kyoto University Hos- pital. Calf thymus was generously supplied by Dr. A. Ikenoue at Kochi Municipal Slaughterhouse.

Poly(ADP-ribose) synthetase was purified from fresh calf thymus by the method of Ito et al. (10) with a slight modification. The purified enzyme was stored at -70 "C in 50 mM Tris-HC1 buffer (pH 8.0) containing 10% glycerol, 1 M NaCl, and 10 mM 2-mercaptoethanol.

DNA-cellulose was prepared according to the method of Albert and Herrick (26). The DNA-cellulose thus prepared contained approxi- mately 3 mg of DNA/ml of packed volume. Fragmented DNA with 20-30 base pairs was prepared by incubating calf thymus DNA (0.5 mg) at 27 'C for 1 min with 20 pg of DNase I in a buffer containing 0.1 M Tris-HC1 (pH 8.0) and 10 mM MgC12 in a total volume of 1 ml.

Preparation of 3-(Bromoacetyl)pyridihe"-(Bromoacetyl)pyridine was prepared by bromination of 3-acetylpyridine as described by Woenckhaus et al. (27), and recrystallized three times from glacial acetic acid. The sample thus obtained was subjected to thin layer chromatography on polyethyleneimine cellulose and found to migrate as a single spot using both solvent systems of 0.25 M LiCl (RF 0.64) and 1-butanol/pyridine/HzO (l:l:l, v/v) (RF 0.67) when detected by 5,5'-dithiobis(2-nitrobenzoic acid) spray for reactive halogen com-

4770

by guest on March 29, 2019

http://ww

w.jbc.org/

Dow

nloaded from

Three Functional Domains of Poly(ADP-Ribose) Synthetase 4771

pounds (28) and by visualization with ultraviolet irradiation. Quan- titative determination of 3-(bromoacety1)pyridine was performed by its reaction with glutathione as described by Hartman (29). Aqueous solutions of 3-(bromoacety1)pyridine were capable of storage frozen at -70 'C for several months without noticeable change.

Assay of Poly(ADP-Ribose) Synthetase-The standard reaction mixture contained 100 mM Tris-HC1 (pH 8.0), 10 mM MgCl,, 2 mM dithiothreitol, 100 p~ [adenine-U-"C]NAD (5500 dpm/nmol), 10 pg each of calf thymus DNA and calf thymus histone H1, and varying amounts of enzyme in a total volume of 0.1 ml. The reaction was carried out a t 25 "C for 3 min unless otherwise specified and termi- nated by the addition of 5 ml of 10% trichloroacetic acid. The radioactivity incorporated into trichloroacetic acid-insoluble material was collected on Whatman GF/C glass fiber filter discs and quanti- tated in a Searle Analytic Mark I11 liquid scintillation spectrophoto- meter using 5 ml of a scintillation fluid containing 0.5%, 2,5-diphen- yloxazole and 0.02% 1,4-bis[2-(4-methyl-5-phenyloxazolyl)]benwne in toluene.

Labeling of the Enzyme-The enzyme (80 pg) was modified at 25 'C with 0.2 mM 3-(bromoacety1)pyridine in 50 mM Hepesl-NaOH buffer (pH 8.0) containing 10% glycerol and 0.2 M NaCl (Buffer A) in the presence and absence of 5 mM NAD in a final volume of 200 pl. In the control experiments, the enzyme was incubated at 25 "C in the same solvent system without the reagent. After 5 min, dithiothreitol was added to the mixture a t a final concentration of 2 mM to terminate the reaction. To the solution was then added 80 pl of 20 mM sodium b~ro[~H]hydride (347 mCi/mmol) and the mixture was allowed to stand at 25 'C for 60 min to reduce enzyme-bound acetylpyridine. When 10 mM nicotinamide was employed in place of 5 mM NAD, the modified enzyme was labeled using 50 p1 of 5 mM sodium b ~ r o [ ~ H ] hydride. The reaction mixture was then dialyzed exhaustively against 50 mM Tris-HC1 buffer (pH 8.0) containing 10% glycerol, 0.1 M NaC1, and 10 mM 2-mercaptoethanol (Buffer B) until no radioactivity was detected in this buffer solution. Under these conditions, exchangeable tritium incorporated into the unmodified enzyme in the control experiments was almost completely removed. The labeled enzyme thus prepared was then diluted with Buffer B to a concentration of 100 pg of protein/ml, and subjected to limited proteolysis with a- chymotrypsin or with papain.

Proteolytic Digestion with a-Chymotrypsin or with Papain-The purified preparation of poly(ADP-ribose) synthetase (100-200 pg/ml) in Buffer B was digested at 25 "C with a-chymotrypsin in a protein ratio of 501. After an appropriate time of digestion, the reaction was terminated by adding either trichloroacetic acid at a final concentra- tion of 20% or phenylmethylsulfonyl fluoride at a final concentration of 1 mM. Digestion of the enzyme with papain was performed under the same conditions as those with a-chymotrypsin except that the incubation was performed at 0 "C and that the reaction was termi- nated by the addition of trichloroacetic acid at a final concentration of 20% or Ep-475 at a final concentration of 2.5 pg/ml.

Polyacrylnmide Gel Electrophoresis-The enzyme sample digested was precipitated with 20% trichloroacetic acid, washed twice with ethyl ether, and dissolved in 30 pl of 5 mM potassium phosphate (pH 7.0) containing 1% SDS, 50 mM dithiothreitol, 10% glycerol, and 0.003% bromphenol blue. The resulting solution was heated at 100 "C for 3 min and subjected to electrophoresis. In the standard analysis, gel electrophoresis in the presence of SDS was carried out by the method of Studier (30) with some modifications; the gel contained 25 mM Tris, 190 mM glycine (pH 8.3), 0.1% SDS, 2 mM EDTA, and 6% acrylamide. After electrophoresis, protein was stained with Coomassie blue as described by Chrambach et al. (31). For the analysis of proteins which had been poly(ADP-ribosy1)ated with [adenine-U-"C]NAD, a gel system of 0.1 M sodium phosphate (pH 7.0), 0.1% SDS, and 7.5% acrylamide was used (32).

Detection of Radioactivity in SDS-Polyacrylamide Gels-The gels, destained with 7% acetic acid, were soaked in 30% methanol for 20 min and then kept in 1 M sodium salicylate solution for 15 min. They were dried on Whatman 3MM filter paper and exposed to K o a X- Omat R film at -70 "C (33). For quantitative determination of radioactivity, SDS-polyacrylamide gels used for fluorography were cut into 5-mm pieces and oxidized with Tri-Carb sample oxidizer C-

The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-pipera- zineethanesulfonic acid; SDS, sodium dodecyl sulfate; 54K and 41K fragments, fragments of M. = 54,000 and 41,000 (other forms are anaiogous).

306, Packard, and the radioactivity was determined using a liquid scintillation spectrometer.

Autopoly(ADP-ribosyUation of Synthetase-The purified prepara- tion of poly(ADP-ribose) synthetase (40 pg) was incubated at 25 "C for 1 min with 1.4 p~ [adenine-U-"CINAD (423 dpm/pmol) in 800 pl of 100 mM Tris-HC1 (pH 8.0) containing 10 mM MgC12, 1 mM dithiothreitol, 10% glycerol, and 100 pg/ml of DNA. The reaction was terminated by the addition of nicotinamide at a final concentra- tion of 10 mM.

Other Methods-Protein was determined by the method of Lowry et al. (34) using bovine serum albumin as a standard after samples were made free of interfering substances (35). Protein concentration of fractions eluted from the DNA-cellulose column was determined by measuring protein-dye complex formation (36). TO 1 ml of Tonein TP, 50 pl of each fraction (0.5-15 pg of protein) was added and the mixture was vortexed. After 5 min, protein was estimated by meas- uring the absorption at 595 nm using bovine serum albumin as a standard. The determination of an average chain length of poly(ADP- ribose) was performed principally by the method of Yamada and Sugimura (37) using thin layer chromatography on a polyethylene- imine cellulose plate as described in the preceding paper (24).

RESULTS

Digestion of Synthetase with a-Chymotrypsin-When poly(ADP-ribose) synthetase was incubated at 25 "C with a- chymotrypsin in a protein ratio of 501, the enzyme was cleaved into a number of fragments which were separated by SDS-polyacrylamide gel electrophoresis (Fig. IA). In the early stage of the digestion, there appeared two major fragments with the simultaneous disappearance of the native enzyme. The molecular weights of these fragments were estimated to be 66,000 and 54,000, respectively, by comparison with refer- ence proteins. Since the molecular weight of the native en- zyme was estimated to be 120,000, it was concluded that a- chymotrypsin cleaved the enzyme into these two fragments. Upon prolonged incubation, the 54K fragment was further cleaved into two fragments of M, = 41,000 and 15,000, re- spectively. The 66K fragment, however, was resistant to fur- ther digestion with a-chymotrypsin. In parallel with the dis- appearance of the intact enzyme, poly(ADP-ribose) synthe- tase activity was lost progressively (Fig. 1B). The addition of calf thymus with highly polymerized or fragmented DNA to the reaction mixture affected neither the rate nor the pattern of digestion as judged by SDS-polyacrylamide gel electropho- resis.

Identification of the Fragment Containing the Substrate- binding Site-When poly(ADP-ribose) synthetase was incu- bated at 25 "C with 0.2 mM 3-(bromoacetyl)pyridine, the enzyme was rapidly inactivated (Fig. 2). However, inactiva- tion by this reagent was significantly prevented by the pres- ence of 5 mM nicotinamide, a potent competitive inhibitor with respect to the binding of the substrate, NAD (38, 39). The addition of fragmented DNA had no effect on the rate of inactivation both in the absence and presence of nicotinamide. These results suggest that nicotinamide protected the enzyme from inactivation by 3-(bromoacety1)pyridine by binding to the catalytic site of poly(ADP-ribose) synthetase.

To locate the substrate-binding site of poly(ADP-ribose) synthetase, the enzyme modified with 3-(bromoacety1)pyr- idine and then labeled with sodium bar~[~H]hydride as de- scribed under "Experimental Procedures," was digested with a-chymotrypsin and subjected to SDS-polyacrylamide gel electrophoresis. As shown in Fig. 3, left, the radioactivity was retained on the four main protein bands which corresponded to the native enzyme (120K) and the 66K, 54K, and 41K fragments, respectively. When the modification was carried out in the presence of nicotinamide, incorporation of tritium into the 54K protein band significantly decreased in compar- ison with that of the 66K fragment. Under these conditions,

by guest on March 29, 2019

http://ww

w.jbc.org/

Dow

nloaded from

4772

A

Three Functional Domains of Poly(ADP-Ribose) Synthetase

15K-

0

- a 1 5 10 30

Time (mid

B

" 0 10 20 30

Time (mid FIG. 1. Digestion and inactivation of poly(ADP-ribose) syn-

thetase with a-chymotrypsin. The enzyme (40 pg) in Buffer B was incubated at 25 'C with 0.8 pg of a-chymotrypsin in the presence and absence of calf thymus DNA (100 pg/ml) in a to t a l volume of 200 pl. At the indicated times, aliquots (35 pl ) were taken and subjected to SDS-polyacrylamide gel electrophoresis. Simultaneously, aliquots (10 pl ) were transferred to the assay mixture to determine the residual enzyme activity as described under "Experimental Pro- cedures." A , gel electrophoretic patterns of poly(ADP-ribose) synthe- tase digested with a-chymotrypsin in the absence of calf thymus DNA. Standard proteins used for molecular weight markers were as follows; p-galactosidase (116,000), phosphorylase a (95,000), bovine serum albumin (68,OOO), ovalbumin (43,000), and lysozyme (14,400). E , time course of inactivation of the enzyme by a-chymotrypsin in the presence (0) or absence (0) of DNA. As a control, the enzyme sample was incubated without a-chymotryspin (A).

the native enzyme and the 41K portion were also protected from modification to a lesser extent. Considering the fact that the 41K fragment is derived from the 54K fragment (Fig. I) , it is reasonable to conclude that the substrate-binding site is located in the 54K fragment.

The labeled enzyme was also digested with papain to further elucidate the localization of the substrate-binding site (Fig. 3, right). After digestion, there appered two predominant frag- ments of M, = 74,000 and 46,000 as shown in Fig. 3C. In this case, incorporation of tritium into the 74K portion of the enzyme was markedly protected by nicotinamide (Fig. 3D), indicating that the substrate-binding site is located in the 74K fragment. Weak protection was also observed in a minor 56K band, which was derived from the 74K fragment (24).

To establish the above lines of evidence, the substrate, NAD, was employed for protecting the active site of the

I I

0 0 5 10 15

T ~ w (mh)

FIG. 2. Inactivation of poly(ADP-ribose) synthetase by 3- (bromoacety1)pyridine in the absence or presence of nicotin- amide. The enzyme (16 pg) was incubated at 25 "C with 0.2 mM 3- (bromoacety1)pyridine in 50 pl of Buffer A in the absence (open symbols) or presence (closed symbols) of 5 mM nicotinamide. The chemical modification was also carried out in the presence (0, W) or absence (0 ,O) of 0.25 pg of fragmented DNA. A t the indicated times, aliquots (10 pl ) were removed from the incubation mixture and assayed for the residual enzyme activity as described under "Experi- mental Procedures." As a control, the enzyme sample was incubated without 3-(bromoacetyl)pyridine (A).

enzyme against modification with 3-(bromoacetyl)pyridine. As shown in Fig, 4, predominant protection from the modifi- cation was observed in the 54K fragment (Fig. 4, left) and in the 74K fragment (Fig. 4, right). Weak protection was also observed in the minor protein bands of M, = 41,000 and 56,000 which were derived from the 54K fragment and the 74K fragment, respectively. On the other hand, the modifi- cation of the 66K and 46K portions were not protected by NAD. These results, taken together, indicate that the site for the binding of the substrate, NAD, is localized in the 54K portion of the native enzyme.

Identification of the Fragment Containing the DNA-binding Site-In order to determine which fragments possess the DNA-binding site, column chromatography on DNA-cellulose was carried out. The enzyme fragents obtained by digestion with a-chymotrypsin was applied to a column of DNA-cellu- lose equilibrated with Buffer B, and eluted with a linear concentration gradient of NaCl from 0.1 to 1.2 M. As shown in Fig. 5A, two protein peaks were detected; one passed through the column when washed with Buffer B (peak I), and the other eluted at approximately 0.6 M NaCl (peak 2). Aliquots of these two peaks were subjected to SDS-polyacryl- amide gel electrophoresis. As shown in Fig. 5B, peak 1 con- tained the 54K and 41K fragments, and peak 2 contained the 66K fragment. When the native enzyme was applied onto the column, the enzyme was eluted at the same salt concentration as that of the 66K fragment. These results indicate that the DNA-binding site was located on the 66K fragment. The 66K fragment was resistant to further digestion with a-chymo- trypsin, but it was cleaved by papain into two fragments of M, = 46,000 and 22,000 (Fig. 6B). To determine which of these two fragments, like the parent intact enzyme, has affin- ity for DNA, chromatography on DNA-cellulose was again carried out. When the two fragments were passed over the column equilibrated with Buffer B, the 22 K fragment was slightly retarded relative to the breakthrough fractions, indi- cating that this fragment has weak affinity for DNA. On the other hand, the 46K fragment was bound tightly to the column under the above conditions. When elution was performed using a linear salt gradient between 0.1 and 1.2 M, the protein peak of the 46K fragment appeared at 0.6 M NaCl (Fig. 6). These findings indicate that the 46K fragment contains the

by guest on March 29, 2019

http://ww

w.jbc.org/

Dow

nloaded from

Three Functional Domains of Poly(ADP-Ribose) Synthetase 120K 66KWK t t t @ - ""

4000 - A

1

30001 66K 54K

t t

I I 0 2 4 6 8 1 0 1 2

Migration Distance ( c d

120K 74K 46K t t t

9 6000-

V

* r .

4000- m V

3000- D 74K 46K t t

I 0 2 4 6 S 1 0 1 2

4113

FIG. 3. Digestion of poly(ADP-ribose) synthetase with a-chymotrypsin or papain after modification with 3-(bromoacety1)pyridine in the presence and absence of nicotinamide. The enzyme (80 pg) in Buffer A was incubated at 25 "C with 0.2 mM 3-(bromoacety1)pyridine in the presence and absence of 10 mM nicotinamide in a total volume of 200 pl. After 5 min, the reaction was terminated by adding dithiothreitol a t a final concentration of 2 mM. To the solution was then added 50 pl of 5 mM sodium b~ro[~H]hydride (347 mCi/mmol) and the mixture kept a t 25 "C for 60 min to reduce enzyme-bound acetylpyridine. The mixture was then dialyzed at 4 'C overnight with two changes against 1 liter of Buffer B. An aliquot (25 pg) of the enzyme thus labeled was digested with 0.5 pg of a-chymotrypsin or 0.5 pg of papain in a total volume of 250 11. The digestion of the enzyme was terminated by the addition of trichloroacetic acid at a final concentration of 2076, and the enzyme fragments thus obtained were subjected to SDS-polyacrylamide gel electrophoresis; left, the enzyme incubated with a-chymotrypsin for 2 min; right, the enzyme digested with papain for 7 min. A and C, quantification of radioactivity in SDS- polyacrylamide gels as described under "Experimental Procedures." The enzyme was modified with 3-(bromoace- ty1)pyridine in the absence (0) or presence (0) of nicotinamide. B and D, the difference between the values denoted by 0 and 0 in A and C was replotted.

DNA-binding site of the enzyme. Identification of the Fragment Containing the Site(s) for

Accepting Poly(ADP-Ribose)-Poly(ADP-ribose) synthetase itself is known to be automodified with poly(ADP-ribose) (21-23). In order to locate the sites for poly(ADP-ribo- sy1)ation in the enzyme molecule, the enzyme previously automodified with ["CINAD was digested with a-chymotryp- sin or with papain, and analyzed by SDS-polyacrylamide gel electrophoresis. In this experiment, the poly(ADP-ribo- sy1)ation reaction was controlled by lowering the concentra- tion of ["CINAD (1.4 PM, 423 dpm/pmol) so that the increase in molecular weight due to the attachment of the polymer might not affect the electrophoretic mobility of the fragments. Under the conditions employed, 1.6 ADP-ribose units were incorporated per molecule of enzyme, and an average chain length of the polymer formed was 3.2 ADP-ribose units. The results of fluorography of an electrophoresed gel is shown in Fig. 7. When the enzyme previously automodified with ["C] NAD was digested with a-chymotrypsin, the radioactivity was detected only on the 66K fragment (Fig. 7b). To elucidate the localization of the site(s) for poly(ADP-ribosyl)ation, the 66K fragment was further digested by papain and again analyzed by SDS-polyacrylamide gel electrophoresis. As shown in Fig. 7c, the radioactivity due to poly(ADP-ribose) was detected exclusively in the protein band corresponding to the M, = 22,000. Upon prolonged digestion with papain, no additional band containing radioactivity appeared up to 120 min. These results indicate that the site(s) for poly(ADP-ribosy1)ation is localized in the 22K fragment.

DISCUSSION

In the present study, three functional domains of poly- (ADP-ribose) synthetase have been separated from each other by limited proteolysis with a-chymotrypsin and papain. The first domain of M, = 54,000 possesses the site for the substrate binding, the second one of M, = 46,000 retains the site for DNA binding, and the third one of M, = 22,000 contains the site(s) for accepting poly(ADP-ribose) (Fig. 8). It may be pointed out, however, that small fragments other than these three fragments are released in the process of limited prote- olysis and not detected on SDS-polyacrylamide gel electro- phoresis under the present conditions, because the primary structure of each fragment is not yet fully determined. Never- theless, total amino acid analyses of these fragments: in addition to our present data on the molecular size of each fragment, support the above conclusion that the enzyme es- sentially consists of these three fragments.

The substrate-binding domain of poly(ADP-ribose) synthe- tase was deduced by chemcal modification with 3-(bromoace- ty1)pyridine in the presence and absence of NAD or its analog, nicotinamide, followed by labeling with sodium b~ro[~H]hy- dride. When the labeled enzyme was subjected to SDS-poly- acrylamide gel electrophoresis after partial proteolytic diges- tion, the radioactivity was found in all of the chymotryptic

*These three fragments are easily separable from each other by chromatography and summation of amino acid compositions of these fragments coincides well with those of the intact poly(ADP-ribose) synthetase (M. Agemori, I. Kameshita, and Y. Shizuta, manuscript in preparation).

by guest on March 29, 2019

http://ww

w.jbc.org/

Dow

nloaded from

4774 Three Functional Domains of Poly(ADP-Ribose) Synthetase 12OK 6 6 K W

e t t t

A

12OK 74K 46K t t t - ,, 03

I I 0 2 4 6 6 1 0 1 2

Mlwatbn Mstance (cm)

FIG. 4. Digestion of poly(ADP-ribose) synthetase with a-chymotrypsin or papain after modification with 3-(bromoacety1)pyridine in the presence and absence of NAD. Modification of the enzyme in the presence of 5 mM NAD was carried out under the same conditions as described in Fig. 3, except that 80 pl of 20 mM sodium b~ro[~H]hydride (347 mCi/mmol) was used for the labeling of the modified enzyme. The enzyme sample (25 pg) thus labeled was digested with 0.5 pg of a-chymotrypsin or 0.5 pg of papain and subjected to SDS- polyacrylamide gel electrophoresis; left, the enzyme digested with a-chymotrypsin for 2 min; right, the enzyme digested with papain for 5 min. A and C, quantification of the radioactivity in SDS-polyacrylamide gels as described under "Experimental Procedures." The enzyme was modified with 3-(bromoacety1)pyridine in the absence (0) or presence (0) of nicotinamide. B and D, the difference between the values denoted by 0 and 0 in A and C was replotted.

A B 0 1 2

loor 1 ' 2 1 I so' t ,

t t 1.2

6 0 . 1 .o . rn , 0.8 d -

' 0.6 y . 0.4

. 0.2

0 5 10 15 20 25 ' 0

+ 66K + 54K

+41K

+15K

Elution Volume (ml)

FIG. 5. DNA-cellulose column chromatography of the en- zyme fragments obtained by digestion with a-chymotrypsin. The enzyme (400 pg) in Buffer B was incubated at 25 "C with 8 pg of a-chymotrypsin in a total volume of 4 ml. After 20 min, phenylmeth- ylsulfonyl fluoride at a final concentration of 1 mM was added to the reaction mixture. The mixture was then applied onto a column (0.7 X 2.6 cm) of DNA-cellulose which was equilibrated with Buffer B. After washing the column with 3 ml of Buffer B, elution was carried out with a linear concentration gradient of NaCI, from 0.1 to 1.2 hi, 10 ml each in Buffer B solution. Protein in each fraction (580 pl) was determined using an aliquot (50 pl) as described under "Experimental Procedures." The concentration of NaCl was estimated by measuring the value of conductivity of each fraction. A , elution profile of DNA- cellulose column chromatography. B, SDS-polyacrylamide gel elec- trophoretic patterns showing the enzyme fragments obtained by digestion with a-chymotrypsin (O), components of peak 1 (I), and of peak 2 (2), respectively.

fragments (54K and 66K) and the papain digests (74K and 46K). Among these fragments, selective protection from the labeling was observed in the 54K and 74K fragments when

A 0 0 1 2

50 1 z ' .

+ 46K

+ 22K

Elution Volume (ml)

FIG. 6. DNA-cellulose column chromatography of the frag- ments obtained by digestion of the 66K fragment with papain. The 66K fragment prepared by DNA-cellulose column chromatogra- phy as shown in Fig. 5 was dialyzed at 4 "C for 3 h against 500 ml of Buffer B. The 66K fragment (140 pg) was incubated at 0 "C for 10 min with 2.8 pg of papain in a total volume of 1.8 ml. To the mixture was then added Ep-475 at a final concentration of 2.5 pg/ml to terminate further digestion. Column chromatography on DNA-cel- lulose and SDS-polyacrylamide gel electrophoresis were carried out in the same manner as described in Fig. 5. A, elution profile of DNA- cellulose column chromatography. B, SDS-polyacrylamide gel elec- trophoretic patterns showing the fragments obtained by digestion of the 66K fragment with papain (01, components of peak 1 (I), and of peak 2 (2), respectively.

NAD or its analog was included in the reaction mixture. The covalent and nonspecific incorporation of tritium observed in the 66K and 46K fragments appears to be derived from acetylpyridine attached to some cysteinyl residues which are not required for the bindinn of the substrate. NAD.3 There-

I. Kameshita and Y. Shizuta, unpublished results.

by guest on March 29, 2019

http://ww

w.jbc.org/

Dow

nloaded from

Three Functional Domains of Poly(ADP-Ribose) Synthetase 4775 F 3

120K*-

6 6 K 4

22K-1

a

c 54K * 46K

b C FIG. 7. Digestion of automodified poly(ADP-ribose) synthe-

tase with a-chymotrypsin and with papain. Poly(ADP-ribose) synthetase (40 pg) was incubated at 25 "C for 1 min with 1.4 p~ [odenine-U-"C]NAD (423 dpm/pmol) in 100 mM Tris-HC1 (pH 8.0) containing 1 mM dithiothreitol, 10 mM MgC12, 10% glycerol, and 100 pg/ml of calf thymus DNA in a final volume of 800 pl. To the mixture was then added nicotinamide at a final concentration of 10 mM. The enzyme (37.5 pg) thus automodified was digested with a-chymotryp- sin (0.75 pg) at 25 "C for 20 min in a to t a l volume of 750 pl. The digestion was terminated by the addition of phenylmethylsulfonyl fluoride at a final concentration of 1 mM. The enzyme (35 pg) treated with a-chymotrypsin was further digested at 0 "C for 5 min with papain (0.7 pg). In each step of digestion, aliquots (50 pl) were removed and subjected to SDS-polyacrylamide gel electrophoresis and the radioactivity was detected by fluorography as described under "Experimental Procedures." a, the automodified enzyme, b, the en- zyme fragment after digestion with a-chymotrypsin, and c, the frag- ment obtained by digestion with papain after a-chymotryptic diges- tion.

a-Chymotrypsin A .I. I 54K 66K I

Substrate Binding DNA Binding & Autanodflcatkn

B Papain .I.

I 74K 46K 1 Substrate Binding 6 Automoaffcation DNA Binding

1

L + . I . I 54K I 22K [ 46K I - -

Substrate Binding DNA Binding

" FIG. 8. Schematic representation for the domain structure

of poly(ADP-ribose) synthetase. The chymotryptic and papain cleavage sites on the enzyme molecule are indicated by arrows. The lejt end may not represent the NH2 terminus of the native enzyme as end group analysis of each fragment was not performed.

fore, it is reasonable to conclude that the substrate-binding domain of the enzyme is located on the 54K portion of the native enzyme (Fig. 8).

We previously determined that the DNA-binding site is localized on the 46K fragment and the acceptor site is on the 74K fragment when the enzyme is partially digested with

papain (24). In the present study, we have demonstrated that both sites for DNA and for the acceptor are localized on the 66K fragment when the enzyme was digested with a-chymo- trypsin (Figs. 5 and 7). Furthermore, the poly(ADP-ribo- sy1)ated 22K fragment was isolated when the automodified enzyme was digested with a-chymotrypsin and then with papain (Fig. 7). The molecular weights of the other products formed and detected so far in this experiment were assigned as 54,000 and 46,000. These results, taken together, indicate that the acceptor domain of M, = 22,000 is located in between the two other domains for the binding of DNA and NAD (Fig. 8C).

When the enzyme was digested with a-chymotrypsin, the enzyme activity was lost progressively in parallel with the disappearance of the native enzyme (Fig. 1). The addition of DNA to the reaction mixture affected neither the rate nor the extent of inactivation of the enzyme by this protease. In contrast, the digestion with papain was somewhat retarded by the presence of DNA and nearly 20% of the initial activity was detected even after the enzyme was completely split into the 74K and 46K fragments (24). These differences might be derived from the difference in the sites of the primary se- quence of poly(ADP-ribose) synthetase which are attacked by these two different proteases. Both the 46K fragment and the 74K fragment as obtained by digestion with papain have affinity for the DNA ligand although the latter fragment retains less weak affinity relative to the former one. Therefore, these two fragments might maintain their original confor- mation to some extent by interacting with DNA and exhibit a weak catalytic function. In contrast, the 66K and 54K fragments as obtained by digestion with a-chymotrypsin ap- pear to lose the original conformation almost completely because the 54K fragment is not able to bind to the DNA ligand.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

REFERENCES Hayaishi, O., and Ueda, K. (1977) Annu. Rev. Biochem. 46,95- 116

Purnell, M. R., Stone, P. R., and Whish, W. J. D. (1980) Biochem. Soc. Trans. 8,215-227

Berger, N. A., Sikorski, W. G., Petzold, S. J., and Kurosawa, K. (1979) J. Clin. Invest. 63, 1164-1171

Durkacz, B. W., Omidiji, O., Gray, D. A., and Shall, S. (1980) Nature (Lord.) 283,593-596

Burzio, L., and Koide, S. S. (1970) Biochem. Biophys. Res. Com- mun. 40,1013-1020

Muller, W. E. G., Tetsuka, A., Nusser, I., Obermeier, J., Rhode, H. J., and Zahn, R. K. (1974) Nucleic Acids Res. 1, 1317-1327

CaDlan. A. I., and Rosenberp, M. J. (1975) Proc. Natl. Acad. Sci. U . S:A. 72, 1852-1857

-. . .

Mandel, P., Okazaki, H., and Niedergang, C. (1977) FEES Lett. 84.331-336

Yoshihara, K., Hashida, T., Tanaka, Y., Ohgushi, H., Yoshihara,

Ito, S., Shizuta, Y., and Hayaishi, 0. (1979) J. Bbl. Chem. 254,

Okayama, H., W o n , C. M., Fukushima, M., Ueda, K., and

Kristensen, T., and Holtlund, J. (1978) Eur. J . Biochem. 88,

Tsopanakis, C., Leeson, E., Tsopanakis, A., and Shall, S. (1978)

Petzold, S. J., Booth, B. A., Leimbach, G. A., and Berger, N. A.

Agemori, M., Kagamiyama, H., Nishikimi, M., and Shizuta, Y.

Ohgushi, H., Yoshihara, K., and Kamiya, T. (1980) J. BwL Chem.

Benjamin, R. C., and Gill, D. M. (1980) J. Bwl. Chem. 255,

Nishizuka, Y., Ueda, K., Honjo, T., and Hayaishi, 0. (1968) J.

H., and Kamiya, T. (1978) J. Biol. Chem. 253,6459-6466

3647-3651

Hayaishi, 0. (1977) J. Bwl. Chem. 252,7000-7005

495-501

Eur. J . Biochem. 90,337-345

(1981) Biochemistry 20, 7075-7081

(1982) Arch. Biochem. Biophys. 215,621-627

255,6205-6211

10502-10508

Biol. Chem. 243,3765-3767

by guest on March 29, 2019

http://ww

w.jbc.org/

Dow

nloaded from

4776 Three Functional Domains of Poly(ADP-Ribose) Synthetase

19.

20.

21.

22.

23.

24.

25.

26.

27.

Okayama, H., Ueda, K., and Hayaishi, 0. (1978) Proc. Natl. Acad. Sci. U. S. A. 75,1111-1115

Ueda, K., Omachi, A., Kawaichi, M., and Hayaishi, 0. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 205-209

Yoshihara, K., Hashida, T., Yoshihara, H., Tanaka, Y., and Ohgushi, H. (1977) Biochem. Biophys. Res. Commun. 78,1281- 1288

Ogata, N., Ueda, K., Kawaichi, M., and Hayaishi, 0. (1981) J. Biol. Chem. 266,4135-4137

Jump, D. B., and Smulson, M. (1980) Biochemistry 19, 1024- 1030

Nishikimi, M., Ogasawara, K., Kameshita, I., Taniguchi, T., and Shizuta, Y . (1982) J. Biol. Chem. 257,6102-6105

Tamai, M., Hanada, K., Adachi, T., Ogawa, K., Kashiwagi, K., Omura, S., and Ohzeki, M. (1981) J. Biochem. (Tokyo) 91,

Albert, B., and Herrick, G. (1971) Methods Enzyml. 21, 198-

Woenckhaus, V. C., Berghauser, J., and Pfleiderer, G. (1969)

255-257

217

Hoppe-Seykr’s Z. Physiol. Chem. 350,473483 28. Schloss, J. V., and Hartman, F. C. (1977) Anal. Biochem. 83,

29. Hartman, F. C. (1981) Biochemistry 20,894-898 30. Studier, F. W. (1973) J. Mol. Biol. 79, 237-248 31. Chrambach, A., Reisfeld, R. A., Wyckoff, M., and Zaccari, J.

32. Zahler, L. W. (1974) Methods Enzyml . 32.70-81 33. Chamberlain, J. P. (1979) Anal. Biochem. 98, 132-135 34. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.

35. Bensadoun, A., and Weinstein, D. (1976) Anal. Biochem. 70,

36. Bradford, M. M. (1976) A w l . Biochem. 72,248-254 37. Yamada, M., and Sugimura, T. (1973) Biochemistry 12, 3303-

38. Stone, P. R., and Shall, S. (1973) Eur. J. Biochem. 38, 146-152 39. Niedergang, C., Okazaki, H., and Mandel, P. (1979) Eur. J.

185-188

(1967) Anal. Biochem. 20,150-154

(1951) J. Biol. Chem. 193,265-275

241-250

3308

Biochem. 102,43-57

by guest on March 29, 2019

http://ww

w.jbc.org/

Dow

nloaded from

I Kameshita, Z Matsuda, T Taniguchi and Y Shizutaautomodification domain.

fragments as the substrate-binding domain, the DNA-binding domain, and the Poly (ADP-Ribose) synthetase. Separation and identification of three proteolytic

1984, 259:4770-4776.J. Biol. Chem. 

  http://www.jbc.org/content/259/8/4770Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/259/8/4770.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on March 29, 2019

http://ww

w.jbc.org/

Dow

nloaded from