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Proc. Natl. Acad. Sci. USA Vol. 76, No. 2, pp. 595-599, February 1979 Biochemistry

Structure of poly( diphosphate ribose): Identification of 2'-[1 "-ribosyl-2"-(or 3"-) (1 "'-ribosyl)]adenosine-5',5",5"'- tris() as a branch linkage (NAD+/ribose-ribose-ribose bond/mass spectrometry) MASANAO MIWA*, NORIKO SAIKAWA*, ZIRO YAMAIZUMIt, SUSUMU NISHIMURAt, AND TAKASHI SUGIMURA* *Virology Division and tBiology Division, National Cancer Center Research Institute, Tokyo 104, Japan Communicated by Paul C. Zamecnik, October 19, 1978

ABSTRACT Poly([14C] ribose) was MATERIALS AND METHODS synthesized from [14C]NAD+ with caf th mus nuclei. The fraction containing poly(adenosine diphosp ate ribose) eluted Poly([14C]ADP-Rib) was synthesized enzymatically from with 0.22-0.40 M phosphate buffer (pH 6.8) from a hydroxyla- [Ade-14C]NAD+ (9.0 X 105 cpm/,gmol of ADP-Rib or 6.4 X 103 patite column, was completely hydrolyzed with venom phos- cpm/,umol of ADP-Rib) with calf thymus nuclei as the phodiesterase, and was separated by DEAE-Sephadex A-25 source (17, 18). column chromatography in 7 M urea. A new compound, which eluted with 0.22-0.40 M constituted 2% of the products from poly(adenosine diphos- Poly([14C]ADP-Rib), phosphate phate ribose), was found in addition to the expected prod- buffer from a hydroxylapatite column (17), was concentrated ucts-i.e., 5'-AMP, 2'41"-ribosyl)adenosine-5,5# SPEW), and hydrolyzed with snake venom phosphodiesterase (EC and its derivatives. This compound was identified as 2'{1"- 3.1.4.1) (Worthington) that had been further purified by the ribosyl-2"-or 3"-X1'-ribosyl)Jadenosine-5',5",5'-tris(phosphate). method of Oka et al. (19). The incubation mixture of 1.5 ml The existence of this compound is evidence of a branching contained 0.4 mg of poly(['4C]ADP-Rib), 30,umol of potassium structure of poly(adenosine diphos hate ribose), which was phosphate buffer (pH 7.5), 1.5 of MgCl2, 525 ,ug of bovine previously thought to be a linear molecule. The content of this Amol compound suggests that the frequency of branching is about 1 serum albumin, and 2 units of snake venom phosphodiesterase. per 20-30 adenosine diphosphate ribose residues of high mo- The reaction mixture was incubated at 37°C for 2-3 hr. The lecular weight poly(adenosine diphosphate ribose). complete hydrolysis was checked by subjecting small samples to chromatography on thin-layer sheets in a mixture A chromatin-bound enzyme located in eukaryotic cell nuclei of isobutyric acid, concentrated ammonium hydroxide, and catalyzes polymerization of NAD+ molecules to form poly- H20 (66:1:33, vol/vol) and confirming the absence of radio- (adenosine diphosphate ribose)[poly(ADP-Rib)] with liberation activity of poly([14C]ADP-Rib) at the origin. of nicotinamide molecules (1-3). The structure of poly(ADP- The snake venom phosphodiesterase hydrolysate was mixed Rib) differs from those of polynucleotides and acid polysac- with urea at a final concentration of 7 M and applied to a charides in having a-(1"- 2')ribose-ribose glycosidic linkages DEAE-Sephadex A-25 column (0.5 X 70 cm) equilibrated with and linkages (4-6). 50 mM Tris-HCI, pH 7.5/7 M urea (20). The peak fractions Poly(ADP-Rib) synthesis may be involved in regulation of were designated as peaks A, B, and C (Fig. 1A) pooled and de- DNA synthesis (7), repair synthesis of DNA (8, 9), cell density salted as described by Rushizky and Sober (21). The recovery (10), cell differentiation (11), transformation (12), and chro- of the material from the column was 90%. matin structure (13). A high titer of antibody activity for pol- The content of was determined from the ultraviolet y(ADP-Rib) was found in the serum of patients with systemic absorption by taking the molar extinction coefficient of the lupus erythematosus (14). It has long been thought that pol- adenine residue as 15,400 at 259 nm and pH 7.5 (22). The y(ADP-Rib) is a linear molecule (1-3). content of was estimated by the orcinol method of Recently, poly(ADP-Rib) has been separated according to Mejbaum (23). The pentose in the peak C compound was its size (up to 65 residues of ADP-Rib) (15, 16). The chain length identified as ribose by the method of Robinson et al. (24). The of the fractions, estimated as the ratio of total residues of material was hydrolyzed with acid, treated with alkaline ADP-Rib to the number of termini after complete hydrolysis phosphomonoesterase, and fractionated by paper chromatog- (15), never exceeded 30 (16). This finding is only explainable raphy with the upper layer of pyridine/ethyl acetate/H20 if poly(ADP-Rib) has a branched structure. Moreover, electron (1:3.6:1.15, vol/vol) (25). The paper chromatogram was then microscopy revealed the presence of a poly(ADP-Rib) popu- stained with alkaline AgNO3. Organic phosphate was deter- lation of high molecular weight with branched structures (K. mined by the method of Ames and Dubin (26). The phloro- Hayashi, M. Tanaka, T. Shimada, M. Miwa, and T. Sugimura, glucinol reaction was carried out by the method of Dische and unpublished data). Therefore, we examined the branching Borenfreund (27). Periodate consumption was assayed by the structure of procedure of Dixon and Lipkin (28). Periodate concentration poly(ADP-Rib). was determined by the procedure of Marinetti and Rouser (29). This paper reports the existence of the structure, 2'-[1"-ri- Reducing was measured by the ferricyanide method of bosyl-2"-(or3"-)(1 "'-ribosyl)]adenosine-5',5",5"'-tris(phos- Park and Johnson (30). phate), as a branch linkage of poly(ADP-Rib). A mixture of the peak C compound (8.8 A260 units) in 140 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: ADP-Rib, adenosine diphosphate ribose; Ado(P)-Rib-P, vertisement" in accordance with 18 U. S. C. §1734 solely to indicate 2'-(1"-ribosyl)adenosine-5', 5"-bis(phosphate) equal to 2'-(5"-phos- this fact. phoribosyl)-5'-AMP; Ado-Rib, 2'-(1"-ribosyl)adenosine. 595 Downloaded by guest on October 1, 2021 596 Biochemistry: Miwa et al. Proc. Natl. Acad. Sci. USA 76 (1979) ,ul and 3 ,l (0.7 unit) of alkaline phosphomo- noesterase (EC 3.1.3.1) was incubated at pH 7.5 for 3 hr at 370C, and then 0.7 unit more enzyme was added and incuba- tion was continued for 1 hr at 370C. Complete hydrolysis was 60- checked by submitting a small sample to thin-layer chroma- aU 40- tography. The reaction product was purified by chromatog- 0 raphy with Whatman no. 40 paper and H20 as solvent. The spot 2020 with ultraviolet absorption corresponding to the dephosphor- ATP Front Ado(P)-Rib-P ylated peak C compound was eluted with H20 and evaporated > \ |Ado(P)-Rib-PAdo AMP to dryness. In this way 6.5 A260 units of material was recov- 80 AMP Ado ATP~ rn ered. 604 Mass spectral data were obtained from the dephosphorylated 40 peak C compound after its conversion to a volatile permethy- 0 r 1 I. . V . lated derivative. Permethylation was performed with 1 A2j0 20 described procedure (31). The unit of the compound by the 0 10 20 30 40 0 10 20 30 40 number of methyl groups introduced was determined by using Distance from origin, cm CH3I and C2H31, respectively, for the permethylation reaction. FIG. 2. Paper chromatography of peak C and peak B compounds Low-resolution mass spectra were obtained on a JEOL-O1SG-2 with two solvent systems. (Upper Left and Right) Peak C compound instrument with a direct inlet system (ionization voltage, 75 eV; (1300 cpm); isobutyric acid/concentrated ammonium hydroxide/water chamber temperature, 250°C; sample temperature, 180°C). (66:1:33, vol/vol). (Lower Left and Right) Peak B compound (1700 About 1/10th of the reaction mixture was introduced into the cpm); 0.1 M sodium phosphate buffer, pH 6.8/ammonium sulfate/ mass spectrometer. n-propanol (100:60:2, vol/wt/vol). RESULTS about -5, representing about 2% of the recovered material. No Isolation of the Branched Structure from Snake Venom peak C, peak A, peak B, nor the minor peak before A was ob- Phosphodiesterase Digestion Products of Poly(ADP-Rib). served when poly(ADP-Rib) was applied to the DEAE Sepha- Poly([14C]ADP-Rib) (1.6 X 106 cpm, 9.0 X 105 cpm/,umol of dex column without venom phosphodiesterase digestion, ADP-Rib residue), digested with snake venom phosphodies- indicating that the peak C compound is an integral part of to DEAE-Sephadex A-25 column chro- poly(ADP-Rib). Additional studies were focused on the struc- terase, was subjected ture of this peak C compound. matography (Fig. 1). Properties of Peak C Compound. The peak C compound If [Ade-14C]poly(ADP-Rib) were to have only a linear has a more negative charge than Ado(P)-Rib-P (Fig. 1), but it structure, the expected digestion products should be 5'-AMP is eluted before ppApp, which should have a negative charge from the termini (peak A), 2'-ribosyl adenosine-5',5"-bis- of -6. Thus it seems to have a negative charge of around -5. (phosphate) [Ado(P)-Rib-P] (peak B), and partially dephos- The peak C compound was distinct from known derivatives of phorylated forms of Ado(P)-Rib-P, namely 2'-(5"phosphori- poly(ADP-Rib), especially Ado(P)-Rib-P, in two solvent systems bosyl)adenosine and 2'-ribosyl-5'-AMP (minor peak before peak (Fig. 2). A). These products were identified by paper chromatography The peak C compound was treated with E. coli alkaline with two different solvent systems (32). Peaks A and B and the phosphomonoesterase and subjected to thin-layer chromatog- minor peak before A constituted 4, 93, and 2%, respectively, raphy (Fig. 3). Like AMP, ADP, ATP, and Ado(P)-Rib-P, un- of the recovered materials. These products have negative treated peak C compound behaved like a substance with a charges of -2 or -4 at pH 7.5. negative charge, and after treatment with alkaline phospho- Careful examination of the chromatograph after peak B monoesterase it moved faster than Ado-Rib. revealed a very small peak (peak C) with a negative charge of ATP 10I O. ADP B Ado-Rib Ado-Rib AMP n 84 Ado 4 Ado 4 > 61 0 Ade 4 ~~~~~~~~~~~Front -15, .1 Ade . 41 1. . Frnt 4 f '-.,. Front 0o 21 0 IO: Front V 10 20 Ado-Rib Ado-Rib 0 8o Ado 4 E 4 Ado o 61o Ade~ 4 140 160 - 2 Ade tcJ5 41 Front %I. 0 A C ,0 21 4 1 I L r A -- 0 410 15 U 5 1U I to 50 100 150 200 Distance from origin, cm Fraction FIG. 3. Peak C and B compounds eluted from the DEAE-Se- FIG. 1. DEAE-Sephadex A-25 column chromatography of hy- phadex A-25 column were desalted and an aliquot (1000 cpm) was drolysis products of poly([14CJADP-Rib) treated with venom phos- digested with 0.3 unit ofE. coli alkaline phosphomonoesterase at 370C phodiesterase. The poly([14C]ADP-Rib) hydrolysate (24 A2Ws units, for 30-190 min. The digests were spotted on PEI-cellulose (Upper 1.6 X 106 cpm) was applied to the column (0.5 X 70 cm). Linear gra- Left and Lower Left) or cellulose (Upper Right and Lower Right) dient elution was achieved with 200 ml of 50 mM Tris-HCl, pH 7.5/7 thin-layer sheets and developed with H20 as treated samples (o). M urea and 200 ml of 0.35 M NaCl/50 mM Tris-HCl, pH 7.5/7 M urea. Untreated peak C (500 cpm) and peak B (1000 cpm) were also de- The peaks eluted are named A, B, and C. 0, cpm X 10-4/ml; 0, veloped as controls (:). (Upper Left and Right) Peak C compound; A260. (Lower Left and Right) peak B compound. Downloaded by guest on October 1, 2021 Biochemistry: Miwa et al. Proc. Natl. Acad. Sci. USA 76 (1979) 597

Peak B compound was confirmed to be Ado(P)-Rib-P by the finding that after alkaline phosphomonoesterase digestion it I uV. I,IOU.45 behaved identically with authentic 2'-(1"-ribosyl)adenosine 80 (Ado-Rib). I0 164 Composition of Peak C Compound. For studies on the 60i 0 composition of peak C, poly([14C]ADP-Rib) (6.4 X 10P cpm/,umol of ADP-Rib) was prepared on a large scale and 16 A260 units of peak C compound was obtained. The peak C 50 100 150 200 250 300 350 compound exhibited a Xmax at 259 nm and Xmin at 227 nm at 202L40- 463981060~1 5 Lj2Jg LA pH 7.0. The purities of the peak A, B, and C compounds were Ir40w X90 434 626 657 confirmed by the fact that their specific radioactivities, ex- pressed as cpm/A260, were identical with that calculated for the residue formed from the [14C]NAD+ used 400 450 500 550 600 650 [14C]ADP-Rib mie as substrate in poly(ADP-Rib) formation. Adenine/pentose/ phosphate of the peak C compound was 1.00:2.71:2.77, indi- FIG. 4. Mass spectrum of permethylated dephosphorylated peak C compound. cating that this compound was a new product, presumably with adenine/pentose/phosphate = 1:3:3 (Table 1). Adenine/pen- tose/phosphate of peak B was 1.00:1.93:1.94, which is consistent Phloroglucinol Reaction. The position of phosphomonoester with the structure of Ado(P)-Rib-P (33-35). The pentose in the bonds attached to ribose was determined by the phioroglucinol peak C and peak B compounds was identified as ribose. reaction for measuring the reactivity of ribose (27). Dische and The yields of inorganic phosphate before and after digestion Borenfreund (27) reported that substitution at 5 of ribose of the peak C compound with alkaline phosphomonoesterase resulted in about a 2-fold increase in the color reaction, whereas were 0.03 and 2.82 mol per mol of adenine, respectively, in- substitution at position 3 had no significant effect on the reac- dicating that all the were removed by alkaline tion. We observed that substitution of phosphate in position 2 phosphomonoesterase digestion. The validity of the assay or 1 also did not significantly change the color intensity (Table procedure was checked by performing the same color reaction 2). When the peak C compound was completely dephosphor- on Ado(P)-Rib-P and 5'-AMP as model compounds. The cor- ylated, isolated by paper chromatography, and treated with responding values for Ado(P)-Rib-P were 0.04 and 2.00, and phloroglucinol, or when the peak C compound was treated with those for standard 5'-AMP were 0.03 and 1.06; these values are alkaline phosphomonoesterase and then directly with phloro- in agreement with the structures of these compounds. glucinol, the color intensity was the same as that with adenosine Mass Spectrometry of Dephosphorylated Peak C Com- pound. Unambiguous determination of the molecular weight of the dephosphorylated peak C compound was achieved by Table 2. Phioroglucinol reaction of ribose and low-resolution mass spectrometry. Because the free compound its phosphate esters was not volatile, it was converted to the volatile permethyl Concen- derivative for mass spectroscopy. Fig. 4 shows the mass spec- tration,cr (A552-A510) Relative trum of the permethylated derivative of the dephosphorylated Substance mM X i03 equiv.t X 1on ratio peak C compound. It showed an abundant molecular ion peak at m/e '657 (m/e, mass-to-charge ratio). Because the peak of Experiment I the permethylated derivative using C2H31 was m/e 684 (data Adenosine 0.18 88 49 1.00 not shown), it was concluded that nine methyl groups had been 2'-AMP 0.18 71 41 0.83 introduced into the compound. Therefore, the molecular 3'-AMP 0.17 71 42 0.86 5'-AMP 0.08 89 113 2.29 weight of the free dephosphorylated peak C compound was to be 531. is consistent 0.16 185 118 2.39 deduced This value with the proposed Ribose-1- structure of the compound (see Discussion) with 1 mol of ad- phosphate 0.09 46 52 1.06 enine and 3 mol of ribose. The other principal diagnostic ions are m/e 163, 164, and 192. These ions were also observed in the Experiment II mass spectrum of permethylated adenosine (base + H, base + Adenosine 0.19 83 44 1.00 2 H, and base + 30 H, respectively) (36), indicating the presence 5'-AMP 0.17 162 94 2.14 of an adenosine moiety in the dephosphorylated compound. 5'-AMPPMased+ 0.18 82 45 1.03 The absence of m/e 220, which is seen in the spectrum of Ado-Rib 0.26 117 45 1.03 permethylated adenosine and thought to be derived from the Ado(P)-Rib-P 0.25 205 83 1.91 base of adenine plus fragments of ribose containing the 2'-OH Ado(P)-Rib-P residue (36), suggested a substitution at 2'-OH of the adenosine + PMase 0.21 92 45 1.02 moiety of the compound. Dephosphorylated peak C Table 1. Molar compositions of peak B and peak C compounds compound 0.27 117 43 0.99 PeakC compound 0.26 220 86 1.97 Peak B Peak C Peak C compound Component 5'-AMP compound compound + PMase 0.22 92 42 0.97 Adenine (1.00) (1.00) (1.00) * The concentration of ribose was calculated by taking the ratios of Ribose (1.00) 1.93 2.71 ribose to adenine for AMP, Ado(P)-Rib-P, and peak C compound Phosphate (1.00) 1.94 2.77 as 1, 2, and 3, respectively (Table 1). t A552 - A510 for substance at 1 mM. The contents of adenine, ribose, and phosphate in authentic 5'- The relative millimolar extinction coefficient of adenosine was taken AMP were each taken as 1.00. The molar composition is expressed as 1.00. relative to adenine. § Phosphomonoesterase. Downloaded by guest on October 1, 2021 598 Biochemistry: Miwa et al. Proc. Natl. Acad. Sci. USA 76 (1979) Table 3. reaction of the phosphomonoester linkages are at the 2' or 3' position, because removal of phosphate from the 2' or 3' position would Concen- generate another cis diol, resulting in additional periodate tration,* A690 Relative consumption. Substance /IM A690 X 103 equiv.t ratio D-Ribose 5 57 11.4 DISCUSSION 10 117 11.7 The structure of the peak C compound, demonstrated among 20 199 9.95 1.001 the phosphodiesterase digestion products of poly(ADP-Rib), 50 495 9.90 is proposed to be 2'-[1"-ribosyl 2"-(or 3"-)(1"'-ribosyl)]adeno- Ado(P)-Rib-P 40* 96 2.40 0.22 sine-5',5",5"'-tris(phosphate), as follows. Ado(P)-Rib-P + PMase§ 40* 77 1.90 0.17 Peak C Ade 2 \ compound 40* 46 1.15 0.11 2' 1" \3# " Peak C Rib Rib Rib compound 5' 5" 5"' + PMase 40* 67 1.68 0.16 J) P J Ribose-1- phosphate 17 33 1.96 0.18 It is still uncertain whether the glycosidic linkage is 1 "'-2" Ribose-1- or 1"'*3", and the anomeric configuration of the ribose-ri- phosphate bose-ribose linkages has not yet been elucidated. + PMase 17 203 12.1 1.13 It is unknown why the peak C compound is eluted before ppApp and apparently has net negative charge of -5. However, * Shown as the concentration of adenine residues. t A690 for substance at 1 mM. it must be remembered in this connection that the positions of The mean value of 10.7 for A690 equivalents of D-ribose is taken as elution of from DEAE-Sephadex are influenced 1.00. not only by their charge but also by their chemical structure § Phosphomonoesterase. (37). The presence of this compound indicates that poly(ADP-Rib) or Ado-Rib. These findings also indicate the presence of 3 mol has a branched structure (Fig. 5). This branched structure is of ribose per mol of adenine in the dephosphorylated peak C unexpected, but we previously detected a fraction of poly- compound. Moreover, the intensity of the color reaction of the (ADP-Rib) with a mean sedimentation coefficient of 12s, which peak C compound was about twice that of the dephospho- was excluded from a Sephadex G-200 column and had a mean rylated peak C compound. This was also found with Ado(P)- chain length of 26 (38). The mean chain length was calculated Rib-P and 5'-AMP, which both have esterified phosphate at the as the ratio of the number of ADP-Rib residues to the number 5' (or 5") position. This strongly suggests that all three molecules of termini (15). If poly(ADP-Rib) with a chain length of 26 has of phosphate of the peak C compound are attached to either a linear structure, it should have a molecular weight of 14,000, the 5', 5", or 5"' position of each ribose. its s value should be about 5s, and it should not be excluded Reducing Sugar Reaction. The peak C compound and its from a Sephadex G-200 column (38). However, if high mo- dephosphorylated compound had no free reducing end of ribose lecular weight poly(ADP-Rib) has branches at intervals of about (Table 3). Therefore, the 1', 1", and 1"' of ribose are 20-30 ADP-Rib units and thus has several termini, it is possible linked either to adenine or to ribose. that its chain length calculated by the ordinary procedure Periodate Consumption. The peak C compound and the would be very short-e.g., 30. dephosphorylated peak C compound both consumed only 1 mol Enzymatic formation of poly(ADP-Rib) may consist of three of periodate per mol of adenine (Table 4). This is consistent with steps: first, ADP-ribosylation of nuclear proteins (initiation); the results of the phloroglucinol reaction, indicating that none second, ADP-ribosylation to 2'-ribose of AMP portions of preexisting ADP-Rib residue (elongation); and third, ADP- ribosylation to 2"- or 3"-ribose of ribose 5'-phosphate portions Table 4. Periodate consumption of preexisting poly(ADP-Rib) chain (branching) and elongation. NaIO4 It would be interesting to know whether formation of the NaIO4 consumption, branched structure and normal elongation are catalyzed by the Amount,* consumption, mol/mol of same enzyme. Nonenzymatic formation of branching of poly- Substance nmol nmol adenine (ADP-Rib) could also be considered by a transacetalization reaction. Experiment I 5'-AMP 18.8 19.1 1.02 Ade Ade 25.2 25.0 0.99 Ado(P)-Rib-P r------Peak C compound 20.4 23.7 1.16 Rib Rib-Rib Rib- 3-NAD+ 9.3 17.2 1.85 PLPI P-P Experiment II P 5'-AMP 18.8 18.9 1.01 Ade I Ade Ade Adenosine 19.3 20.2 1.05 Ado-Rib 21.3 20.4 0.96 -Rib,' Rib-Rib:, Rib-Rib Rib- Dephosphorylated peak C compound 21.2 23.8 1.12 P- P P-I-P P-P l_____.____- * Amount calculated from the absorbance at 259 nm in 50 mM sodium phosphate buffer (pH 7.0) and expressed as the amount of adenine FIG. 5. Proposed branching structure of poly(ADP-Rib).L:,, peak residue. C compound. Downloaded by guest on October 1, 2021 Biochemistry: Miwa et al. Proc. Natl. Acad. Sci. USA 76 (1979) 599 Poly(ADP-Rib) is hydrolyzed by poly(ADP-Rib) glycohy- 14. Kanai, Y., Kawaminami, Y., Miwa, M., Matsushima, T., Sug- drolase to yield ADP-Rib (39). It is not known whether there imura, T., Moroi, Y. & Yokohari, R. (1977) Nature (London) 265, 175-177. is a debranching enzyme to yield linear molecules of poly- 15. Tanaka, M., Miwa, M., Hayashi, K., Kubota, K., Matsushima, T. (ADP-Rib). & Sugimura, T. (1977) Biochemistry 16, 1485-1489. The antigenic determinant of poly(ADP-Rib) is thought to 16. Tanaka, M., Hayashi, K., Sakura, H., Miwa, M., Matsushima, T. have a ribose-ribose structure, because Ado(P)-Rib-P caused & Sugimura, T. (1978) Nucleic Acids Res. 5, 3183-3194. significant inhibition of the binding of poly(ADP-Rib) to the 17. Sugimura, T., Yoshimura, N., Miwa, M., Nagai, H. & Nagao, M. antipoly(ADP-Rib) antibody (40). 2'-[1"-ribosyl 2"-(or 3"-)- (1971) Arch. Biochem. Biophys. 147,660-665. 18. Sakura, H., Miwa, M., Tanaka, M., Kanai, Y., Shimada, T., (1"'-ribosyl)]adenosine-5',5",5"'-tris(phosphate) may also be Matsushima, T. & Sugimura, T. (1977) Nucleic Acids Res. 4, involved in the antigenic site of high molecular weight poly- 2903-2915. (ADP-Rib). 19. Oka, J., Ueda, K. & Hayaishi, 0. (1978) Biochem. Biophys. Res. The finding of this compound indicating a branched struc- Commun. 80, 841-848. ture of poly(ADP-Rib) introduces new concepts of the structure 20. Rushizky, G. W., Bartos, E. M. & Sober, H. A. (1964) Biochem- and function of poly(ADP-Rib). istry 3, 626-629. 21. Rushizky, G. W. & Sober, H. A. (1962) Biochim. Biophys. Acta We thank Dr. F. Harada and Dr. M. Maeda of this Institute and Dr. 55,217. S. M. Hecht of the Massachusetts Institute of Technology for valuable 22. Bock, R. M., Ling, N., Morell, S. A. & Lipton, S. H. (1956) Arch. suggestions. We also thank Mr. H. Fukushima of Mitsubishi-Kasei Biochem. Biophys. 62,253-263. Institute of Life Sciences, Japan, for supplying calf thymus. This work 23. Mejbaum, W. (1939) Hoppe-Seyler's Z. Physiol. Chem. 258, was supported by a Grant-in-Aid for Scientific Research and a 117-120. Grant-in-Aid for Cancer Research from the Ministry of Education, 24. Robinson, J. B., Jr., Singh, M. & Srere, P. A. (1976) Proc. Natl. 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