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[ 1 "-Ribosyl-2"-(Or 3"-) (1"'-Ribosyl)]Adenosine-5',5

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[ 1 Proc. Natl. Acad. Sci. USA Vol. 76, No. 2, pp. 595-599, February 1979 Biochemistry Structure of poly(adenosine diphosphate ribose): Identification of 2'-[1 "-ribosyl-2"-(or 3"-) (1 "'-ribosyl)]adenosine-5',5",5"'- tris(phosphate) 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]adenosine diphosphate 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 enzyme 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 cellulose 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 pyrophosphate 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 adenine 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 pentose 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 sugar 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 Escherichia coli 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.
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