Poly ADP-ribose synthesis and DNA replication in synchronized mouse L-cells

Colyer, Robert A.; Burdette, Kristen; Kidwell, William R.

doi:10.1016/0006-291x(73)90185-x

Cited by 80 publications

(19 citation statements)

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“…Chemical identification ofnaturally occurring poly(ADP-ribose) has been reported (6)(7)(8)(9). In 1974, we obtained a specific antibody to poly(ADP-ribose) (10), and this prompted several groups to measure the in vivo content of poly(ADP-ribose) in different experimental systems (11)(12)(13)(14).…”

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confidence: 99%

Immunofluorescent staining of poly(ADP-ribose) in situ in HeLa cell chromosomes in the M phase.

Kanai¹,

Tanuma²,

Sügimura³

1981

Proc. Natl. Acad. Sci. U.S.A.

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Randomly and synchronously growing HeLa cells were tested for poly(ADP-ribose) by direct and indirect immunofluorescent antibody techniques. Fluorescence ofpoly(ADPribose) was seen only in the nuclei of intact cells when the direct immunofluorescent antibody technique was used but in both the nuclei and cytoplasm when the indirect immunofluorescent antibody technique was used; fluorescence in the cytoplasm was nonspecific. When It has been suggested that poly(ADP-ribose), a unique biopolymer synthesized from NAD' by a nuclear enzyme(s) (1), is related to DNA polymerase activity in nuclei (2, 3), DNA repair (4, 5), and some regulatory processes in gene expressions (2, 3). Chemical identification ofnaturally occurring poly(ADP-ribose) has been reported (6-9). In 1974, we obtained a specific antibody to poly(ADP-ribose) (10), and this prompted several groups to measure the in vivo content of poly(ADP-ribose) in different experimental systems (11)(12)(13)(14). The presence of naturally occurring antibodies to poly(ADP-ribose) in patients who have systemic lupus erythematosus indicates that poly(ADPribose) is also present in humans (15, 16). As the poly(ADP-ribose) polymerase activity of HeLa cells has been studied extensively (11, 17), we chose these cells for studies on fluorescent antibody staining ofpoly(ADP-ribose). In this work, the following results were obtained by incubation of randomly or synchronously growing HeLa cells with the antibody: (i) poly(ADPribose) synthesis from endogenous NAD', even in acetonefixed cells, occurred during incubation with antibody; (ii) poly(ADP-ribose) synthesis was enhanced by pretreatment of the cells with DNase I, which causes a 4-to 6-fold stimulation of poly(ADP-ribose) polymerase in chromatin; (iii) poly(ADPribose) synthesis was inhibited by 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) polymerase; and (iv) the fluorescence of chromosomes of HeLa After incubation, the coverslips were rinsed four times with phosphate-buffered saline (10 mM phosphate/0. 14 M NaCl, pH 7.4) and once with distilled water and dried in air. They were subjected to fluorescent antibody staining as described below. Antisera and Fluorescent Antibody. Antisera against poly(ADP-ribose) were raised in rabbits as described (10). Antisera were absorbed with methylated bovine serum albumin, which was used as a carrier for antibody production. Crude gamma globulin was obtained from antisera diluted twice with phosphate-buffered saline by two precipitations with 50% saturated ammonium sulfate. It was dialyzed against 2.7 mM Tris/ 1 mM phosphate buffer (pH 7.8) and then applied to a column of DE52 (Whatman) that had been equilibrated with the same buffer. On stepwise elution with 80 mM Tris/30 mM phosphate buffer (pH 8.0), two bands of IgG were obtained. Both had strong poly(ADP-ribose) binding activity, but the one that eluted first, designated as IgGlIa, had greater antigen-binding activity than the one that eluted second, designated as IgGlIb.

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Immunofluorescent staining of poly(ADP-ribose) in situ in HeLa cell chromosomes in the M phase.

Kanai¹,

Tanuma²,

Sügimura³

1981

Proc. Natl. Acad. Sci. U.S.A.

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“…9 and 10), there is no report that has demonstrated the existence of the protein-bound polymer in vivo. Doly and Mandel (11) [3H]adenosine, with no reference made of any binding to proteins (12). Smith and Stocken (13) and Dietrich et al (14), on the other hand, briefly described the occurrence of proteinbound ADP-ribose in rat liver nuclei.…”

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Natural occurrence of poly(ADP-ribosyl) histones in rat liver.

Ueda¹,

Omachi²,

Kawaichi³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

126

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Poly(ADP-ribose) bound to histones has been isolated from rat liver. When [14Clribose was administered intraperitoneally to rats at a dosage of 300-750Aig (100-250 A&Ci)/100 g, approximately 1% of the radioactivity was recovered in the acid (5% CLCCOOH)-insoluble material of the liver nuclei 2 hr after injection.Of the acid-insoluble radioactivity, 4. Poly(ADP-ribose), a macromolecule synthesized from NAD in chromatin (1-5), has been assumed to be covalently bound to nuclear proteins, principally histones (6-8). Although this poly(ADP-ribosyl)ation of proteins has been well established in vitro using various nuclear preparations (for reviews, see refs. 9 and 10), there is no report that has demonstrated the existence of the protein-bound polymer in vivo. Doly and Mandel (11) (14), on the other hand, briefly described the occurrence of proteinbound ADP-ribose in rat liver nuclei. Neither of these reports, however, presented any evidence for the existence of polymerized t ADP-ribose attached to nuclear proteins.Recently, we carried out a series of experiments to determine the conditions for labeling NAD in vivo and, thereby, poly(ADP-ribose). The results, which have been presented proved to be useful for following adenine-derived compounds. In this communication, new data obtained by employing these precursors are presented which enable us to report the first unequivocal evidence for the natural existence of poly-(ADP-ribosyl) histones in mammalian tissues. spectively. In the main experiments reported herein, 0.5 ml of the ribose solution and 0.25 ml of the adenine solution were mixed and injected intraperitoneally into each rat. The animal was put in a metabolic chamber and maintained for 2 hr under ventilation with CR2-free air. The animal was then etherized and the liver was quickly removed. The liver was homogenized in either 3 volumes of ice-cold 20% Cl3CCOOH (or 5% HCl04) or 5 volumes of 0.25 M sucrose containing 3.3 mM CaCl2 with the aid of a Potter-Elvehjem type tissue grinder. The acid homogenate was centrifuged for 10 min at 10,000 X g. The precipitate was treated with 20% C13CCOOH and centrifuged as before. This washing procedure was repeated three more times so that the final material was contaminated by less than 0.5% of the total acid-soluble radioactivity. The sucrose homogenate was centrifuged for 5 min at 800 X g, and the precipitate obtained (crude nuclei) was subjected to acid washing as was applied to the acid homogenate. The acid-insoluble material prepared by either of these procedures was extracted with 5 volumes (per original liver weight) of 0.25 N HCl by stirring for 30 min at 4°. After the mixture was centrifuged for 15 min at 15,000 X g, the supernatant fraction was collected and the precipitate was reextracted as above. Five extracts' obtained in this way were combined, concentrated approximately 50-fold with the aid of a Diaflo (Amicon) ultrafiltration device equipped with a UM-2 membrane, and centrifuged for 60 min at 105,000 X g. The final, slightly yellow supernatant fracti...

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“…Poly(ADP-ribose) of various sizes, with chain length varying from 1-40, is bound to nuclear proteins, both histones and non-histones [4,5]. Although the biological role of the polymer is not yet understood, its involvement in the control of DNA synthesis [6][7][8], DNA repair [9-121, cell proliferation [13-151 and cell differentiation has been reported.Recently, several procedures for the isolation of highly purified poly(ADP4bose) polymerase from different sources have been reported [I9 -221. The availability of purified polymerase may be of great importance, both in elucidating the biological role of poly(ADP-ribose) and in other kinds of studies.…”

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Poly (ADP‐ribose) Polymerase from Ehrlich Ascites Tumor Cells

Kristensen

Holtlund

1978

European Journal of Biochemistry

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Poly(ADP-ribose) polymerase from Ehrlich ascites tumor cells, partially purified by chromatography on DNA-agarose, was obtained as a more than 80 ' %, homogeneous preparation by isoelectric focusing in a sucrose gradient. The polymerase activity was shown to be associated with the major protein in the preparation. Results obtained by electrophoresis in the presence of sodium dodecylsulfate indicated that poly(ADP-ribose) polymerase consists of a polypeptide chain with a molecular weight of 130 000. Ultracentrifugation at non-denaturating conditions indicated that the active enzyme may be an oligomeric form of this polypeptide chain. The isoelectric point of the polymerase was 9.40. The effects of various additions to the assay mixture on the synthesis of poly(ADPribose) as well as some kinetic data, are given. It is shown that poly(ADP-ribose) is a highly efficient inhibitor of its own synthesis, and results are presented which suggest that the well-known stimulatory effect of DNA on the synthesis is due to reduction of this inhibitory effect of the product.Eukaryotic cell nuclei contain an enzyme which incorporates the ADP-ribose moiety of NAD into poly(ADP-ribose) [l -31. Poly(ADP-ribose) of various sizes, with chain length varying from 1-40, is bound to nuclear proteins, both histones and non-histones [4,5]. Although the biological role of the polymer is not yet understood, its involvement in the control of DNA synthesis [6][7][8], DNA repair [9-121, cell proliferation [13-151 and cell differentiation has been reported.Recently, several procedures for the isolation of highly purified poly(ADP4bose) polymerase from different sources have been reported [I9 -221. The availability of purified polymerase may be of great importance, both in elucidating the biological role of poly(ADP-ribose) and in other kinds of studies. It is, for instance, not known how the cell regulates the degree of ADP-ribosylation of the different acceptor proteins and whether there exist several polymerases with different specificities towards the acceptor proteins. Also, polymerase purification will make it possible to determine whether the higher rate of synthesis of poly(ADP-ribose) reported in transformed cells [23] is due to higher polymerase concentrations or to the stances, has the major protein present after purification been shown to be responsible for the polymer synthesis. In the present work we will describe a relatively fast and simple method for obtaining an almost homogeneous preparation of poly(ADP-ribose) polymerase from Ehrlich ascites tumor cells. The major protein present is shown to be able to synthesize poly-(ADP-ribose). Some properties of the polymerase are given, as well as a possible explanation of the wellknown DNA requirement of the polymer synthesis [24,25].

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Poly ADP-ribose synthesis and DNA replication in synchronized mouse L-cells

Cited by 80 publications

References 14 publications

Immunofluorescent staining of poly(ADP-ribose) in situ in HeLa cell chromosomes in the M phase.

Immunofluorescent staining of poly(ADP-ribose) in situ in HeLa cell chromosomes in the M phase.

Natural occurrence of poly(ADP-ribosyl) histones in rat liver.

Poly (ADP‐ribose) Polymerase from Ehrlich Ascites Tumor Cells

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