Methods have been developed and applied to determine the size and branching frequency of polymers of ADP-ribose synthesized in nucleotide-permeable cultured mouse cells and in intact cultured cells. Polymers were purified by affinity chromatography with a boronate resin and were fractionated according to size molecular sieve high-performance liquid chromatography. Fractions were enzymatically digested to nucleotides, which were separated by strong anion exchange high-performance liquid chromatography. From these data, average polymer size and branching frequency were calculated. A wide range of polymer sizes was observed. Polymers as large as 190 residues with at least five points of branching per molecule were generated in vitro. Polymers of up to 67 residues containing up to two points of branching per molecule were isolated from intact cells following treatment with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Cells treated with hyperthermia prior to DNA damage contained polymers of an average maximum size of 244 residues containing up to six points of branching per molecule. The detection of large polymers of ADP-ribose in intact cells suggests that alterations in chromatin organization effected by poly(ADP-ribosylation) may extend beyond the covalently modified proteins and very likely involve noncovalent interactions of poly(ADP-ribose) with other components of chromatin.

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We have characterized the covalent poly(ADP-ribosyl)-ation of p53 using an in vitro reconstituted system. We used recombinant wild type p53, recombinant poly-(ADP-ribose) polymerase-1 (PARP-1) (EC 2.4.2.30), and ␤NAD ؉ . Our results show that the covalent poly(ADPribosyl)ation of p53 is a time-dependent protein-poly-(ADP-ribosyl)ation reaction and that the addition of this tumor suppressor protein to a PARP-1 automodification mixture stimulates total protein-poly(ADP-ribosyl)ation 3-to 4-fold. Electrophoretic analysis of the products synthesized indicated that short oligomers predominate early during hetero-poly(ADP-ribosyl)ation, whereas longer ADP-ribose chains are synthesized at later times of incubation. A more drastic effect in the complexity of the ADP-ribose chains generated was observed when the ␤NAD ؉ concentration was varied. As expected, increasing the ␤NAD ؉ concentration from low nanomolar to high micromolar levels resulted in the slower electrophoretic migration of the p53-(ADP-ribose) n adducts. Increasing the concentration of p53 protein from low nanomolar (40 nM) to low micromolar (1.0 M) yielded higher amounts of poly(ADP-ribosyl)ated p53 as well. Thus, the reaction was acceptor protein concentrationdependent. The hetero-poly(ADP-ribosyl)ation of p53 also showed that high concentrations of p53 specifically stimulated the automodification reaction of PARP-1. The covalent modification of p53 resulted in the inhibition of the binding ability of this transcription factor to its DNA consensus sequence as judged by electrophoretic mobility shift assays. In fact, controls carried out with calf thymus DNA, ␤NAD ؉ , PARP-1, and automodified PARP-1 confirmed our conclusion that the covalent poly-(ADP-ribosyl)ation of p53 results in the transcriptional inactivation of this tumor suppressor protein.The covalent poly(ADP-ribosyl)ation of DNA-binding proteins in eucaryotes is a post-translational modification reaction that has been implicated in the modulation of chromatin structure and function in DNA-damaged and apoptotic cells (1-3). The immediate synthesis of poly(ADP-ribose) from ␤NAD ϩ in response to DNA strand break formation in vivo is mostly catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) 1 (1-3).This enzyme was believed for some time to be the only nuclear DNA-dependent enzyme (EC 2.4.2.30) responsible for the synthesis of chromatin-bound ADP-ribose chains (1-3). However, over the last 4 years, and since the last International Symposium on protein-poly(ADP-ribosyl)ation (4), other novel and less abundant PARP-like proteins have been identified and reported (5-8). The physiological existence of other proteins with ADP-ribose-polymerizing activity explains why PARP-1 (Ϫ/Ϫ) knockout cells still display a positive immunofluorescent nuclear signal when exposed to a fluorescently tagged monoclonal antibody specific for this unique nucleic acid (9). Nevertheless, it appears that about 90% of the total protein-bound polymers synthesized in DNA-damaged cells are assembled by PARP-1. Although most of the...

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Here, we performed a time-course analysis of (i) poly(ADP-ribose) synthesis and degradation as well as (ii) the subnuclear localization of PARP and its fragments by using confocal laser scanning immunofluorescence microscopy. PARP was activated within 15 min post-treatment, as revealed by nuclear immunostaining with antibody 10H (recognizing poly-(ADP-ribose)). This was followed by a late, time-dependent, progressive decline of 10H signals that coincide with the time of PARP cleavage. Strikingly, nucleolar immunostaining with antibodies 10H and C-II-10 (recognizing the 85-kDa PARP fragment) was lost by 15 min post-treatment, whereas F-I-23 signals (recognizing the 29-kDa fragment) persisted. We hypothesize that the 85-kDa PARP fragment is translocated, along with covalently bound poly(ADP-ribose), from nucleoli to the nucleoplasm, whereas the 29-kDa fragment is retained, because it binds to DNA strand breaks. Our data (i) provide a link between the known time-dependent bifunctional role of PARP in apoptosis and the subcellular localization of PARP fragments and also (ii) add to the evidence for early proteolytic changes in nucleoli during apoptosis.Higher eucaryotic organisms have developed a sophisticated signaling system to eliminate nonfunctional cells in a highly coordinated sequence of "suicidal" events. This tightly regulated process is known as "programmed cell death" or "apoptosis" (1-3). Superfluous cell populations that undergo apoptosis include genetically damaged and/or aging cells. The death program is executed in three chronologically distinct phases (4). First to occur is "the condemned phase" where a fully reversible sequence of metabolic and cell cycle adaptation(s) take place with mitochondrial components, such as cytochrome c and the bcl-2 gene produced, playing a decisive role. The second phase of "commitment" is irreversible and derives from unleashing a cascade of proteolytic signals emanating from the mitochondrion to the nucleus. Finally, the "execution phase" of apoptosis is manifested by the macromolecular degradation of chromosomal DNA catalyzed by caspase (cysteine-aspartase)-activated death-factor (5, 6). In this phase, key nuclear proteins are degraded by caspase-3, the main "executioner" of nuclear disassembly (7), and this leads to the disintegration of the nucleus. One of the primary targets for caspase-3 is poly(ADPribose)polymerase (PARP) 1 (E.C. 2.4.2.30) (8 -10). PARP is a nuclear DNA-binding protein of 1,014 amino acid residues (113 kDa) (11, 12) that is constitutively expressed in eucaryotes and comprises up to 1% of the total nuclear protein. This enzyme displays a multimodular domain structure that can be dissected into three functionally distinct domains from the amino terminus to the carboxyl terminus (13). The first module is the "DNA-binding domain" (DBD), a fragment of 46-kDa that contains two zinc fingers, which mediate binding to DNA strands breaks as well as a bipartite nuclear localization signal (14). The two karyophilic regions of the nuclear localization signa...