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 report the biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase (PARP) (EC 2.4.2. 30). PARP was effectively mono(ADP-ribosyl)ated both in solution and via an activity gel assay following SDS-PAGE with 20 microM or lower concentrations of [32P]-3'-dNAD+ as the ADP-ribosylation substrate. We observed the exclusive formation of [32P]-3'-dAMP and no polymeric ADP-ribose molecules following chemical release of enzyme-bound ADP-ribose units and high-resolution polyacrylamide gel electrophoresis. The reaction in solution (i) was time-dependent, (ii) was activated by nicked dsDNA, and (iii) increased with the square of the enzyme concentration. Stoichiometric analysis of the reaction indicated that up to four amino acid residues per mole of enzyme were covalently modified with single units of 3'-dADP-ribose. Peptide mapping of mono(3'-dADP-ribosyl)ated-PARP following limited proteolysis with either papain or alpha-chymotrypsin indicated that the amino acid acceptor sites for chain initiation with 3'-dNAD+ as a substrate are localized within an internal 22 kDa automodification domain. Neither the amino-terminal DNA-binding domain nor the carboxy-terminal catalytic fragment became ADP-ribosylated with [32P]-3'-dNAD+ as a substrate. Finally, the apparent rate constant of mono(ADP-ribosyl)ation in solution indicates that the initiation reaction catalyzed by PARP proceeds 232-fold more slowly than ADP-ribose polymerization.