Regulation of p53 sequence-specific DNA-binding by covalent poly(ADP-ribosyl)ation

We have characterized the covalent poly(ADP-ribosyl)ation of p53 using an i n vitro reconstituted system. We used recombinant wild type p53, recombinan t poly(ADP-ribose) polymerase-1 (PARP-1) (EC 2.4.2.30), and beta NAD(+). Ou r results show that the covalent poly(ADP-ribosyl)ation of p53 is a time-de pendent protein-poly(ADP-ribosyl)ation reaction and that the addition of th is tumor suppressor protein to a PARP-1 automodification mixture stimulates total protein-poly(ADP-ribosyl)ation 3- to 4-fold. Electrophoretic analysi s of the products synthesized indicated that short oligomers predominate ea rly 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 beta NA D(+) concentration was varied. As expected, increasing the beta NAD(+) conc entration from low nanomolar to high micromolar levels resulted in the slow er electrophoretic migration of the p53-(ADP-ribose). adducts. Increasing t he concentration of p53 protein from low nanomolar (40 nm) to low micromola r (1.0 mum) yielded higher amounts of poly(ADP-ribosyl)ated p53 as well. Th us, the reaction was acceptor protein concentration-dependent. The hetero-p oly(ADP-ribosyl)ation of p53 also showed that high concentrations of p53 sp ecifically stimulated the automodification reaction of PARP-1. The covalent modification of p53 resulted in the inhibition of the binding ability of t his transcription factor to its DNA consensus sequence as judged by electro phoretic mobility shift assays. In fact, controls carried out with calf thy mus DNA, beta NAD(+), PARP-1, and automodified PARP-1 confirmed our conclus ion that the covalent poly(ADP-ribosyl)ation of p53 results in the transcri ptional inactivation of this tumor suppressor protein.