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 in vitroreconstituted system. We used recombinant wild type p53, recombinant poly(ADP-ribose) polymerase-1 (PARP-1) (EC 2.4.2.30), and βNAD+. Our results show that the covalent poly(ADP-ribosyl)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 βNAD+concentration was varied. As expected, increasing the βNAD+ 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 μm) yielded higher amounts of poly(ADP-ribosyl)ated p53 as well. Thus, the reaction was acceptor protein concentration-dependent. 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, βNAD+, 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.

• Publication year

  2001

• Venue

  Journal of Biological Chemistry

• Publication date

  2001-09-28

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1074/JBC.M105215200PMID 11477085
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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