Up-Regulation of Two Distinct p53-DNA Binding Functions by Covalent Poly(ADP-ribosyl)ation: Transactivating and Single Strand Break Sensing

Abstract

We used a [³²P] p53 sequence-specific oligodeoxynucleotide and Electrophoretic-Mobility-Shift-Assays to monitor p53 DNA sequence-specific binding with p53-R267W, a nonbinding point mutant; and p53-Δ30, a deletion-mutant which lacks the carboxy-terminus that recognizes DNA-strand-breaks. Recombinant p53 and poly(ADP-ribose)polymerase-1 (PARP-1) were incubated with labeled βNAD⁺ with/without DNA. The poly(ADP-ribosyl)ation of each protein increased with incubation-time and βNAD⁺ and p53 concentration(s). Since p53-Δ30 was efficiently labeled, poly(ADP-ribosyl)ation target site(s) of wt-p53 must reside outside its carboxy-terminal-domain. The poly(ADP-ribosyl)ation of p53-Δ30 did not diminish its DNA binding; Instead, it enhanced DNA-sequence-specific-binding. Therefore, we conclude that DNA-sequence-specific-binding and DNA-nick-sensing of mutant-p53 are differentially regulated by poly(ADP-ribosyl)ation.

Keywords: :

• Tumor suppressor
• Poly(ADP-ribose) polymerase-1 [PARP-1]
• Electrophoretic mobility shift assay [EMSA]
• Poly(ADP-ribosyl)ation