title: 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 [(32)P] 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.
authors.affiliation: Centennial Alumnus, University of North Texas System, TX, USA, 1.
authors.forename: Rafael
authors.initials: R
authors.lastname: Alvarez-Gonzalez
authors.forename: Hilda
authors.initials: H
authors.lastname: Mendoza-Alvarez
authors.forename: Manfred
authors.initials: M
authors.lastname: Frey
authors.forename: Hanswalter
authors.initials: H
authors.lastname: Zentgraf
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D053904:DNA Breaks, Single-Stranded; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D009243:NAD / Q000378:metabolism; D000071137:Poly (ADP-Ribose) Polymerase-1; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013997:Time Factors; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D011994:Recombinant Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D009243:NAD; D011064:Poly Adenosine Diphosphate Ribose; D004247:DNA; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases