RESUMEN
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 ßNAD(+) with/without DNA. The poly(ADP-ribosyl)ation of each protein increased with incubation-time and ßNAD(+) 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.
Asunto(s)
Roturas del ADN de Cadena Simple , ADN/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismo , Sitios de Unión , Ensayo de Cambio de Movilidad Electroforética , Humanos , NAD/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Factores de Tiempo , Proteína p53 Supresora de Tumor/genética , Regulación hacia ArribaRESUMEN
The 40 kDa carboxy-terminal catalytic domain (CD) of avian poly(ADP-ribose) polymerase (PARP-1) was cloned, expressed in a baculovirus expression system, and purified to homogeneity by affinity chromatography. The purified polypeptide synthesized covalent CD-poly(ADP-ribose) conjugates in the absence of DNA. Electrophoretic analysis of the ADP-ribose chain length distribution generated indicated that recombinant CD was able to catalyze the initiation, elongation, and branching reactions of poly(ADP-ribose) synthesis, although at a 500-fold lower efficiency than wild-type PARP-1. Kinetic evaluation of poly(ADP-ribose) synthesis showed that the enzymatic activities of CD increased for up to 60 minutes in a time-dependent manner. Moreover, the rates of CD auto-poly(ADP-ribosyl)ation increased with second-order kinetics as a function of the protein concentration with either betaNAD(+) or 3'-deoxyNAD(+) as a substrate. Furthermore, the formation of catalytically competent CD-[PARP-1] heterodimers was also observed in specific ultrafiltration experiments. Thus, we conclude that the 40 kDa carboxy terminus of PARP-1 forms a competent catalytic dimer in the absence of DNA, and that its automodification reaction is intermolecular.
