We have examined the consequences of p53 domain-specific poly(ADP-ribosyl)ation [1] on its ability to bind to its consensus DNA-sequence by electrophoretic mobility shift assays (EMSA). We first poly(ADP-ribosyl)ated wt p53 and a p53 point-mutant (p53mt267) where Arg 267 was changed to Trp by site directed mutagenesis. We selected p53mt267 because it does not efficiently bind to the canonical p53 consensus DNA-sequence. We also poly(ADP-ribosyl)ated a p53 mutant (p53-Δ30), where the last 30 amino acids from its carboxy-terminus were deleted. We chose p53-Δ30 because this form does not efficiently bind DNA strand-breaks. We confirmed that the poly(ADP-ribosyl)ation of each p53 form increases as a function of time as well as theβNAD+ and p53 protein concentrations. Our control experiments also showed that the poly(ADP-ribosyl)ation of wt-p53 completely abolished its sequence-specific DNA-binding [2]. By contrast, the poly(ADP-ribosyl)ation of p53mt267 lead to enhanced DNA-binding, probably via its intact carboxy-terminal domain. Unexpectedly, the poly(ADP-ribose) modification of p53-Δ30 also enhanced the mobility shift signal. Therefore, we conclude that p53 transactivation is down regulated by poly(ADP-ribosyl)ation more efficiently when the primary structure of p53 is intact. Interestingly, since p53-Δ30 was efficiently poly(ADP-ribosyl)ated, we also conclude that the poly(ADP-ribose) covalent target site(s) of p53 must be upstream from its DNA-nick sensing carboxy-terminal domain. Our data are consistent with the conclusion that the physiological functions of p53, either as a transactivator, or as a DNA-strand break sensor, are distinctly regulated by domain-specific poly(ADP-ribosyl)ation.

1.Kumari SR, Mendoza-Alvarez H, Alvarez-Gonzalez R. (1998) Cancer Research 58: 5075-5078

2.Mendoza-Alvarez H, Alvarez-Gonzalez R. (2001) Journal of Biological Chemistry 276: 36425-36430

key words: p53 • DNA-binding • EMSA • PARP-1 • Poly(ADP-ribosyl)ation

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA