02 May 2003
DNA-binding specificity of wild type and two mutant forms of human poly(ADP-ribosyl)ated-p53
R. Alvarez-Gonzalez, H. Zentgraf, M. Frey, H. Mendoza-AlvarezMed Sci Monit 2003; 9(1): 1-0 :: ID: 14985
Abstract
We examined the consequences of peptide domain-specific poly (ADP-ribosyl)ation [1] on the ability of p53 to selectively bind to its consensus DN -sequence by electrophoretic mobility shift assays (EMSA). Protein-poly(ADP-ribosyl)ation was performed with recombinant poly (ADP-ribose) polymerase-1 (PARP-1) and bNAD+. We poly(ADP-ribosyl)ated wild type p53 (wt-p53) and a p53 point-mutant (p53mt267) where Arg 267 was mutated 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 deletion-mutant (p53-D30) that lacks the last 30 amino acids from its carboxy-terminus and does not efficiently bind DNA strand-breaks. We observed that the poly(ADP-ribosyl)ation of each p53 form increases as a function of time as well as bNAD+ and p53 concentrations. As expected, EMSA showed that the poly (ADP-ribosyl) ation of wt-p53 completely abolished sequence-specific DNA-binding [2]. By contrast, the modification of p53-D30 did not diminish, but enhanced the mobility shift signal. Thus, transactivation is down regulated by poly(ADP-ribosyl)ation more effectively when the primary structure of p53 is intact. Analogously, the poly (ADP-ribosyl)ation of p53mt267 also enhanced non-specific DNA-binding via its carboxy-terminal domain. Thus, non-specific interactions between p53mt267 and broken DNA are more stable when the tumor suppressor protein contains a ‘hot spot’ mutation. Finally, since p53-D30 is efficiently poly (ADP-ribosyl) ated, we conclude that the p53-poly(ADP-ribosyl)ation target site(s) are outside its carboxy-terminal domain.In summary, our data are consistent with a complex model of regulation where the physiological p53 functions, either as a transactivator or as a broken DNA-sensor, are differentially controlled by peptide domain-specific poly(ADP-ribosyl)ation. References: 1.Kumari SR, Mendoza-Alvarez H, Alvarez-Gonzalez R: Functional interactions of p53 with poly(ADP-ribose) polymerase (PARP) during apoptosis following DNA damage: Covalent poly (ADP-ribosyl) ation of p53 by exogenous PARP and non-covalent binding of p53 to the M(r) 85, 000 proteolytic fragment. Cancer Research, 1998; 58: 5075-5078 2.Mendoza-Alvarez H, Alvarez-Gonzalez R: Regulation of p53 sequence-specific DNA binding by covalent poly(ADP-ribosyl)ation. Journal of Biological Chemistry, 2001; 276: 36425-36430
Keywords: p53, DNA-binding, EMSA, PARP-1, Poly(ADP-ribosyl)ation
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