ADP-RIBOSYLATION OF WILD-TYPE P53 IN-VITRO - BINDING OF P53 PROTEIN TO SPECIFIC P53 CONSENSUS SEQUENCE PREVENTS ITS MODIFICATION

We have recently reported that mutant but not wild-type (wt) p53 prote in was ADP-ribosylated in primary rat cells overexpressing the tempera ture-sensitive murine p53(val135) gene. To examine whether the lack of susceptibility to modification is a specific feature of p53(val135) a dopting wt conformation or rather a general property of this tumor sup pressor protein, we have studied ADP-ribosylation of wt p53 of differe nt origin in vitro using semi-purified poly(ADP-ribose) transferase (p ADPRT). In vitro pADPRT modified human and mouse wt p53 and p53(val135 ). Under limiting substrate concentration, the molar mass of ADP-ribos ylated p53 was only slightly altered. Chase experiments with high NAD concentration resulted in the formation of poly(ADP-ribosyl)ated p53 p rotein shifted to 64 kD. However, preincubation of wt p53 proteins wit h a p53 consensus sequence resulting in complex formation abolished th e modification of wt p53. This indicates that in the cellular environm ent the specific DNA binding of wt p53 prevents its covalent modificat ion by poly(ADP-ribose). (C) 1996 Academic Press, Icc.