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 noncovalent binding of p53 to the M(r) 85,000 proteolytic fragment.

We have examined the domain-specific interactions between p53 and poly(ADP-ribose)polymerase (PARP) (E.C. 2.4.2.30) in apoptotic HeLa cells. Apoptosis was induced by exposing cells to 50 microM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for increasing lengths of time and was confirmed by: (a) oligonucleosomal fragmentation of chromatin; (b) increase in p53 levels; and (c) degradation of PARP into the characteristic M(r) 85,000 (COOH-terminal catalytic domain) and M(r) 29,000 (DNA-binding domain) peptide fragments. We also immunodetected p53 in immunoprecipitates obtained with a PARP-specific antibody. However, intact PARP coimmunoprecipitated with a p53-specific antibody during the initial 30 min of MNNG treatment. After 60 min, only the COOH-terminal fragment coimmunoprecipitated with p53, indicating that PARP noncovalently binds p53 via its M(r) 85,000 catalytic domain. Therefore, we next examined p53 as a covalent target for poly(ADP-ribosyl)ation. Although p53 was not endogenously poly (ADP-ribosyl)ated in situ, incubation of cell extracts with full-length PARP from calf thymus and [32P]beta NAD+ resulted in its time-dependent poly(ADP-ribosyl)ation. In summary, our results are consistent with the conclusion that PARP and p53 are activated with nonoverlapping kinetics during apoptosis.