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 Mr 85,000 Proteolytic Fragment1

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 jtiM .Y-mi-lliyl-.Vnitro-JV-nitrosoguanidine (MNNG) for increasing lengths of time and was confirmed by: (a) oligonucleosomal fragmentation of chromatin; (In in crease in p53 levels; and (r l degradation of PARP into the characteristic .»/,. 85,000 (COOH-terminal catalytic domain) and .I/,. 29,000 (DMA-bind ing domain) peptide fragments. We also immunodetected p53 in immnnoprecipitates obtained with a PARP-speciflc 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, 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]/3NAD+ re sulted 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.