We have focused on the roles of PARP and poly(ADP-ribosyl)ation early in apoptosis, as well as during the early stages of differentiation-linked DNA replication. In both nuclear processes, a transient burst of PAR synthesis and PARP expression occurs early, prior to internucleosomal DNA cleavage before commitment to apoptosis as well as at the round of DNA replication prior to the onset of terminal differentiation. In intact human osteosarcoma cells undergoing spontaneous apoptosis, both PARP and PAR decreased after this early peak, concomitant with the inactivation and cleavage of PARP by caspase-3 and the onset of substantial DNA and nuclear fragmentation. Whereas 3T3-L1, osteosarcoma cells, and immortalized PARP +/+ fibroblasts exhibited this early burst of PAR synthesis during Fas-mediated apoptosis, neither PARP-depleted 3T3-L1 PARP-antisense cells nor PARP -/- fibroblasts showed this response. Consequently, whereas control cells progressed into apoptosis, as indicated by induction of caspase-3-like PARP-cleavage activity, PARP-antisense cells and PARP -/- fibroblasts did not, indicating a requirement for PARP and poly(ADP-ribosyl)ation of nuclear proteins at an early reversible stage of apoptosis. In parallel experiments, a transient increase in PARP expression and activity were also noted in 3T3-L1 preadipocytes 24 h after induction of differentiation, a stage at which approximately 95% of the cells were in S-phase, but not in PARP-depleted antisense cells, which were consequently unable to complete the round of DNA replication required for differentiation. PARP, a component of the multiprotein DNA replication complex (MRC) that catalyzes viral DNA replication in vitro, poly(ADP-ribosyl)ates 15 of approximately 40 MRC proteins, including DNA pol alpha, DNA topo I, and PCNA. Depletion of endogenous PARP by antisense RNA expression in 3T3-L1 cells results in MRCs devoid of any DNA pol alpha and DNA pol delta activities. Surprisingly, there was no new expression of PCNA and DNA pol alpha, as well as the transcription factor E2F-1 in PARP-antisense cells during entry into S-phase, suggesting that PARP may play a role in the expression of these proteins, perhaps by interacting with a site in the promoters for these genes.