Studies on proteolytic cleavage of poly(ADP-ribose) polymerase by caspase during apoptosis

Abstract

The roles of proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) by caspase were examined in cultured HeLa cells undergoing apoptosis. Both of the cleavage products, N-terminal fragment (N214) and C-terminal fragment (C215) of PARP played positive roles in triggering apoptosis. Ultrastructural analysis of cells expressing a caspase resistant PARP which contains Ala in place of Asp214 $(PARP^{D214A})$ revealed typical features of necrotic cell death after UV irradiation, while cells co-expressing $PARP^{D214A}$ either with N214 or C215 showed features consistent with apoptosis. PARPD214A was constitutively activated after UV irradiation, depleted cellular $NAD^+$ and ATP, and ultimately induced necrotic cell death. The binding of N214 to DNA breaks was an absolute requirement for the execution of apoptosis. Failure of N214 binding to DNA breaks in cells expressing $PARP^{D214A}$ results in necrosis. The C215 associated to PARP through bZip motif in auto-modification domain and disturbed PARP activation leading necrosis. These results suggested that caspases cleave PARP to maintain the cellular energy to escape necrosis and to trigger apoptosis. The N214 contributed to apoptosis by inhibiting DNA repair as well as maintaining cellular energy. The N214 cleaved by caspase was isolated and inhibition of DNA repair synthesis was by N214 was observed in UV irradiated HeLa cell lysates. The recovery of UV damage was inhibited by the expression of N214 in HeLa cells with evident increase in apoptosis. The biological consequences of decreased poly(ADP-ribosyl)ation of nuclear substrates proteins by PARP cleavage were examined. The p53, which has been reported to be stabilized by poly(ADP-ribosyl)ation, was found to inhibit DNA repair during apoptosis. The expression of p53 dependent genes, p21 and Bax, were not changed during the course of UV induced apoptosis but C-terminal nonspecific DNA binding domain of p53, self-cleavage product, was required for the inducti...