THE LARGE SUBUNIT OF REPLICATION FACTOR-C IS A SUBSTRATE FOR CASPASE-3 IN-VITRO AND IS CLEAVED BY A CASPASE-3-LIKE PROTEASE DURING FAS-MEDIATED APOPTOSIS

Caspase-3 is an ICE-like protease activated during apoptosis induced b y different stimuli, Poly(ADP-ribose) polymerase (PARP), the first cha racterized substrate of caspase-3, shares a region of homology with th e large subunit of Replication Factor C (RF-C), a five-subunit complex that is part of the processive eukaryotic DNA polymerase holoenzymes. Caspase-3 cleaves PARP at a DEVD-G motif present in the 140 kDa subun it of RF-C (RFC140) and evolutionarily conserved, We show that cleavag e of RFC140 during Fas-mediated apoptosis in Jurkat cells and lymphocy tes results in generation of multiple fragments, Cleavage is inhibited by the caspase-3-like protease inhibitor Ac-DEVD-CHO but not the casp ase-1/ICE-type protease inhibitor Ac-YVAD-CHO. In addition, recombinan t caspase-3 cleaves RFC140 in vitro at least at three different sites in the C-terminal half of the protein, Using amino-terminal microseque ncing of radioactive fragments, we identified three sites: DEVD(723)G, (DLVDS)-S-922 and IETD(1117)A. We did not detect cleavage of small su bunits of RF-C of 36, 37, 38 and 40 kDa by recombinant caspase-3 or by apoptotic Jurkat cell lysates, Cleavage of RFC140 during apoptosis in activates its function in DNA replication and generates truncated form s that further inhibit DNA replication, These results identify RFC140 as a critical target for caspase-3-like proteases and suggest that cas pases could mediate cell cycle arrest.