The Large Subunit of the DNA Replication Complex C (DSEB/RF-C140) Cleaved and Inactivated by Caspase-3 (CPP32/YAMA) during Fas-induced Apoptosis*

We report the identification of the large subunit of the DNA replication factor, DSEB/RF-C140, as a new substrate for caspase-3 (CPP32/YAMA), or a very closely related protease activated during Fas-induced apoptosis in Jurkat T cells. DSEB/RF-C140 is a multifunctional DNA-binding protein with sequence homology to poly(ADP-ribose) polymerase (PARP). This similarity includes a consensus DEVD/G cleavage site for caspase-3. Cleavage of DSEB/RF-C140 is predicted to occurs between Asp706 and Gly707, generating 87-kDa and 53-kDa fragments. An antiserum raised against the amino-terminal domain of DSEB/RF-C140 detects a new 87-kDa protein in Jurkat T cells in which apoptosis is activated by a monoclonal antibody to Fas. This cleavage occurs shortly after PARP cleavage. In vitro translated DSEB/RF-C140 is specifically cleaved into the predicted fragments when incubated with a cytoplasmic extract from Fas antibody-treated cells. Proteolytic cleavage was prevented by substituting Asp706 by an alanine in the DEVD706/G caspase-3 cleavage site. The cleavage of DSEB/RF-C140 is prevented by iodoacetamide and the specific caspase-3 inhibitor, tetrapeptide aldehyde Ac-DEVD-CHO, but not by the specific ICE (interleukin-1-converting enzyme) inhibitors: CrmA and Ac-YVAD-CHO, indicating that the protease responsible for the cleavage of DSEB/RF-C140 during Fas-induced apoptosis in Jurkat cells is caspase-3, or a closely related protease. This conclusion is reinforced by the fact that recombinant caspase-3 but not caspase-1 reproduced the “in vivo” cleavage. Inasmuch as the cleavage of DSEB/RF-C140 separates its DNA binding from its association domain, required for replication complex formation, we propose that such a cleavage will impair DNA replication. Recent in vitromutagenesis support this proposal (Uhlmann, F., Cai, J., Gibbs, E., O’Donnel, M., and Hurwitz, J. (1997) J. Biol. Chem. 272, 10058–10064).

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