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Volume 272, Number 31, Issue of August 1, 1997 pp. 19562-19568
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, May 2, 1997)

From the Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, and Harvard Medical School, Boston, Massachusetts 02114

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 vitro mutagenesis 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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