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

EMBO J. 1997 Nov 3;16(21):6346-54. doi: 10.1093/emboj/16.21.6346.


Caspase-3 is an ICE-like protease activated during apoptosis induced by different stimuli. Poly(ADP-ribose) polymerase (PARP), the first characterized substrate of caspase-3, shares a region of homology with the 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 subunit of RF-C (RFC140) and evolutionarily conserved. We show that cleavage of RFC140 during Fas-mediated apoptosis in Jurkat cells and lymphocytes results in generation of multiple fragments. Cleavage is inhibited by the caspase-3-like protease inhibitor Ac-DEVD-CHO but not the caspase-1/ICE-type protease inhibitor Ac-YVAD-CHO. In addition, recombinant caspase-3 cleaves RFC140 in vitro at least at three different sites in the C-terminal half of the protein. Using amino-terminal microsequencing of radioactive fragments, we identified three sites: DEVD723G, DLVD922S and IETD1117A. We did not detect cleavage of small subunits 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 inactivates its function in DNA replication and generates truncated forms that further inhibit DNA replication. These results identify RFC140 as a critical target for caspase-3-like proteases and suggest that caspases could mediate cell cycle arrest.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / physiology*
  • Caspase 3
  • Caspases*
  • Cysteine Endopeptidases / metabolism*
  • Cysteine Proteinase Inhibitors / pharmacology
  • DNA Replication / drug effects
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / metabolism*
  • Homeodomain Proteins*
  • Humans
  • Leukemia-Lymphoma, Adult T-Cell / pathology
  • Lymphocytes / drug effects
  • Lymphocytes / metabolism
  • Minor Histocompatibility Antigens
  • Neoplasm Proteins / metabolism
  • Oligopeptides / pharmacology
  • Proto-Oncogene Proteins c-bcl-2*
  • Recombinant Fusion Proteins / pharmacology
  • Replication Protein C
  • Repressor Proteins*
  • Saccharomyces cerevisiae Proteins*
  • Substrate Specificity
  • Tumor Cells, Cultured
  • fas Receptor / physiology*


  • BCL2-related protein A1
  • Cysteine Proteinase Inhibitors
  • DNA-Binding Proteins
  • Homeodomain Proteins
  • MATA1 protein, S cerevisiae
  • Minor Histocompatibility Antigens
  • Neoplasm Proteins
  • Oligopeptides
  • Proto-Oncogene Proteins c-bcl-2
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • Saccharomyces cerevisiae Proteins
  • acetyl-aspartyl-glutamyl-valyl-aspartal
  • fas Receptor
  • L 709049
  • CASP3 protein, human
  • Caspase 3
  • Caspases
  • Cysteine Endopeptidases
  • Replication Protein C