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Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis
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The large subunit of the DNA replication complex C (DSEB/RF-C140) cleaved and inactivated by caspase-3 (CPP32/YAMA)

M Ubeda1, J F Habener

  • 1Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, and Harvard Medical School, Boston, Massachusetts 02114, USA.

The Journal of Biological Chemistry
|August 1, 1997
PubMed

Insights

The DNA replication factor DSEB/RF-C140 is cleaved by caspase-3 during Fas-induced apoptosis. This cleavage, occurring at a specific site, generates fragments that impair DNA replication, as confirmed by in vitro studies.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Apoptosis is a critical cellular process involving programmed cell death.
  • Caspases are key proteases that execute apoptosis.
  • DNA replication factors are essential for cell division and integrity.

Purpose of the Study:

  • To identify substrates of caspase-3 during Fas-induced apoptosis.
  • To investigate the cleavage of the DNA replication factor DSEB/RF-C140.
  • To determine the functional consequences of DSEB/RF-C140 cleavage on DNA replication.

Main Methods:

  • Western blot analysis using an antiserum against DSEB/RF-C140.
  • In vitro translation and cleavage assays with cytoplasmic extracts.
  • Inhibition studies using specific caspase inhibitors (Ac-DEVD-CHO, CrmA, Ac-YVAD-CHO).
  • Site-directed mutagenesis of the caspase cleavage site.

Main Results:

  • DSEB/RF-C140 was identified as a novel substrate for caspase-3 (CPP32/YAMA).
  • Cleavage occurred at a predicted DEVD/G site, generating 87-kDa and 53-kDa fragments.
  • Cleavage was specifically mediated by caspase-3, not caspase-1, and inhibited by Ac-DEVD-CHO.
  • Mutagenesis of the cleavage site prevented proteolytic processing.

Conclusions:

  • Caspase-3 directly cleaves DSEB/RF-C140 during Fas-induced apoptosis in Jurkat T cells.
  • Cleavage separates the DNA-binding and replication complex association domains of DSEB/RF-C140.
  • This cleavage is proposed to inhibit DNA replication, contributing to apoptotic processes.

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