Caspase-mediated Cleavage of DNA Topoisomerase I at Unconventional Sites during Apoptosis

doi:10.1074/jbc.274.7.4335

Caspase-mediated Cleavage of DNA Topoisomerase I at Unconventional Sites during Apoptosis*

From the ^tOaInstitute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, United Kingdom, the ^tObDivision of Oncology Research, Mayo Clinic, Rochester, Minnesota 55905, ^tOcAthena Neurosciences, Inc., South San Francisco, California 94080, the ^tOhJefferson Cancer Institute, Philadelphia, Pennsylvania 19107-5541, the ^tOgCentre Hospitalier de l’Université Laval Research Center and Laval University, Sainte-Foy, Quebec G1V 4G2, Canada, and the ^tOdBiomolecular Structure Center and the Departments of ^tOeBiological Structure and ⁱMicrobiology, School of Medicine, University of Washington, Seattle, Washington 98195

Abstract

Previous studies have demonstrated that topoisomerase I is cleaved late during apoptosis, but have not identified the proteases responsible or examined the functional consequences of this cleavage. Here, we have shown that treatment of purified topoisomerase I with caspase-3 resulted in cleavage at DDVD¹⁴⁶↓Y and EEED¹⁷⁰↓G, whereas treatment with caspase-6 resulted in cleavage at PEDD¹²³↓G and EEED¹⁷⁰↓G. After treatment of Jurkat T lymphocytic leukemia cells with anti-Fas antibody or A549 lung cancer cells with topotecan, etoposide, or paclitaxel, the topoisomerase I fragment comigrated with the product that resulted from caspase-3 cleavage at DDVD¹⁴⁶↓Y. In contrast, two discrete topoisomerase I fragments that appeared to result from cleavage at DDVD¹⁴⁶↓Y and EEED¹⁷⁰↓G were observed after treatment of MDA-MB-468 breast cancer cells with paclitaxel. Topoisomerase I cleavage did not occur in apoptotic MCF-7 cells, which lack caspase-3. Cell fractionation and band depletion studies with the topoisomerase I poison topotecan revealed that the topoisomerase I fragment remains in proximity to the chromatin and retains the ability to bind to and cleave DNA. These observations indicate that topoisomerase I is a substrate of caspase-3 and possibly caspase-6, but is cleaved at sequences that differ from those ordinarily preferred by these enzymes, thereby providing a potential explanation why topoisomerase I cleavage lags behind that of classical caspase substrates such as poly(ADP-ribose) polymerase and lamin B₁.

Footnotes

↵* This work was supported in part by National Institutes of Health Grants AG13487 (to E. S. A.), CA69008 (to S. H. K. and W. C. E), and GM49156 (to L. S. and J. J. C.) and by a principal research fellowship from the Wellcome Trust (to W. C. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵FNf Present address: Emerald BioStructures Inc., 7865 Northeast Day Rd. W., Bainbridge Island, WA 98110.
↵FNj Leukemia Society of America Scholar.
↵FNk These authors contributed equally to this work.
↵FNl Principal Research Fellow of the Wellcome Trust. To whom correspondence should be addressed: Inst. of Cell and Molecular Biology, University of Edinburgh, Michael Swann Bldg., The King’s Bldgs., Mayfield Rd., Edinburgh EH9 3JR, Scotland, UK. Tel.: 44-131-650-7101; Fax: 44-131-650-7100; E-mailbill.earnshaw{at}ed.ac.uk.
Abbreviations:

topo I

DNA topoisomerase I

YVAD-cmk

acetyltyrosinylvalinylalanylaspartyl chloromethyl ketone

PAGE

polyacrylamide gel electrophoresis.
- Received June 22, 1998.
- Revision received November 9, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.