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

Kumiko Samejima,Phyllis A Svingen,Guriqbal S Basi,Timothy Kottke,Peter W Mesner,Lance Stewart,Françoise Durrieu,Guy G Poirier,Emad S Alnemri,James J Champoux,Scott H Kaufmann,William C Earnshaw

doi:10.1074/jbc.274.7.4335

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 downward arrowY and EEED170 downward arrowG, whereas treatment with caspase-6 resulted in cleavage at PEDD123 downward arrowG and EEED170 downward arrowG. 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 downward arrowY. In contrast, two discrete topoisomerase I fragments that appeared to result from cleavage at DDVD146 downward arrowY and EEED170 downward arrowG 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.

Highlights

Clear enzyme (105-106 copies/nucleus) involved in the regulation of DNA topology and the control of gene expression, is emerging as a protein of considerable medical significance
Compared with apoptotic cleavage of other caspase targets, e.g. poly(ADP-ribose) polymerase [30] or lamin B1 [31], topoisomerase I (topo I) cleavage appeared to be a later event and was typically incomplete [8, 28]. In contrast to this result, topo I fragments of 70 kDa have been reported in HeLa cells exposed to UV-B irradiation [28], HL-60 cells treated with etoposide [32], and Jurkat cells exposed to anti-CD95 antibody [29, 32]
1) Which proteases are responsible for topo I cleavage during apoptosis? 2) Where are the cleavage sites located within the topo I molecule? 3) Are the topo I fragments generated during apoptosis enzymatically active? 4) Are the same topo I fragments invariably generated in different cells undergoing apoptosis? In this study, we have mapped the sites at which caspase-3 and caspase-6 cleave topo I, compared the resulting fragments with those generated in situ in several cell types undergoing apoptosis, demonstrated that the major topo I cleavage fragment retains enzymatic activity, and probed the location of the cleaved fragment within apoptotic cells

Summary

Introduction

Clear enzyme (105-106 copies/nucleus) involved in the regulation of DNA topology and the control of gene expression, is emerging as a protein of considerable medical significance. Cleavage of Topoisomerase I in Jurkat Cells Undergoing Apoptosis—Initial studies were performed to confirm that topo I is cleaved to a detectable fragment during apoptosis and to assess the timing of this cleavage relative to other proteolytic events.

Results

Conclusion