Caspase-mediated cleavage of DNA topoisomerase I at unconventional sites during apoptosis

Previous studies have demonstrated that topoisomerase I is cleaved late dur ing apoptosis, but have not identified the proteases responsible or examine d the functional consequences of this cleavage. Here, we have shown that tr eatment of purified topoisomerase I with caspase-3 resulted in cleavage at DDVD(146)down arrow Y and EEED(170)down arrow G, whereas treatment with cas pase-6 resulted in cleavage at pEDD(123)down arrow G and EEED(170)down arro w G. After treatment of Jurkat T lymphocytic leukemia cells with anti-Fas a ntibody or A549 lung cancer cells with topotecan, etoposide, or paclitaxel, the topoisomerase I fragment comigrated with the product that resulted fro m caspase-3 cleavage at DDVD(146)down arrow Y. In contrast, two discrete to poisomerase I fragments that appeared to result from cleavage at DDVD(146)d own arrow Y and EEED(170)down arrow G; were observed after treatment of MDA -MB-468 breast cancer cells with paclitaxel. Topoisomerase I cleavage did n ot occur in apoptotic MCF-7 cells, which lack caspase-3. Cell fractionation and band depletion studies with the topoisomerase I poison topotecan revea led that the topoisomerase I fragment remains in proximity to the chromatin and retains the ability to bind to and cleave DNA. These observations indi cate 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 topoisomera se I cleavage lags behind that of classical caspase substrates such as poly (ADP-ribose) polymerase and lamin B-1.