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

J Biol Chem. 1999 Feb 12;274(7):4335-40. 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Apoptosis*
  • Caspase 3
  • Caspase 6
  • Caspases / metabolism*
  • Catalytic Domain
  • DNA Topoisomerases, Type I / metabolism*
  • Humans
  • Jurkat Cells
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Recombinant Proteins / metabolism
  • Spodoptera

Substances

  • Peptide Fragments
  • Recombinant Proteins
  • CASP3 protein, human
  • CASP6 protein, human
  • Caspase 3
  • Caspase 6
  • Caspases
  • DNA Topoisomerases, Type I