Association of human DNA topoisomerase II alpha with mitotic chromosomes in mammalian cells is independent of its catalytic activity

DNA topoisomerase (topo) II is an essential nuclear enzyme that plays an im portant role in DNA metabolism and chromosome organization. In the present study, we expressed human topo II alpha in mammalian cells by fusion to an enhanced green fluorescent protein (EG;FP). Decatenation assays indicated t hat the EGFP-topo II alpha is catalytically active in vitro. Assays for ban d depletion, growth inhibition, and cytotoxicity by topo II inhibitors sugg ested that the fusion protein is also functional in vivo. By following its subcellular localization throughout. the cell cycle in living cells, we fou nd that the fusion protein is localized to the nucleus and nucleolus at int erphase, and it is bound to chromosomal DNA at every stage of mitosis. Of i mportance, a mutant EGFP-topo II alpha, in which the active Tyr 805 is repl aced by Phe (Y805F) and is catalytically inactive, still binds to chromosom al DNA throughout the cell cycle like the wild-type enzyme. Together, our r esults suggest that the ability of topo II alpha to bind to chromosomal DNA in the cell, a presumed requirement for its structural role, can be separa ted from its catalytic activity. (C) 1999 Academic Press.