A wide range of anti-cancer drugs work by inhibiting topoisomerase II (topoII) activity. Much of their cytotoxic activity is based on the formation of inhibitor/topoII/DNA complexes that cause DNA damage and cell death. It is also known, however, that loss of one vertebrate topoII isoform (a), but not the other (b), leads to aberrant chromosome segregation and cell death. Although this is likely to contribute to the cytotoxicity of topoII inhibitors, to minimising associated drug-resistance and to secondary cancers, its analysis in topoII-inhibited cells is complicated by the pleiotropic effects of inhibitors. We therefore aim to characterise the essential function of topoIIa in a human cell line, recently developed in our lab, in which topoIIa expression can be silenced. In one approach we will determine which parts of the topoIIa molecule, distinct from those in topoIIb, are required to rescue these cells from lethal topoIIa-depletion. Any proteins that specifically interact with such regions will be identified. In a complementary approach, we will investigate chromosomal aberrations resulting from topoIIa-depletion, including the timing and location of newly discovered double strand breaks in improperly segregating chromosomes, and any aberrant homologous recombination between sister chromatids.