Topoisomerase II is a key player in both the etiology and treatment of many cancers, including both primary and metastatic diseases. Normally, Topo II creates a transient gate in a DNA duplex by breaking both strands via a covalent protein-DNA complex that is then reversed to reseal the break. The enzyme is a target of potent anti-cancer drugs such as etoposide, which trap the protein when it is bound to broken DNA and thus turn Topo II into a DNA-damaging toxin. Topo II can also promote tumor formation, as secondary leukemias occur in a fraction of patients treated with etoposide, and spontaneous Topo II errors are implicated in tumorigenic gross chromosomal rearrangements. However, despite widespread use of Topo II poisons in the clinic and the long-time appreciation that this enzyme has critical functions in genome integrity, remarkably little is known about how mammalian cells respond to and repair Topo II-generated double-strand breaks (DSBs). We are developing innovative approaches to address this lack of knowledge, exploiting novel mutant versions of Topo II that behave as if constitutively poisoned. We are pursuing studies in the yeast S. cerevisiae and in cultured mammalian cells to define genetic pathways for repair and/or tolerance of Topo II DSBs, and we aim to generate a conditional mouse model that will be a powerful system to explore tissue-specific mutagenesis and tumorigenesis caused by Topo II errors. This work will develop novel biological and molecular methods for exploring a critical but understudied aspect of genome integrity.