Abstract Chromosome segregation errors result in aneuploidy which causes birth defects and cancer. We have defined a new mitotic Topo II-responsive control (TRC) in yeast that delays the cell cycle when Topo II activity is insufficient for accurate chromosome segregation. This TRC mechanism is conserved in human cells, but has not been extensively studied. Activation of the TRC is triggered by defects in the strand passage reaction of Topo II, when the enzyme transits too slowly through its structural conformations. TRC activation also requires the catalytically inert C-terminal domain of Topo II, which contains novel protein- protein interaction motifs that bind to TRC signaling proteins. The central model is that aberrant strand passage may structurally transfer from the catalytic core of Topo II to the C-terminal domain which functions as a signal-generating scaffold to halt the cell cycle. The conservation between the human and yeast TRC responses provides unique opportunities to identify the TRC components and to reveal the mechanism of TRC activation. We aim to determine the cellular/genomic sites of TRC activation, the signaling molecules involved and the mechanism of TRC activation by the C-terminal domain of Topo II. The results of these studies will impact opportunities for translational research because we will identify new potential therapeutic targets. Our findings will also impact the use of widely prescribed therapeutic drugs that target Topo II because we will gain mechanistic insight into cellular responses to Topo II inhibition. The preliminary data and newly developed experimental tools place us in a unique position to determine the conserved mechanism of this scarcely studied mitotic control.