Abstract: Monitoring Editor: Kerry S. Bloom The completion of chromosome segregation during anaphase requires the hypercondensation of the approximately 1Mb rDNA array, a reaction dependent on condensin and Cdc14 phosphatase. Using systematic genetic screens, we identified 29 novel genetic interactions with budding yeast condensin. Of these, FOB1, CSM1, LRS4 and TOF2 were required for the mitotic condensation of the tandem rDNA array localized on chromosome XII. Interestingly, while Fob1 and the monopolin subunits Csm1 and Lrs4 function in rDNA condensation throughout M phase, Tof2 was only required during anaphase. We show that Tof2, which shares homology with the Cdc14 inhibitor Net1/Cfi1, interacts with Cdc14 phosphatase and its deletion suppresses defects in Mitotic Exit Network (MEN) components. Consistent with these genetic data, the onset of Cdc14 release from the nucleolus was similar in TOF2 and tof2Delta cells, however the magnitude of the release was dramatically increased in the absence of Tof2, even when the MEN pathway was compromised. These data support a model whereby Tof2 coordinates the biphasic release of Cdc14 during anaphase by restraining a population of Cdc14 in the nucleolus after activation of the CdcFourteen Early Anaphase Release (FEAR) network, for subsequent release by the MEN.