Chromosome double strand breaks (DSBs) that evade DNA damage checkpoints can persist into mitosis. These DSBs are in danger of forming highly detrimental structures connected to genome shattering and tumor progression, collectively referred to as micronuclei. To find mechanisms that prevent micronuclei, we discovered that Drosophila papillar cells naturally inactivate DNA damage checkpoints, and as a result frequently exhibit DNA fragments in mitosis. These fragments lack centromeres (acentric DNA), yet remarkably segregate during papillar mitosis. This process prevents micronuclei and tissue development defects. The distinctive dependence of papillar tissue development on acentric DNA segregation holds promise to reveal fundamental responses to DSBs that persist into mitosis. From our combination of in vivo genetic screens, live imaging, and complementary biochemistry approaches with collaborators, we are poised to make unique conceptual advances in this area. This proposal leverages our expertise, new findings, and a genetically amenable Drosophila model to uncover regulation of broken chromosome segregation. The significance of our proposed work is evident in the frequent contribution of micronuclei to genome instability and the evolutionary conservation of the molecules studied, including the Alternative End Joining (Alt-EJ) repair protein DNA Polymerase Theta, conserved monoubiquitination of the DNA repair scaffold FancD2, and the ubiquitin ligase CRL4CDT2. The innovation of our approach derives from our model system that is evolutionarily wired to solve the challenge of frequent persistent broken chromosomes, and the enhanced in vivo genetic screening capability of our system. These advantages led to the preliminary data presented in this proposal. In Aim1, we will define the pathway leading to poleward segregation of acentric DNA. In this Aim, we will identify Pol Theta domains that function in acentric DNA segregation, pinpoint the extent to which Alt-EJ occurs in papillar cells with DSBs, and assess the role of FancD2 in regulating Pol Theta after DSBs. In Aim2, we will define the signaling pathway that promotes the transition from lagging to segregating acentric DNA. In this Aim, we will determine if CRL4CDT2 functions together with Pol Theta/FancD2 to promote acentric DNA segregation, uncover whether critical regulation of CRL4CDT2 activity or in papillar cells with DSBs, and assess if inactivity of interphase checkpoints leads to a requirement for CRL4CDT2 in segregating acentric DNA in a non-papillar cell context (wing cells). In Aim 3, we will determine how regulation of mitotic chromosome condensation contributes to segregation of acentric DNA fragments. We will build on biochemical, genetic, and protein localization data connecting CRL4CDT2 to the mitotic complex Condensin I. We will assess the role of CRL4CDT2 in regulating Condensin I localization during acentric DNA segregation and determine the role of a conserved CRL4CDT2 re...