PROJECT SUMMARY/ABSTRACT Genome instability and cell cycle dysregulation are two important yet unique hallmarks of cancer, as they can accelerate acquisition of other tumorigenic mutations and thus the development of cancer. While the molecular functions and signaling pathways of both hallmarks have been extensively investigated, the quantitative and continuous interplay between DNA damage and the cell cycle is unclear. Understanding the direct interrelationship requires precise quantitative measurements of cell cycle transitions and DNA damage throughout the cell cycle in the same cell. Gaining this quantitative understanding will yield translational insights, as the efficacy of many chemotherapeutics and radiation therapies require precise dosage levels (to induce appropriate amounts of DNA damage) and treatment schedules (to target specific phases of the cell cycle). My overall objective is to understand how DNA double strand breaks (DSBs), the most harmful form of DNA lesions, affect cell cycle progression; and how the cell cycle program conversely regulates repair of DSBs. To achieve this objective, I propose to combine quantitative dynamic measurements of DSBs and cell cycle progression in single cells. I will use live-cell imaging, computational modeling, and manipulation of DNA damage to elucidate the molecular mechanisms that interlink DSB dynamics and cell cycle outcomes. I will focus my investigation specifically on DNA damage that arises from loss of CUL9, an E3 ubiquitin ligase and tumor suppressor. We previously found that loss of CUL9 increases the frequency of spontaneous DSBs in a subpopulation of unperturbed cells. How DSBs arise in this context and how they affect cell cycle progression is not known. Using time-lapse fluorescence microscopy with a 53BP1 live-cell reporter for DSBs, I discovered a striking trend in which 53BP1 foci are absent ~6 hours before cytokinesis in unperturbed cells. This pattern was disrupted in CUL9-deficient cells. Based on my preliminary data, my central hypothesis is that CUL9 facilitates the coordination of DSB repair and cell cycle progression. To test my hypothesis, I propose two specific aims: Aim 1: Determine how the dynamics of DSBs affect cell cycle progression and define the role of CUL9 in this relationship. This aim uses quantitative approaches and computational modeling to understand how DSBs slows cell cycle progression in a phase-specific way and determine how this regulatory relationship depends on CUL9. Aim 2: Determine the cell cycle regulation of DSB repair protein 53BP1 recruitment to DSBs, and if this regulation depends on CUL9. This aim characterizes 53BP1 recruitment to sites of DSBs at the late S/G2 phase and asks how CUL9 governs this process. This proposed research is innovative in its quantitative, single live-cell approach and is significant because the computational model to be developed will provide insights into novel treatment strategies against cancer. In the context of the extraordinary training environment at UNC Chapel Hill and an integrative training plan, this fellowship will strengthen my professional development as a physician-scientist.