SUMMARY Advancing our knowledge of pericentromeric heterochromatin repair is a high impact investment for improving human health: heterochromatin is a poorly characterized region that comprises nearly a third of the human genome; double-strand break (DSB) repair failures in this region affect not just specific genes but also genome-wide stability; and failures here are a high risk because of the abundance of repeated sequences that characterizes this domain. In spite of the foundational importance of characterizing these processes, DSB repair mechanisms in heterochromatin are mostly unknown. We recently discovered a specialized pathway that promotes faithful homologous recombination (HR) repair in heterochromatin while preventing massive genome instability. We discovered a critical role of the Smc5/6 complex in this pathway, but how this complex participates in heterochromatin repair is unknown. Deregulation of heterochromatin repair is likely one of the most underestimated and powerful sources of tumorigenesis, and identifying the components involved is essential for understanding cancer etiology and developing more effective strategies for therapeutic intervention. To gain insight into the role of Smc5/6 in heterochromatin repair, we used mass spectroscopy to identify new interactors of this complex, which will be further investigated in this proposal. Our central hypothesis is that repair occurs in three steps: an initial phase when abnormal progression of HR is suppressed inside the heterochromatin domain; a second phase when repair sites relocalize to the nuclear periphery; and a third phase characterized by the removal of the block to HR progression at the nuclear periphery. We will combine a wealth of imaging, genetic and biochemical approaches in Drosophila cells and organisms to identify the molecular targets involved in these steps, and determine their role in the spatial and temporal regulation of heterochromatin repair. Expected positive outcomes of this research include the first systematic identification of the molecular machinery that protects heterochromatin from massive genome rearrangements, enabling successful completion of HR repair. These studies are also expected to illuminate missing links between nuclear architecture and dynamics, repair progression, RNAi silencing pathways, and the stability of repeated DNA sequences. These results will have an important positive impact by identifying crucial safeguard mechanisms used by normal cells to protect the genome from environmental threats. Mutations in these pathways result in genome instability, tumorigenesis, and reduced life span. Thus, we expect that the proposed studies and future research will trigger exciting advancements in the prevention, early detection, and treatment of cancer and other human diseases associated with genome instability and aging- related disorders.