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 the likelihood of failures is high because of the many repeated sequences that characterize this domain. Despite the foundational importance of characterizing these processes, DSB repair mechanisms in heterochromatin are largely understudied. We discovered a specialized pathway that promotes faithful homologous recombination (HR) repair in heterochromatin while preventing aberrant recombination, effectively isolating heterochromatic repair sites to the nuclear periphery before strand invasion. We have recently identified several components required for this process, including nuclear actin filaments (F-actin) an myosins, and chromatin-associated nucleoporins, but the regulation and function of these components remain poorly understood. 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. Our central hypothesis is that F-actin, myosins, nucleoplasmic nucleoporins, and phase separation are essential regulators of heterochromatin repair dynamics, and that SUMOylation participates in coordinating their function repair progression. We will combine a wealth of super resolution imaging, genetic and biochemical approaches to investigate the molecular mechanisms involved in these process. Expected positive outcomes of this research include the 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, phase separation, repair progression, 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.