Humans inevitably age, and often develop cancer. A fundamental cause of aging and cancer is the loss of genome stability (abnormalities in nuclear DNA or chromosomes). Hence, understanding mechanisms regulating genome stability has huge impact on biology and human health. Hypothesis related to genomic instability generation and cancer evolution has long been revolved around the DNA damage response and repair programs that control DNA metabolism. Our recent findings indicate an important role of liquid phase separation, an emerging biological process by which proteins and nucleic acids form membraneless liquid droplets/condensates inside cells to respond to environmental and internal cues, in regulating genome stability. We demonstrated that this activity preserves the structure and the function of heterochromatin through a protein called 53BP1; importantly, this new protective activity of 53BP1 is independent of its widely known role in DNA double strand break (DSB) repair (Nat Communications, 2022). Hence, our results open a new research paradigm in studying genome stability regulation and stress response. The overarching goal of this application is to establish this research paradigm by understanding the mechanism and the impact of 53BP1 liquid phase separation. Two Aims are proposed. Aim 1 will determine how exactly 53BP1 phase separates at heterochromatin. Aim 2 will determine the biological significance of 53BP1 phase separation. By assembling an interdisciplinary team consisting of experts on molecular and cellular biology, proteomics, computational bioinformatics, and next generation sequencing, we will use a combination of cell biological, molecular, biochemical, genetic, morphological, and chemical approaches to answer these questions. Upon completion, our studies will illustrate a previously uncharacterized function of 53BP1 and a novel interplay between 53BP1 and heterochromatin and determine their impact on genome stability, facilitating our understanding of fundamental cellular information processing, cellular homeostasis, and cellular physiology. To the best of my knowledge, no other investigators are working on this research direction.