DNA damage constitutes a major threat to genetic integrity, and has thus been implicated in the pathogenesis of cancer. Elucidating the mechanism safeguarding genome stability is important for understanding the mechanism underlying carcinogenesis. Genome integrity is constantly threatened by endogenous and exogenous agents arising from cellular metabolic processes as well as environmental exposure, many of which impede normal DNA replication and cause replication fork stalling. Stalled forks need to be properly repaired and rescued to prevent DNA lesions and genome instabilities that contribute to tumorigenesis. To repair and rescue stalled replication, a network of proteins regulating DNA damage response, DNA repair, replication, and cell cycle checkpoints are activated in response to stalled replication in order to stabilize and restart stalled forks. However, the mechanism underlying fork repair is poorly understood. Recent studies have revealed that the balance and dynamics of RAD51 and RPA at stalled forks are crucial for fork stabilization and restart. Yet how RAD51 and RPA activities are modulated remains largely elusive. Our recent findings suggest that the high-affinity single-stranded DNA binding protein complex known as CST may be a new modulator for RPA and RAD51 at GC-rich repetitive sequences in response to replication stress. The objective of this proposal is to understand the molecular relationship between CST, RAD51 and RPA in fork rescue, with the goal to provide novel insights into how cells counteract DNA damage caused by genotoxins. We propose to integrate advanced biochemical, cell biological, cell imaging, and next-gen sequencing techniques to examine how CST may regulate RPA binding at stalled sites and maintain fork progression and stability under stress (Aim 1), define how CST may modulate RAD51 activity at GC-rich stalled sites (Aim 2), and characterize the regulatory role of a surface-exposed region of CST in regulating RAD51 and RPA activities (Aim 3). Findings from the proposed research will provide novel information on the mechanism of genome stability maintenance of rescuing stalled replication and preserving genome stability.