Project Summary Werner Syndrome (WS) is an autosomal recessive disorder characterized by premature development of aging features. In addition, WS individuals have an increased predisposition to cancers that are mesenchymal in origin. Primary cells derived from WS patients exhibit elevated levels of chromosomal translocations, inversions, and deletions of large segments of DNA and have a high spontaneous mutation rate. The Werner syndrome protein (WRN), mutated in WS, is unique among the RecQ family proteins as it possesses exonuclease and 3′ to 5′ helicase activities. Thesetwo enzymatic activities have coordinated functions on a variety of structured DNA substrates. Additionally, WRN also has nuclease-independent functions during DNA replication and repair, although these non-enzymatic activities are not well understood. WRN forms dynamic sub-complexes with different factors involved in multiple biological processes.Though accumulating evidence suggests that WRN plays a crucial role in genome stability maintenance pathways, the exact contribution of WRN in preventing genome instability is unclear. Our hypothesis is that WRN suppresses genomic instability by facilitating faithful repair of collapsed replication forks upon replication stress. To accomplish our goals, three specific aims are proposed: Aim 1. Verify that a single phosphorylation site dictates other post- translational modifications in WRN and its function in genome stability maintenance upon replication stress; Aim 2. Determine the mechanism used by WRN to stabilize Rad51 at collapsed replication forks; and Aim 3. Verify that WRN interacts with specific-genomic loci and suppresses mis-joining of collapsed replication forks. Innovative aspects of this proposal include: a. Development of a novel system to identify cell cycle-specific biological functions of WRN. In this system, expression of WRN can be regulated in a cell cycle-specific manner. b. Establishment of a new tool to map genome interaction domains of WRN and precisely identify fidelity of replication-associated DNA double-strand breaks at a single nucleotide level. Results from this genomic sequencing study will provide information on molecular biomarker(s) contributing to the initiation of carcinogenic events in WS. Significantly, deciphering the molecular choreography of WRN, its biochemical activities, post-translational modifications and interaction partners in the fidelity of replication fork processing will provide new insight into the molecular origin of cancer in WS individuals. Our results will ultimately advance our understanding of the pathophysiology of WS.