Genome instability is a hallmark of cancer. Yet genome maintenance and mechanisms of genome instability are not clearly understood. This proposal continues the collaboration between Dr. Paul Hasty at the Health Science Center at San Antonio and Dr. Cristina Montagna at Albert Einstein College of Medicine in New York. They will explore the involvement of TREX2 in both the suppression and development of genomic instability. TREX2 is a 3'?5' exonuclease that removes 3' mismatches in DNA. However, its biological function is not known. We found that TREX2-deletion increased the level of isochromatid breaks and gross chromosomal rearrangements (GCRs) demonstrating that TREX2 maintains intact chromosome structure; however, we also found that TREX2 fused mismatch inverted repeats to induce palindromic chromosomes that were unstable and susceptible to continual rearrangements that involve multiple chromosomes. In addition, we found that TREX2 enables genomic instability in cancer prone cells. Thus, TREX2 both maintains and alters genome integrity. Yet just how TREX2 accomplishes these antithetical outcomes is not known. Since TREX2 deletion caused breaks and rearrangements, we thought it participated in DNA double strand break (DSB) repair. However, TREX2-deleted cells have elevated DSB repair capacity. Therefore, we hypothesized that TREX2 suppressed the formation of DSBs by maintaining replication forks (RFs). To support this hypothesis, we found TREX2 associated with proteins in the error-free postreplication repair (EF-PRR) pathway and enabled PCNA (proliferating cell nuclear antigen) ubiquitination; a key EF-PRR function that suppresses RF stalling through two mechanisms: translesion synthesis and template switch. We wish to further these investigations. In Aim 1 we will address our hypothesis that TREX2 suppresses DSB formation and GCRs though EF-PRRmediated lesion bypass and in Aim 2 we will address our hypothesis that TREX2 enables genomic instability through EF-PRR and its genetic integration with other tumor suppressor pathways. Finally in Aim 3 we propose to alter TREX2 in mice to observe its biological function in the whole animal. These studies will elucidate the dual role that TREX2 plays in preserving and altering genomic integrity and will provide mechanistic insight to both the suppression and creation of genomic instability that causes cancer. Such knowledge will be useful to evaluate TREX2 as an anti-cancer drug target.