Project Summary The faithful repair of DNA damage is critical to maintain genome stability. Lack of repair or inappropriate DNA repair can lead to mutations, uncontrolled cell growth, cell death, or growth arrest. We have identified FAN1 nuclease as a potential effector for maintaining genome stability. FAN1 has been implicated in interstrand crosslink (ICL) repair as a nuclease responsible for unhooking and releasing the crosslinked or damaged DNA base. Mutations in ICL repair proteins usually give rise to bone marrow failure or cancer; however, mutations in FAN1 cause a form of chronic kidney disease termed Karyomegalic Interstitial Nephritis (KIN). KIN patients ultimately develop renal failure requiring dialysis or kidney transplantation. The underlying mechanism as to how FAN1 mutations gives rise to KIN still remains unknown. It may be that kidney dysfunction occurs through defective repair of endogenously produced ICLs or it could be due to independent functions of FAN1 outside of canonical ICL repair. More recently, it was shown that mutations in FAN1 confer a greater susceptibility to colorectal and pancreatic cancer, and are genetic modifiers for trinucleotide repeat expansion diseases, such as Huntington's disease. Together, these findings suggest that FAN1 may have functions outside of the canonical ICL repair. In the proposed research, we aim to discern the various molecular mechanisms of FAN1 function in vivo as well as to identify the causative endogenous lesions driving KIN. In Aim 1, the goal is to use a multi-pronged approach determine the role of FAN1 in somatic repeat instability. Using a mouse model of trinucleotide repeat expansion we will assess if the absence of Fan1 drives repeat expansion in a tissue- specific manner or through germline inheritance. We will also determine if FAN1 affects microsatellite repeat instability (MSI), a common phenotype observed in colon cancer. In Aim 2, we will determine the function of FAN1 in DNA replication, the mechanism of polyploidization in KIN, and the predisposition to tumorigenesis. We will use a Fan1 knockout mouse model that recapitulates the human KIN phenotype. By isolating cells from this model and performing various DNA replication assays in the presence/absence of DNA replication stress or damage, we will shed light on the in vivo role for Fan1. We will also perform experiments to better understand the role for FAN1 in KIN and tumorigenesis. Finally, in Aim 3 we will identify the causative lesion(s) driving KIN in the absence of FAN1. The findings from this proposal have the potential to uncover the fundamental principles of how FAN1 acts to suppress dysfunction of the kidney and digestive system.