Project Summary/Abstract PRMT5 is an arginine methyltransferase with key roles in cancer. The gene has pleiotropic functions ranging from gene regulation and development to modulation of DNA double strand break (DSBs) repair. In S-phase and mitosis, DSBs are repaired primarily by homologous recombination (HR) a process in which missing genetic information is copied from another undamaged chromosomal region. In humans, error-free HR is mediated by BRCA1, BRCA2 and RAD51. RAD52, an accessory gene, facilitates an error-prone HR sub-pathway that can produce intrachromosomal deletions (ICDs). PRMT5 appears to modulate chromatin remodeling at the DSB through the error-free BRCA1/2-RAD51 pathway. In fission yeast (S. pombe), we identified physical and genetic interactions between PRMT5 and RAD52. Deletion of PRMT5 increases the frequency of ICDs while deletion of RAD52 decreases ICDs suggesting that they have opposite functions. In cancer cells PRMT5 mutations increases the size of ICDs. Using artificial intelligence algorithms, we discovered multiple likely pathogenic mutations including three driver mutations in the active site of PRMT5 that are likely to destabilize the function of the enzyme. We hypothesize that PRMT5 inhibits ICDs by biasing repair of DNA double strand breaks toward conservative pathways. We developed innovative in vivo assays to probe HR repair mechanisms that produce ICDs. The repair mechanisms and factors involved are conserved from yeast to humans making the S. pombe model system highly tractable. A homology analysis reveals that all identified human mutated residues are present in yeast. In Aim1 we will place PRMT5 in the DNA damage repair epistatic pathway using mutational analysis and sensitivity to various DNA damage drugs. Additionally, we will employ in vivo repair assays to understand the HR pathways by which ICDs are produced in the absence of the PRMT5 function. In Aim 2 we will analyze PRMT5 mutations identified in cancer cells using the Catalogue of Somatic Mutations in Cancer (COSMIC). We will employ modeling techniques and enzymatic assays to test how these mutations affect the enzymatic function of PRMT5 and DSB repair. Most of these mutations are conserved in yeast making this analysis tractable. The findings will be further validated in human cells.