Chromosomal rearrangements are a hallmark of cancer cells and constitute a major pathway by which genes that affect tumor initiation and progression become mutated. Such aberrancies can result from defects in double-stranded break (DSB) repair. There are three major pathways of DNA repair in mammalian cells – the well-studied homology recombination (HR) and non-homologous end joining (NHEJ) pathways, and the poorly characterized, yet highly error-prone alt-NHEJ (alternative-NHEJ) pathway. Genetic rearrangements consistent with alt-NHEJ have been noted both in spontaneous and therapy-related tumors. In this proposal we will focus on polymerase theta (Polθ), a low-fidelity enzyme that we recently identified as a key factor that mediates DSB repair by alt-NHEJ. Given that HR-defective tumors are “addicted” to repairing DSBs via the alt-NHEJ pathway, we hypothesize that the mutagenic activity of Polθ help establish a genomic landscape that is conducive for aggressive tumor behavior. Additionally, we predict that deleting Polθ in tumors with mutations in the breast cancer susceptibility (BRCA) genes will sensitize cells to DNA damage- inducing therapeutic agents, including radiation therapy, cisplatin and PARP inhibitors. In the second aim, we will investigate the impact of ATM and PARP1 on Polθ is recruited to break sites and how the polymerase modulates damage sites to promote erroneous repair. In addition, we will pursue a proteomic-based approach to highlight the full spectrum of molecular players involved in alt-NHEJ. Ultimately, a full understanding of the mechanistic basis of alt-NHEJ will provide a better understanding of the source of genomic instability during the course of malignancy and guide more effective treatment strategies for the increasing number of patients with HR mutated tumors.