PROJECT SUMMARY Repair of chromosome breaks by Nonhomologous end joining (NHEJ) determines the effectiveness of many cancer therapies, especially ionizing radiation. NHEJ is also essential for assembly of antigen receptor genes (immunoglobulins and T cell receptors) required for adaptive immunity. Here we propose to investigate the fundamental mechanism of this pathway, with a focus on aspects relevant to the effectiveness of radiation therapy. We’ve previously shown repair by NHEJ is often initiated by Polymerases that add RNA to the 3’ end of one strand of a chromosome break, which stimulates ligation of that strand. In Aim1 we will investigate the next steps, repair of the complementary strand, and replacement of RNA embedded in the initially repaired strand. Preliminary data argues the RNA embedded in the first step of NHEJ is not always metabolized well, and thus may contribute to genome instability. RNA incorporation also has a role in determining the choice of DSB repair pathway. Synthesis initiated from a 11-15 nucleotide RNA primer at the 5’ ends of the DSB has been shown to re-direct repair to the NHEJ pathway. This mechanism helps determine of the effectiveness of therapies, including radiation therapy, that specifically target hereditary breast cancers. However, our preliminary data indicates the RNA primer must be removed for NHEJ to occur; in Aim 2 we will investigate a candidate mechanism for removal of the primer. Targeted inhibition of DNA repair pathways holds great promise as a means of improving the effectiveness of radiation therapy. In Aim 3 we will systematically explore the consequences of combined DNA pathway inhibition on genome stability. We will address the impact of separate and combined inhibition of the two end joining pathways, and how pathway inhibition in both cases differs from genetic deficiency, as well as the impact of impaired incorporation of RNA by NHEJ polymerases.