Project Summary DNA double strand breaks (DSBs), an extremely toxic form of DNA damage, arise both spontaneously and in a programmed manner during antigen receptor development. Failure to properly repair these breaks leads to a variety of deleterious outcomes including cell death and gross chromosomal rearrangements that are a major driver of cancer. The primary DSB repair pathway in humans is non-homologous end joining (NHEJ). Deficiencies in NHEJ result in several severe genetic diseases that are characterized by immune deficiency, sensitivity to ionizing radiation and developmental abnormalities. Conversely overactive NHEJ in tumors provides resistance to DSB-inducing treatments and is correlated with poor clinical prognosis. Given these observations, inhibitors of NHEJ are a focus of therapeutical development. To fully exploit the clinical potential of these inhibitors it is critical to understand the molecular mechanism of NHEJ and how its various steps are regulated. During NHEJ, DNA ends are held together by a multiprotein synaptic complex and ultimately ligated back together. As DNA ends are often not initially compatible for ligation, several NHEJ-associated end processing enzymes act on ends to enable ligation. Some of these end processing enzymes are error-prone; their activity must be regulated to minimize genome alterations at the repair junction. In this project, we will further elucidate the molecular mechanism of DNA end synapsis and how it is coordinated with end processing to maximize the fidelity of NHEJ. To do this we will employ novel single-molecule imaging approaches developed by my laboratory to probe the structural dynamics of the NHEJ machinery in a physiologically complex cell-free system. These approaches will allow us to observe how DNA ends are brought together during repair and how protein factors are recruited to the NHEJ machinery and gain access to DNA ends. We will pursue the following three aims. In Aim 1, we will determine how ligation is coordinated with end processing within the Short-Range (SR) synaptic complex. Formation of the SR complex requires end binding of the ligase which likely competes with end processing factors for DNA ends. In Aim 2, we will elucidate how PAXX contributes to DNA end synapsis. Finally, in Aim 3, we will determine how end processing factors are regulated within the SR complex. Completion of these aims will provide rich molecular insight into how the NHEJ machinery carries out DNA end synapsis and processing to properly repair DSBs.