Mechanisms of Error-Prone DNA Repair - Abstract DNA double strand breaks (DSBs) are an extremely toxic form of DNA damage that arise spontaneously and in a programmed manner during antigen receptor development. Failure to properly repair these DSBs 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 cancerous tumors provides resistance to DSB-inducing treatments and is correlated with poor clinical prognosis. Given the important role of NHEJ in disease, inhibitors of the pathway 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. Loss of DNA end synapsis can lead to mispairing and chromosome translocations that are a driver of cancer. 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. Therefore, their activity must be regulated to minimize genome alterations at the repair junction. In this project, we will elucidate the molecular mechanism of DNA end synapsis and how it is coordinated with end processing to maximize the fidelity of NHEJ. To accomplish these goals, 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 investigate key open questions in the field including: (i) how core and accessory factors cooperate to tether DNA ends, (ii) how synapsis is maintained during end processing, and (iii) how end processing enzymes are prioritized to minimize mutagenesis. Overall, our studies will provide rich molecular insight into how the NHEJ machinery carries out DNA end synapsis and processing to repair DSBs.
