SUMMARY - Project 2: DSBR: Mechanisms of DNA double strand break repair and pathway selection Understanding how cells detect and repair DNA double strand breaks (DSBs) is critical to improving patient responses to radiation therapy and many forms of chemotherapy. The major pathway for repair of DSBs in normal cells is non-homologous end joining (NHEJ), which is active throughout interphase but error prone. However, in S and G2 phases, the more accurate homology directed repair (HDR) pathway is also active. Moreover, cancer cells sometimes use highly error-prone, mutagenic alternative-end joining (Alt-EJ) pathways for DSB repair. While we know a significant amount about the proteins involved in each pathway, there are wide knowledge gaps in understanding how these pathways intersect and how they are regulated in normal and cancer cells. Project 2 brings together leading investigators with expertise in NHEJ, HDR and Alt-EJ to determine how DNA end resection directs DSB repair pathway choice. In Aim 1 we will determine how NHEJ proteins tether broken DSB ends in synaptic complexes prior to processing to remove damaged end groups, and how DNA ligase IV uniquely catalyzes rejoining of DSB ends. We will also determine how the nuclease Artemis and the end-processing enzyme PNKP interact with synaptic complexes, providing the first comprehensive view of how NHEJ complexes function at DSBs. In Aim 2, we will explore how Artemis interacts with the MRN complex to regulate the initial stages of resection, how the short- and long-range resection machineries are coupled and how the tumor suppressor BRCA1-BARD1 complex stimulates long range resection by DNA2/BLM. In Aim 3 we will determine how XRCC1/DNA ligase III interacts with MRN and DNA polymerase to link DNA resection, gap-filling DNA synthesis and ligation during Alt-EJ and how DNA polymerase interacts with the key HDR protein, RAD51. Our studies will yield unprecedented molecular level insights into fundamentally important DNA repair pathways, and will also paint a comprehensive picture of how DSB repair pathways act together as an integrated network to repair radiation and chemotherapy-induced DSBs in cancer cells and non-malignant cells. The information generated by Project 2 will pave the way for novel approaches to better target tumors in the clinic. Indeed, small molecule inhibitors of NHEJ protein DNA-PKcs are in clinical trials and inhibitors of Artemis, PNKP, MRE11 and DNA ligases are being evaluated in pre-clinical cancer models, Moreover, novel Pol inhibitors with the potential to selectively target cancer cells that are dependent upon Alt-EJ will be identified in Project 2 by our structure-based approaches. EMB core expertise in protein expression and cutting-edge biophysical approaches of the SCB core are critical for the success of our project, which is fully synergistic with Project 4 on replication fork preservation, Project 3 on the role of PARP1 and PARylation in DSB repair, and P...