Project Summary/Abstract The DNA dependent protein kinase catalytic subunit (DNA-PKcs) is a key regulator of the canonical non- homologous end-joining (NHEJ) pathway for repair of DNA double strand breaks (DSBs) and resistance to ionizing radiation (IR). DNA-PKcs is recruited by the Ku70/80 heterodimer to the DSB ends to form the DNA-PK holoenzyme to initiate NHEJ mechanism. DNA-PKcs also plays an important role in cellular resistance to replication stress. It coordinates with and is rapidly phosphorylated by the ATR (ataxia telangiectasia mutated and Rad3 related) kinase at stalled replication forks upon UV, although the mechanism is not well understood. Our recent work has focused on identifying the player(s) required for the recruitment of DNA-PKcs to stalled replication forks. We have identified that PIDD (p53-induced protein with a death domain), a known apoptosis mediator for assembling the PIDDosome complex and Caspase-2 activation, is required for DNA-PKcs recruitment to stalled replication forks and its association with ATR. Disrupting the interaction between DNA- PKcs and PIDD not only compromised ATR dependent DNA-PKcs phosphorylation at the Thr2609 cluster but also attenuated the ATR signaling pathway and intra-S checkpoint. To assist our investigation, we have created cell lines and a mouse model expressing a DNA-PKcsPL mutant protein unable to interact with PIDD. Our results showed that DNA-PKcsPL cells were highly sensitive to both UV and IR. Based on these preliminary findings, we hypothesize that PIDD, but not the Ku70/80 heterodimer, mediates DNA-PKcs recruitment to stalled replication forks and promotes its association with the ATR pathway. We also hypothesize that PIDD facilitates DNA-PKcs kinase activation and DSB repair. Finally, we hypothesize that the interaction of DNA-PKcs with PIDD will affect cell death regulation and cancer development. In this project, we will determine the coordination between DNA- PKcs, PIDD, and ATR in the cellular response to replication stress. Our specific aims are: 1) To test the hypothesis that PIDD modulates DNA-PKcs kinase activation at stalled replication forks and at IR-induced DNA lesions that are not bound by Ku70/80, 2) To test the hypothesis that PIDD is required for DNA-PKcs to properly function at stalled replication forks upon UV irradiation, and 3) To test the hypothesis that the DNA-PKcs-PIDD association affects cell fate determination upon DNA damage and cancer development.