The repair of DNA double strand breaks (DSBs), induced by ionizing radiation (IR) or generated during the process of V(D)J recombination, is of paramount importance to the cell, as misrepair of DSBs can lead to cell death or promote tumorigenesis. The DNA-dependent protein kinase (DNA-PK) is the key component of the non-homologous end-joining (NHEJ) pathway of DNA DSB repair in mammalian cells. We have previously shown that the kinase activity of the DNA-PK catalytic subunit (DNA-PKcs) is absolutely essential for the repair of DSBs by NHEJ. However, nothing is known about the in vivo targets of DNA-PKcs (whether itself and/or other factors), and about the mechanisms underlying the requirement of DNA-PKcs kinase activity for NHEJ. We propose, in this grant, to define the phosphorylation events mediated by DNA-PK and to understand the mechanisms by which these modifications modulate NHEJ. Towards this goal we propose to 1) Identify relevant phosphorylation sites on DNA-PKcs, demonstrate in vivo phosphorylation at these sites, and understand the biological consequences of abrogation of phosphorylation; 2) Examine the localization of phospho-DNA-PKcs with respect to DSBs, determine the mechanism by which phosphorylation might modulate DNA-PK function, and understand how DNA-PKcs phosphorylation is coordinated with other early events at a break; 3) Identify phosphorylation sites and the function of phosphorylation of two known targets of DNA-PK, XRCC4 and WRN, and identify novel targets of DNA-PKcs kinase activity. We have recently made a breakthrough, by identifying one of the sites on DNA-PKcs that it is phosphorylated in vivo in response to DNA damage and by demonstrating that abrogation of phosphorylation leads to radiation sensitivity. We have also identified putative phosphorylation sites on XRCC4 and WRN, and are pursuing a novel method to identify new targets of DNA-PKcs. The work proposed in this grant should provide us with a mechanistic basis for the requirement of the kinase activity of DNA-PKcs in NHEJ.