We propose to study the impact of loss-of-function mutations of DNA DSB (double-strand break) repair genes on end joining processes in human cells. We will focus heavily, but not exclusively, on the heterodimeric (Ku70 and Ku86 subunits) Ku DNA end-binding complex and LIGIII (DNA ligase III). Ku has a storied history and it has been known for decades that it is required for most of the generalized DSB repair occurring in somatic cells as well as being critical for the proper development of the vertebrate immune system. Ku performs these duties as part of a repair pathway referred to as C-NHEJ (classic-non-homologous end joining). In work published this year, we demonstrated that Ku has a hitherto underappreciated function as a suppressor of the other competing DSB repair pathways, namely A-NHEJ (alternative-NHEJ) and HR (homologous recombination). Thus, when the levels of Ku are reduced, A-NHEJ and HR, which normally comprise only a small fraction of the end joining activity in human cells, become more active. This observation has potential clinical implications. Thus, the vast majority of human cancer patients are treated either with radio- or chemotherapy (or both) and the efficacy of these anti-tumor modalities resides in their ability to generate DNA DSBs. Thus, strategies designed to increase the efficacy of tumor cell killing by radio- or chemotherapy that involve reducing the levels of Ku are likely to fail due to the up-regulation of A-NHEJ and/or HR. This is a hypothesis that we will directly test in this grant application. Moreover, the molecular mechanism by which Ku mediates this suppression is unknown and we propose genetic and reverse genetic experiments to elucidate this process. Finally, we have begun to genetically tackle the A-NHEJ pathway. A number of genes have been implicated in A-NHEJ, but the important genes remain poorly defined. One of the more compelling A- NHEJ candidates is LIGIII (DNA ligase III). To experimentally test the importance of LIGIII we have constructed a viable LIGIII-null human cel line and herein propose to characterize the impact of the loss-of- function of LIGIII on A-NHEJ. In particular, one characterization involves a novel and powerful assay system by which we can measure ionizing radiation-induced gross chromosomal rearrangements in human cells lacking LIGIII (or Ku86). To our knowledge, we are one of only a few laboratories in the world utilizing genetic, loss-of-function approaches to study DNA DSB in human cells and thus we are well positioned to gain insights into the mechanism of DSB repair that cannot be obtained elsewhere.