Non-homologous end joining (NHEJ) catalyzes joining of unrelated DNA ends and is one of the principal DNA double-strand break (DSB) repair mechanisms. The major factors required for NHEJ have been identified and characterized. Yet, we do not understand how NHEJ proteins are coordinated for efficient joining with relatively minimal errors. Our goal is to identify the mechanisms that regulate events at DNA ends during NHEJ. We previously demonstrated that the yeast end-processing factor tyrosyl-DNA phosphodiesterase (Tdp1) participates in NHEJ. Tdp1 was identified through its role in repair of topoisomerase-mediated DNA damage and later shown to be mutated in a human neuropathy termed SCAN1. Tdp1-deficient cells are sensitive to topoisomerase independent DSB-inducing agents, implicating Tdp1 in DSB repair. We reported that mutations of yeast Tdp1 altered fidelity and efficiency of NHEJ and that the role of Tdp1 differed depending on DNA end structure. We hypothesize that Tdp1, which also has 3' nucleosidase activity, regulates events at DNA ends during mammalian NHEJ by generating a 3' PO4, thereby temporarily limiting further end processing. Our new results show that Tdp1 interacts with human NHEJ factors and influences their interaction with DNA, providing additional support a role for Tdp1 in mammalian NHEJ. In this application, we propose experiments to test the hypothesis that Tdp1 participates in mammalian NHEJ, and to test how the structure of DNA ends affects NHEJ reactions. In Aim 1, we will study the role of Tdp1 in NHEJ on a variety of DNA end structures, using a plasmid repair assay. This assay uses plasmid DNA linearized in vitro, has minimal background due to uncut DNA, and can be readily modified to assess the influence of different end structures, such as ends with 3' or 5' extensions, compatible and incompatible DNA ends, and the presence of various DNA adducts. These substrates will be tested in cells proficient or deficient in Tdp1, and will also be tested in cells expressing Tdp1- H493R, which causes accumulation of the Tdp1-DNA covalent reaction intermediate. These experiments will clarify the role of Tdp1 in NHEJ, and may shed insight on how protein:DNA adducts influence NHEJ. Aim 2 will use chromosomally integrated repair substrates to examine the role of Tdp1 in repair of DSBs with both 3' and 5' extensions in a normal chromosomal context. While breaks with 3' extensions are commonly used substrates in the study of DSB repair, breaks with 5' extensions have not been studied in detail. To study repair of breaks carrying 5' extensions, we will develop a new GFP-based reporter system with recognition sites for transcription activator-like nucleases (TALENs). Successful completion of these aims will improve our understanding of the mechanistic details of NHEJ and factors affecting the mutagenic potential of this repair pathway. A thorough understanding of the mechanism of NHEJ in human cells will provide important insights into cancer etiology, cellular response to cytotocxic anti-cancer agents, and the ageing process, thereby contributing fundamental information relevant to human health.