Abstract Double strand breaks (DSBs) in DNA pose a serious threat to genomic integrity and cell survival. These breaks arise from endogenous and exogenous sources and promote tumorigenesis. Non-homologous end joining (NHEJ) is active throughout the cell cycle and is responsible for repairing the majority of DSBs in higher eukaryotes. Previous work from our lab identified two key intermediates in the end joining reaction, termed the long-range complex (LRC) and short-range complex (SRC). Rapid binding of Ku to DSBs followed by recruitment of DNA-PKcs mediates LRC formation where the two dsDNA ends are tethered together but not aligned closely for ligation. Subsequent DNA-PK kinase activity and binding of XRCC4/LigIV and XLF promote the transition from the LRC to the SRC, where opposing DNA ends are poised to be ligated. DNA ends, however, are frequently chemically damaged and thus incompatible for ligation. The NHEJ machinery utilizes a vast array of end processing factors to correct this damage which enable end joining. Our lab has demonstrated that end processing is largely restricted to the SRC which prioritizes ligation over processing and also minimizes the extent of processing to what is necessary to enable ligation. Moreover, data from our laboratory suggests that end processing favors damage-correction (i.e. de/phosphorylation) over error-prone (i.e. polymerases and nucleases) processing factors to maintain genomic stability. The means by which the SRC favors less mutagenic repair remain unclear. In this proposal, I aim to understand how two end processing NHEJ factors - PNKP (damage-correction) and polymerase λ (error-prone) - act to create ligatable ends utilizing a Xenopus egg extract system combined with single-molecule fluorescence experiments. Building on preliminary data demonstrating that processing factors gain access to DNA ends within the SRC, I will ask how factor recruitment is regulated and how a hierarchy of access to DNA ends is maintained. To this end, I have generated fluorescently labeled NHEJ processing factors to directly monitor recruitment to the SRC using three-color FRET single-molecule imaging. Additionally, I will determine if competition between or recruitment of damage-correction and error-prone factors explains how minimal processing is achieved. Nucleases are the most mutagenic end processing factors as their activity always leads to loss of genetic information. Though many nucleases are implicated in NHEJ, there is little physiologically relevant data for many of the claims. To identify nucleases acting during NHEJ, I developed a crosslinking assay to pull-down flap associated proteins for identification via mass spectroscopy using the egg extract system. The activity of these nucleases will then be validated by testing their activity in a depletion and add-back assay. Overall, this proposal will advance our mechanistic understanding of how NHEJ regulates error-prone end processing which may inform effort...