DNA Double strand breaks (DSBs) are highly genotoxic lesions induced by external agents (ionizing radiation or chemotherapeutic drugs), cellular processes (DNA replication defects, reactive oxygen species, recombination intermediates in B/T cell development), or through increasingly prevalent gene editing techniques (CRISPR/Cas9). Mammals have evolved two major pathways for repairing DSBs: Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ). While NHEJ is frequently referred to as the “error prone” pathway, recent biochemical and in vitro single-molecule imaging studies have revealed mechanisms to protect ends from excessive end-processing through a two-stage synaptic model. First, DNA ends are synapsed by NHEJ factors in a long-range complex (LRC) with ends held ~115 Å apart. The LRC recruits downstream factors and only permits highly regulated end processing of bulky adducts. Second—following the recruitment of the NHEJ-associated DNA Ligase IV (L4)—ends are brought into direct proximity for final end-processing and ligation. In this dissertation, I report that catalytically inactive L4 promotes significant amounts of end joining in cell models, supporting the recent two-stage synaptic model for NHEJ. Furthermore, I characterize repair products from cells expressing catalytically inactive L4 showing that repair is significantly more mutagenic than in cells expressing active L4. Finally, I identify multiple interfaces in L4’s DNA binding domain critical for maintaining promoting repair, providing insight into how L4 structurally supports synapsis of ends.
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