Electronic Thesis and Dissertation Repository


Master of Science




Dr. Caroline Schild-Poulter


DNA double strand breaks (DSBs) are a toxic and dangerous form of DNA damage repaired primarily by non-homologous end joining (NHEJ) in mammals. The Ku70/80 heterodimer rapidly responds to DSBs, stimulating recruitment of downstream NHEJ factors to protect, process, and ligate broken DNA ends. Work in our lab has shown that a D192A/D195R mutation in helix five of the Ku70 von Willebrand A (vWA) domain leads to an NHEJ defect. However, little is known about the function of this region in NHEJ. We hypothesized that helix five of the Ku70 vWA domain mediates a protein-protein interaction that is crucial for DNA repair. We optimized a laser microirradiation protocol and used microirradiation and in vitro binding assays to assess helix five’s ability to recruit and interact with a variety of NHEJ factors. Surprisingly, the D192A/D195R mutation did not prevent recruitment of NHEJ factors to laser-induced DNA damage, nor did it impede several protein-protein interactions that we assessed in vitro. Overall, while the role of helix five of the Ku70 vWA domain in DNA repair remains unknown, our investigations have ruled out several possible binding partners and suggest that this helix could interact with a yet unidentified DNA repair factor.

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