Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dr. Caroline Schild-Poulter

Abstract

Ku is an abundant, highly conserved DNA binding protein found in both prokaryotes and eukaryotes that plays essential roles in the maintenance of genome integrity. In eukaryotes, Ku is a heterodimer comprised of two subunits, Ku70 and Ku80, and is best characterized for its central role as the initial DNA end binding factor in the “classical” non-homologous end joining (C-NHEJ) pathway, the main DNA double-strand break (DSB) repair pathway in mammals. At the break, Ku directly and indirectly interacts with several C-NHEJ factors and processing enzymes, serving as the scaffold for the entire DNA repair complex. In this work we aim to characterize the function of the Ku70 von Willebrand A-like (vWA) domain, a protein-protein interaction domain, in Ku’s role in the response to DSBs.

In this study we identified a requirement for the Ku70 vWA domain in both NHEJ and signaling to the DNA damage response (DDR) pathway to determine cell fate decisions. We demonstrated that mutation of residues D192A/D195R in helix 5 of the Ku70 vWA domain resulted in decreased DNA repair efficiency and extremely low survival in cells treated with ionizing radiation (IR), indicating a role for these residues in NHEJ. We also identified a novel phosphorylation event at Ku70 S155 in response to DNA damage. Cells expressing a mutant Ku70 bearing an alanine substitution of this residue displayed increased survival and decreased activation of apoptosis following IR treatment, indicating a defect in the DDR pathway to relay signals to the cell death machinery. The phosphomimetic mutant of Ku70 (S155D) exhibited the opposite phenotype, very low survival after IR, and the constitutive activation of DDR markers and cell cycle arrest even in the absence of any DNA damage. We found that Ku70 S155 phosphorylation after IR was required for Ku to interact with and inhibit the Aurora Kinase B, to induce cell cycle arrest. Overall we propose that the Ku70 vWA domain functions to both facilitate the repair of breaks by NHEJ, and to relay the signal of unsuccessful repair to the DDR in order to activate cell cycle arrest and apoptosis.


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