Exploring the implications of Ku70 interactions in response to DNA damage

Abstract

The Ku protein, composed of the subunits Ku70 and Ku80, is a highly abundant heterodimer essential for the repair of double stranded breaks through the non-homologous end joining pathway (NHEJ), functioning as a recruitment hub for various repair factors to ensure repair of the break. Previously, we identified the D192/D195 residues in the Ku70 vWA domain as essential for NHEJ, as mutation of these residues (D192A/D195R) grossly impaired DSB repair. Due to its role as a recruitment hub, we hypothesized that the NHEJ defect observed with the Ku70 D192A/D195R mutants was due to an abolished interaction with known or novel repair factors. Thus, we employed a proximity labelling technique called BioID miniTurbo to identify Ku70 and Ku70 D192A/D195R proximity based interacting partners and found LigIV, a core component of the NHEJ machinery, to be enriched in our wildtype screen and underrepresented in our mutant expressing constructs. We further validated that upon mutation of these residues, the LigIV-XRCC4 complex interaction with Ku70 was abolished, suggesting a critical requirement for these residues in NHEJ. We further explored the role of another proximity based interacting partner of Ku70 called TRIP12. Our lab previously identified the Ku70 S155 residue as a novel phosphorylation site in the DDR and conducted proximity labelling with BioID2 with the phosphomimetic mutant Ku70 S155D. These experiments identified the E3 ligase TRIP12 as a high confidence proximal interactor. I further validated complex formation between Ku70 and TRIP12 and demonstrated the Ku70-TRIP12 complex tracks with active DSBs repair using proximity ligation assays and γH2Ax signalling. Due to its role as a recruitment hub, we hypothesized that Ku70 may be responsible for recruiting TRIP12 to DSB sites. Using domain deletion mutants with microlaser irradiation, we found that TRIP12 is recruited to sites of DSBs in a Ku70- and -WWE dependent manner. Because WWE domains recognize and bind PAR chains, we also treated our cells with the PARP inhibitor Olaparib and observed the loss of TRIP12 recruitment to sites of damage, consistent with our previous observations. In summary, this work documents the first comprehensive Ku70 proximal proteome in response to DNA damage and validated the LigIV interaction with the D192/D195 residues. We also demonstrate novel complex formation between Ku70 and TRIP12 and further demonstrate that TRIP12 recruitment to sites of DSBs is WWE and Ku dependent. More work is needed to fully elucidate the functional implications of the Ku70-TRIP12 interaction. However, the work in this thesis lays the foundation for studying how Ku70 interacts with DDR regulators to promote faithful DSB repair.