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Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Schild-Poulter, Caroline

Abstract

The Ku heterodimer, composed of subunits Ku70 and Ku80, is a highly abundant protein complex, known for its affinity for double-stranded DNA ends. Accordingly, Ku is most well-studied for repairing double-stranded DNA breaks through the non-homologous end-joining (NHEJ) DNA repair pathway. Aside from NHEJ, Ku has also been implicated and studied in various other cellular processes including V(D)J recombination and telomere maintenance.

Numerous protein interactions have been mapped to the C-terminal region of Ku70, although few have been mapped to its N-terminal von Willebrand A-like (vWA) domain. Here, we used the high-throughput proteomic technique, proximity-dependent biotin identification (BioID, BioID2) to identify proteins that interact with full-length Ku70, the Ku70 vWA domain, and with a novel phosphorylation site, serine 155 (S155), found within the Ku70 vWA domain.

Using BioID, a proximity-based in vivo protein labelling technique, and a second high-throughput proteomics technique, affinity purification coupled to mass spectrometry (AP-MS), we were able to establish the Ku protein interactome and infer potential protein complexes and cellular pathways that may involve Ku.

Using BioID2 with various segments of the Ku70 protein including full-length Ku70, Ku70 ΔvWA, and the vWA domain targeted to the nucleus alone, we were able to map domain-specific interactions, test the specificity of the BioID2 technique, and validate RNF113A and Spindly as proximal and/or transient Ku70 vWA-specific interactors. Finally, we also used the BioID2 technique to identify Ku70 phospho-S155-specific interactors by comparing the BioID2 proteomic datasets for Ku70 S155A to S155D. We identified TRIP12, CHD3, and Borealin as candidate proximal interactors of Ku70 S155D, and we were able to further verify a proximity-based association between Ku70 S155D and TRIP12.

In sum, this research is the first to utilize the BioID and BioID2 techniques to identify in vivo candidate proximity-based interactors for the Ku heterodimer, the Ku70 vWA domain, and Ku70 S155, allowing us to infer novel functions that may involve Ku70. In addition, we demonstrated the versatility of the BioID2 technique by using it to identify full-length, domain-specific, and residue-specific candidate interactors, laying the foundation for future studies.

Summary for Lay Audience

Proteins represent the functional units within a cell and work together in groups, referred to as complexes or networks, to complete tasks. The interactions formed by groups of proteins can provide context and clues about the function(s) of a specific interactor protein. The Ku70 protein is best known for working with other factors to repair double-stranded DNA breaks. However, like most proteins, Ku70 is multi-functional, meaning it has been implicated in more than one cellular process. In order to identify the other processes that may involve Ku70, we were the first to use a proximity-based detection technique called BioID that is capable of identifying all proteins that come in close proximity to a protein of interest within cells. BioID and BioID2, a newer variation of the original technique, were both used in this study. Both techniques rely on fusing the protein of interest, Ku70, to a promiscuous biotin ligase enzyme isolated from bacteria. With the biotin ligase attachment acting as a proximity-sensor capable of biotin-tagging proteins that come near, proteins tagged within cells over a 24-hour period can be isolated and identified using mass spectrometry.

Using BioID and BioID2 with Ku70, we observed many known Ku70 interactors, confirming that the technique works. Most of the identified candidates were previously unknown and allowed us to infer new networks, processes, and complexes that could involve the Ku complex. In addition, we were able to validate three proteins, RNF113A, Spindly, and TRIP12 as novel proteins found in close proximity to Ku70. As the first to conduct BioID with Ku70, this research lays the groundwork for future studies to identify interactors under additional contexts, such as at double-stranded DNA breaks.

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

Available for download on Sunday, July 30, 2023

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