Williams, GarethLa, Justin2023-01-232023-01-232023-01-16La, J. (2023). Biochemical and structural approaches toward determining the role of the DEK protein in homologous recombination repair (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.http://hdl.handle.net/1880/115712https://dx.doi.org/10.11575/PRISM/40625The DEK proto-oncogene is a nuclear protein important in regulating many cellular processes including DNA double-strand break repair and chromatin biology, through protein-protein and protein-nucleic acid interactions. Mutations or altered expression of DEK result in the development of various human diseases including acute myeloid leukemia and many other types of cancer. However, despite its importance in biology, there is a lack in knowledge on the mechanistic and structural basis for how DEK interactions regulate the numerous cellular processes it is involved in. Here, I developed purification protocols for full-length and subdomains of DEK, and developed biochemical assays to investigate DEK-DNA interactions and role of DEK in regulating homologous recombination repair. The DNA binding results showed that DEK can bind a wide variety of DNA substrates with a range of affinities between ⁓130-1300 nM. Phosphorylation of full-length DEK blocked DNA binding, consistent with and extending published work. The preliminary homologous recombination repair results showed that DEK directly binds to the RAD51 recombinase in vitro and may promote RAD51-mediated double-stranded DNA displacement during strand invasion. The results and protocols developed from this thesis provide a platform for future efforts to further characterize DEK’s role in HRR, to test the effects of disease-associated mutations, and to determine structures of DEK complexes with DNA, RAD51, and other interacting proteins including the nucleosome. This future work will address the gap in knowledge regarding the mechanistic basis for DEK’s role in homologous recombination repair and may provide new insights into its functions in human diseases.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.DNA repairDEKHomologous recombination repairBiologyBiology--MolecularBiochemistrymaster thesis