Williams, GarethPepper, Jordan Tan2025-02-212025-02-212025-02-19Pepper, J. T. (2025). The RAD51C-XRCC3 Homologous recombination repair complex: structural basis for its interactions and cancer associated mutations (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.https://hdl.handle.net/1880/120806The RAD51 paralogs are crucial proteins involved in the repair of double-stranded DNA breaks (DSBs) via homologous recombination repair (HRR). There are six human RAD51 paralogs, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and SWSAP1, and mutations in these genes are associated with cancer and other diseases. The RAD51 paralogs operate as discrete multi-protein complexes whose functional roles and structures are not fully understood. A key RAD51 paralog complex is the RAD51C-XRCC3 heterodimer (CX3), which is the focus of this thesis. I used biochemical, structural, and computational approaches to 1) profile disease and cancer-linked missense mutations found in RAD51C in the context of CX3, 2) determine the structural details of human CX3, and 3) identify and characterize potential CX3 protein interaction partners. I used the Alvinella pompejana ortholog of CX3 to investigate the impact of 5 different cancer associated missense mutations of human RAD51C on protein stability and biochemistry using heterologous expression and purification, mass photometry, fluorescence polarization assays, molecular dynamics simulations, and in silico mutation stability calculators. To investigate human CX3, I used a combination of small-angle X-ray scattering, cross-linking mass-spectrometry, structural prediction models and analysis to uncover features of how RAD51 family members interact with one another. Finally, I used affinity-purification and mass-spectrometry proteomics to detect possible CX3 protein-protein interaction partners, which I then attempted to verify using fluorescence-detection size-exclusion chromatography, with post-hoc analysis of candidate interaction partners using AlphaFold 3 and in silico calculation of interprotein energetics and affinity. From this work, I uncover insights in the structure of the CX3 complex, its possible roles in HRR, and a pathway towards characterizing it in association with interaction partners.enUniversity 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.RAD51 ParalogHomologous RecombinationDNA RepairProteinDNASAXSXL-MSStructural BiologyBiology--CellBiochemistrydoctoral thesis