Biochemical and towards structural characterization of RAD51 and RAD51AP1 complexes
Date
2024-01-02
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Abstract
All living organisms must maintain accurate functioning and propagation of their genetic information. This is under persistent threat from DNA damaging events arising from cellular processes or exposure to DNA damaging agents. Amongst the most toxic forms of DNA damage are DNA double-strand breaks (DSBs). When unresolved or misrepaired DSBs can cause chromosomal breakages, fusions and chromosomal loss that drives cell death or genomic instability, which are hallmarks and drivers of cancer. The homologous recombination repair (HRR) pathway is an essential process that maintains genomic stability through the accurate repair of DSBs. RAD51 is the eukaryotic HRR recombinase that promotes HRR by assembling onto ssDNA to form nucleoprotein filaments and catalyses key HRR steps of strand invasion and homology search in the sister chromatid. A large number of accessory factors and RAD51 binding proteins, with inherited mutations in many of these predisposing to cancer, regulate the activities of RAD51 and the RAD51 filament to promote or inhibit HRR. The proper balance between pro- and anti-recombination factors is clearly essential to execute HRR only when necessary, in the correct cellular context. Despite this, there is a lack of high-resolution structures of RAD51 in complex with its protein partners, leading to large gaps in knowledge about structure-based mechanisms for RAD51 regulation in HRR. Here, I developed purification protocols and biochemical assays to quantify the activities of RAD51, which when applied in the context of RAD51 interactors will inform on the mechanistic basis for how they interact with and regulate RAD51 and RAD51 nucleoprotein filaments. I used these assays to study the interaction of RAD51AP1 with RAD51 and investigated a potential novel RAD51 interaction site within RAD51AP1. Finally, the reagents and protocols I developed, in addition to new methodologies, were used to investigate RAD51 interactions with other RAD51 regulatory proteins and the impact of small molecule inhibitors on Ku biochemistry.
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Keywords
RAD51, RAD51AP1, DNA repair, Homologous recombination
Citation
McCoy, C. J. (2024). Biochemical and towards structural characterization of RAD51 and RAD51AP1 complexes (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.