Evaluating Chemical Crosslinking as a Tool for Enhancing DNA Damage Repair Interactome Analysis
Date
2022-03-03
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Abstract
Eukaryotic cells can repair DNA Double-Strand Breaks (DSBs) through a number of mechanisms, including end resection-mediated pathways and non-homologous end joining (NHEJ). An End-Bridging Complex (EBC) comprised of elements from both end resection and NHEJ may be the first complex recruited to DSB sites and play a key role in repair pathway selection. The protein composition of the EBC or how it structurally supports repair pathway choice is not fully understood. Conceptually, Affinity Purification-Mass Spectrometry (AP-MS) methods can be used to determine the composition and subunit stoichiometry of such complexes. However, none of the existing AP-MS workflows combine isolation of complexes with high temporal sampling, both of which are essential for characterizing complexes involved in transient repair processes. In this thesis, I have investigated whether chemical crosslinking incorporated into a DNA-based AP-MS workflow can improve the coverage of DSB repair interactome in S. cerevisiae. To this end, I first evaluated and optimized the RIME protocol for isolating affinity-tagged putative EBC members after DSB-induction. Further, I tested various strategies for incorporating chemical crosslinking into this protocol and increasing protein coverage, using the homo-bifunctional reagent BS3. Lastly, I investigated networks of interaction within the EBC interactome based on the RIME method and evaluated the identified hits for biological significance. My results indicate that the epitope-tagged system I used (hemagglutinin-based) prohibits the incorporation of standard crosslinkers, suggesting that a new strategy is required to take advantage of crosslinking. Nevertheless, the optimized RIME-based AP-MS protocol could successfully isolate known and potentially novel interactors of the EBC using a strategy incorporating multiple baits. Studying the novel interactors identified in these analyses could help in complementing our current knowledge of eukaryotic DSB repair pathway choice and understanding the extent of the DNA damage repair interactome.
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Systems Biology, Proteomics, DNA Damage Repair, DNA Double-Strand Break, Affinity Purification, Mass Spectrometry, Chemical Crosslinking
Citation
Shariat-Panahi, A. (2022). Evaluating Chemical Crosslinking as A Tool for Enhancing DNA Damage Repair Interactome Analysis (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.