Evaluating Chemical Crosslinking as a Tool for Enhancing DNA Damage Repair Interactome Analysis

Simple item page
dc.contributor.advisorSchriemer, David C.
dc.contributor.authorShariat-Panahi, Ali
dc.contributor.committeememberAntonie, Dufour
dc.contributor.committeememberCobb, Jennifer
dc.date2022-06
dc.date.accessioned2022-03-17T13:43:30Z
dc.date.available2022-03-17T13:43:30Z
dc.date.issued2022-03-03
dc.description.abstractEukaryotic 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.en_US
dc.identifier.citationShariat-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.en_US
dc.identifier.doihttp://dx.doi.org/10.11575/PRISM/39648
dc.identifier.urihttp://hdl.handle.net/1880/114488
dc.language.isoengen_US
dc.publisher.facultyCumming School of Medicineen_US
dc.publisher.institutionUniversity of Calgaryen
dc.rightsUniversity 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.en_US
dc.subjectSystems Biologyen_US
dc.subjectProteomicsen_US
dc.subjectDNA Damage Repairen_US
dc.subjectDNA Double-Strand Breaken_US
dc.subjectAffinity Purificationen_US
dc.subjectMass Spectrometryen_US
dc.subjectChemical Crosslinkingen_US
dc.subject.classificationBiology--Molecularen_US
dc.subject.classificationBiochemistryen_US
dc.titleEvaluating Chemical Crosslinking as a Tool for Enhancing DNA Damage Repair Interactome Analysisen_US
dc.typemaster thesisen_US
thesis.degree.disciplineMedicine – Biochemistry and Molecular Biologyen_US
thesis.degree.grantorUniversity of Calgaryen_US
thesis.degree.nameMaster of Science (MSc)en_US
ucalgary.item.requestcopytrueen_US
Files
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
ucalgary_2022_shariat-panahi_ali.pdf
Size:
18.96 MB
Format:
Adobe Portable Document Format
Description:
Master's Thesis
Download
License bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
license.txt
Size:
2.62 KB
Format:
Item-specific license agreed upon to submission
Description:
Download
Collections
Open Theses and Dissertations