Functional Applications of Systems Biology Tools: Identification of Novel DNA Repair Factors and Peptide Design
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The highly annotated budding yeastSaccharomyces cerevisiaehas emerged as the primary model for systems biology, the study of how individual cellular components function within the context of a dynamic cellular system. Several genome/proteome-scale tools developed using theS. cerevisiaemodel have produced extensive information on gene function and interaction networks that is stored in publicly accessible databases. Bioinformatic tools can exploit these databases to infer novel biological activity but these predictions must be tested in functioning cellular systems to assess the effectiveness of any method. The work herein uses systems-based computational tools to make predictions on novel protein/gene function that are tested using yeast functional genomic approaches. This thesis describes the development and validation of a new tool to design synthetic binding proteins that bind to and inhibit targeted yeast proteins Psk1 and Pin4 as well as the identification and functional analysis of three yeast DNA repair genes,PSK1,ARP6, andDEF1. Thein-silicoprotein synthesizer, InSiPS successfully engineered two synthetic proteins known as anti-Psk1 and anti-Pin4. This demonstrated the ability of our approach to translate from computational prediction, to a specific biological interaction and importantly, a functionally significant phenotype. Chemical-genetic interaction analysis showed that cells expressing α-Psk1 and α-Pin4 phenocopyΔpsk1andΔpin4mutants and yeast-two-hybrid confirmed binary interactions in vivo whilein vitroassays verify that binding is occurring at predicted loci. Further analysis of the anti-Psk1/Psk1 interaction motif showed strong, specific binding. Psk1 was inferred to participate in yeast non-homologous end-joining (NHEJ) repair of double-strand breaks (DSB), an essential DNA repair pathway. Our functional genetic analysis showed thatPSK1is an important novel NHEJ gene that contributes to repair fidelity while appearing to function throughRAD27activity. We also report thatARP6, affects NHEJ through the RSC chromatin remodeling complex. Lastly, we identify new properties of the Def1 DNA repair protein in yeast NHEJ and a physical and genetic interaction between Yku80 and Def1. Together, these findings demonstrate the ability to predict novel gene/protein function using computational tools and expand our understanding of eukaryotic DSB repair.
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Copyright © 2019 the author(s). Theses may be used for non-commercial research, educational, or related academic purposes only. Such uses include personal study, research, scholarship, and teaching. Theses may only be shared by linking to Carleton University Institutional Repository and no part may be used without proper attribution to the author. No part may be used for commercial purposes directly or indirectly via a for-profit platform; no adaptation or derivative works are permitted without consent from the copyright owner.
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burnside-functionalapplicationsofsystemsbiologytools.pdf | 2023-05-05 | Public | Download |