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Insights into the Ubiquitylation System and DNA Damage Repair
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- Author / Creator
- Abou Farraj, Rabih
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DNA serves as a detailed blueprint for cellular functions, but continuous exposure to damaging agents poses a threat to genomic stability, especially through DNA double-strand breaks. Cells utilize two main repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR), to mitigate this risk. Ubiquitylation, a widespread cellular process, is vital for DNA damage repair, involving the attachment of ubiquitin to proteins. This mechanism relies on E2 conjugating enzymes and E3 ligases, particularly RING E3 ligases, which target substrates and activate E2 enzymes for ubiquitin transfer. The E2 ubiquitin conjugating enzyme Ubc13, along with its binding partner Mms2, construct ubiquitin chains with distinctive lysine 63 (K63) linkage. These chains are responsible for recruiting downstream DNA repair proteins. Another notable RING E3 ligase, RNF138, plays a crucial role in ubiquitylating target proteins to promote HR. Here we investigate Ubc13 and RNF138. Ubc13 has been implicated in several different cancer pathways. A new structure of Ubc13 revealed a novel conformation of its active site pointing towards a dynamic active site loop. We suggest that distinctive structural characteristics of the active site loop of Ubc13 may serve as a foundation for the rational development and design of targeted Ubc13 inhibitors. We characterized two compounds, one which showed covalent reactivity with Ubc13’s active site, but also inhibited a mutant of Ubc13 which resembles other E2 conjugating enzymes, suggesting it is a non-specific inhibitor. The second compound, a small fragment, is believed to non-covalently bind to Ubc13's active site, selectively inhibiting the wildtype form of the enzyme. We suggest that this small molecule fragment represents a promising starting point for developing next-generation inhibitors. Our studies continued characterizing RNF138, a RING E3 ligase, also implicated in disease pathways. We first characterized the DNA binding of RNF138, which showed strong preference for long single stranded 3’ and 5’ DNA overhangs. AlphaFold2 structural modeling unveiled an overall flexible architecture, with N-terminal domains, including the RING domain and the first zinc finger, crucially involved in E2-ubiquitin coordination. Finally, we characterized the regulation of RNF138. We showed RNF138 is ubiquitylated at K158 and phosphorylated by CDK2, and ATM kinase at residues T27, and S124 respectively. Interfering with the three post-translational modifications significantly diminishes RNF138's capacity to facilitate HR. Overall, our study provides significant insights into the mechanism underlying the essential roles of the E2 enzyme Ubc13 and the RING E3 ligase RNF138.
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- Graduation date
- Fall 2024
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.