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postgraduate thesis: Role of ring finger protein 169 in DNA double strand break signaling and repair
Title | Role of ring finger protein 169 in DNA double strand break signaling and repair |
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Authors | |
Advisors | |
Issue Date | 2018 |
Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
Citation | An, L. [安利伟]. (2018). Role of ring finger protein 169 in DNA double strand break signaling and repair. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. |
Abstract | DNA double strand breaks (DSBs) are highly cytotoxic DNA lesions. Cells have evolved a sophisticated DNA damage response (DDR) network to recognize, transduce and repair DSBs. The activation and propagation, as well as the spatiotemporal restriction, of DDRs are important for the maintenance of genomic stability.
Ring finger protein 169 (RNF169) is a DNA damage responsive protein that accumulates at the vicinity of DSBs. Functionally, RNF169 limits the deposition of p53-binding protein 1 (53BP1), a key regulator of DDR, at the damaged chromatin. However, how this RNF169-53BP1 interplay is regulated and how this balance controls DSB repair remains elusive. In the first part of my study, a dual functional nuclear localization signal (NLS) in RNF169 was identified. The NLS not only shuttles RNF169 into nucleus, but also promotes its stability in nucleus by mediating a direct interaction with ubiquitin specific protease 7 (USP7). Guided by the crystal structure of USP7 in complex with the NLS, I uncoupled USP7 binding from its nuclear import function, and found that perturbing the USP7-RNF169 complex impaired RNF169 ionizing radiation induced foci formation (IRIF), compromised high-fidelity homologous recombination (HR) repair, and hyper-sensitized cells to PARP inhibition. Taken together, my findings uncover an NLS-mediated bipartite mechanism that supports the functions of RNF169 at DSBs.
In the second part of this study, the functional outcomes of the RNF169-53BP1 interplay in repair of DSBs were investigated. To this end, I employed an endonuclease-based DSB-induction system and used chromatin immunoprecipitation (ChIP) approach to decipher the equilibrium distribution of RNF169 and 53BP1 at DSBs. Results showed that 53BP1 mainly occupies DSB-flanking distal chromatin regions, whereas RNF169 robustly accumulates at DSB end-proximal regions and preferentially targets resected, RPA-bound DSBs. Accordingly, RNF169 promotes DSB resection process and favors homology-mediated DSB repair pathways. Importantly, RNF169 stimulates single-strand annealing (SSA) repair by counteracting 53BP1. These results highlight the RNF169-53BP1 interplay in fine-tuning choice of DSB repair pathways.
Collectively, the findings presented in this thesis provide new insights to understand the regulation of RNF169 and DSB repair pathway choice.
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Degree | Doctor of Philosophy |
Subject | DNA-binding proteins |
Dept/Program | Biomedical Sciences |
Persistent Identifier | http://hdl.handle.net/10722/267735 |
Award | Li Ka Shing Prize, The Best PhD Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), 2017-2018 |
DC Field | Value | Language |
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dc.contributor.advisor | Huen, MSY | - |
dc.contributor.advisor | Chen, H | - |
dc.contributor.author | An, Liwei | - |
dc.contributor.author | 安利伟 | - |
dc.date.accessioned | 2019-03-01T03:44:40Z | - |
dc.date.available | 2019-03-01T03:44:40Z | - |
dc.date.issued | 2018 | - |
dc.identifier.citation | An, L. [安利伟]. (2018). Role of ring finger protein 169 in DNA double strand break signaling and repair. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. | - |
dc.identifier.uri | http://hdl.handle.net/10722/267735 | - |
dc.description.abstract | DNA double strand breaks (DSBs) are highly cytotoxic DNA lesions. Cells have evolved a sophisticated DNA damage response (DDR) network to recognize, transduce and repair DSBs. The activation and propagation, as well as the spatiotemporal restriction, of DDRs are important for the maintenance of genomic stability. Ring finger protein 169 (RNF169) is a DNA damage responsive protein that accumulates at the vicinity of DSBs. Functionally, RNF169 limits the deposition of p53-binding protein 1 (53BP1), a key regulator of DDR, at the damaged chromatin. However, how this RNF169-53BP1 interplay is regulated and how this balance controls DSB repair remains elusive. In the first part of my study, a dual functional nuclear localization signal (NLS) in RNF169 was identified. The NLS not only shuttles RNF169 into nucleus, but also promotes its stability in nucleus by mediating a direct interaction with ubiquitin specific protease 7 (USP7). Guided by the crystal structure of USP7 in complex with the NLS, I uncoupled USP7 binding from its nuclear import function, and found that perturbing the USP7-RNF169 complex impaired RNF169 ionizing radiation induced foci formation (IRIF), compromised high-fidelity homologous recombination (HR) repair, and hyper-sensitized cells to PARP inhibition. Taken together, my findings uncover an NLS-mediated bipartite mechanism that supports the functions of RNF169 at DSBs. In the second part of this study, the functional outcomes of the RNF169-53BP1 interplay in repair of DSBs were investigated. To this end, I employed an endonuclease-based DSB-induction system and used chromatin immunoprecipitation (ChIP) approach to decipher the equilibrium distribution of RNF169 and 53BP1 at DSBs. Results showed that 53BP1 mainly occupies DSB-flanking distal chromatin regions, whereas RNF169 robustly accumulates at DSB end-proximal regions and preferentially targets resected, RPA-bound DSBs. Accordingly, RNF169 promotes DSB resection process and favors homology-mediated DSB repair pathways. Importantly, RNF169 stimulates single-strand annealing (SSA) repair by counteracting 53BP1. These results highlight the RNF169-53BP1 interplay in fine-tuning choice of DSB repair pathways. Collectively, the findings presented in this thesis provide new insights to understand the regulation of RNF169 and DSB repair pathway choice. | - |
dc.language | eng | - |
dc.publisher | The University of Hong Kong (Pokfulam, Hong Kong) | - |
dc.relation.ispartof | HKU Theses Online (HKUTO) | - |
dc.rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works. | - |
dc.rights | This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. | - |
dc.subject.lcsh | DNA-binding proteins | - |
dc.title | Role of ring finger protein 169 in DNA double strand break signaling and repair | - |
dc.type | PG_Thesis | - |
dc.description.thesisname | Doctor of Philosophy | - |
dc.description.thesislevel | Doctoral | - |
dc.description.thesisdiscipline | Biomedical Sciences | - |
dc.description.nature | published_or_final_version | - |
dc.identifier.doi | 10.5353/th_991044081523603414 | - |
dc.date.hkucongregation | 2019 | - |
dc.description.award | Li Ka Shing Prize, The Best PhD Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), 2017-2018 | - |
dc.identifier.mmsid | 991044081523603414 | - |