MRE11-RAD50-NBS1 complex disassembly from DNA double-strand breaks after ionising radiation
<p>The complex formed by MRE11 –RAD50 and –NBS1, designated the MRN complex is essential for the DNA damage response (DDR). The MRN complex has a well-studied role in DNA end resection as part of the homologous recombination (HR) pathway. HR repairs double-strand breaks (DSB), the most toxic l...
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
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2022
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| _version_ | 1797110633789915136 |
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| author | Koukouravas, SAR |
| author2 | Ramadan, K |
| author_facet | Ramadan, K Koukouravas, SAR |
| author_sort | Koukouravas, SAR |
| collection | OXFORD |
| description | <p>The complex formed by MRE11 –RAD50 and –NBS1, designated the MRN complex is essential for the DNA damage response (DDR). The MRN complex has a well-studied role in DNA end resection as part of the homologous recombination (HR) pathway. HR repairs double-strand breaks (DSB), the most toxic lesions that can arise in cells. The ATPase, p97, also called VCP, is a major component in the ubiquitin-proteasome system (UPS) and has an intrinsic unfoldase activity. It has been shown that loss of p97 strongly radiosensitises cells to ionising radiation (IR). Previous work shows that p97 provides timely removal of MRE11 from DSB sites thus preventing excessive end resection. Although the crucial role of the MRN complex in repairing DSBs has been well studied, the mechanism by which p97 controls the activity of the complex remains unknown. A better characterisation of the crosstalk between the MRN complex and p97 could lead to improving cancer prevention, diagnosis, and treatment. Indeed, drugs targeting the MRN complex and p97 are currently under clinical trial as sole or as combination (radiosensitiser) therapy (NCT03921021 and NCT04402541). This thesis aims to generate tools to answer the vital biological question of, <strong>how is p97 directed to disassemble the MRN complex from DNA after DSBs</strong>. To achieve this aim, it is intended to elucidate both the binding partners and post-translational modification (PTMs) of the MRN complex, using APEX2 proximity ligation assays in combination with mass spectrometry analysis. To this end, molecular cloning techniques were used to synthesise plasmids expressing fusion proteins of MRE11, RAD50 and NBS1 tagged with either APEX2 or SSH. The expression and functionality of these fusion proteins were validated using western blotting and co-immunoprecipitation, respectively. Subsequent stable cell lines were generated using these plasmids via the Flp-In system.</p>
<p>Additional studies identified post-translational modifications of the MRN complex using co-immunoprecipitation where ubiquitination of MRN complex was observed.</p> |
| first_indexed | 2024-03-07T07:57:34Z |
| format | Thesis |
| id | oxford-uuid:780f235b-230c-4b74-b48a-78905c3dd52d |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T07:57:34Z |
| publishDate | 2022 |
| record_format | dspace |
| spelling | oxford-uuid:780f235b-230c-4b74-b48a-78905c3dd52d2023-08-25T15:21:10ZMRE11-RAD50-NBS1 complex disassembly from DNA double-strand breaks after ionising radiationThesishttp://purl.org/coar/resource_type/c_bdccuuid:780f235b-230c-4b74-b48a-78905c3dd52dEnglishHyrax Deposit2022Koukouravas, SARRamadan, K<p>The complex formed by MRE11 –RAD50 and –NBS1, designated the MRN complex is essential for the DNA damage response (DDR). The MRN complex has a well-studied role in DNA end resection as part of the homologous recombination (HR) pathway. HR repairs double-strand breaks (DSB), the most toxic lesions that can arise in cells. The ATPase, p97, also called VCP, is a major component in the ubiquitin-proteasome system (UPS) and has an intrinsic unfoldase activity. It has been shown that loss of p97 strongly radiosensitises cells to ionising radiation (IR). Previous work shows that p97 provides timely removal of MRE11 from DSB sites thus preventing excessive end resection. Although the crucial role of the MRN complex in repairing DSBs has been well studied, the mechanism by which p97 controls the activity of the complex remains unknown. A better characterisation of the crosstalk between the MRN complex and p97 could lead to improving cancer prevention, diagnosis, and treatment. Indeed, drugs targeting the MRN complex and p97 are currently under clinical trial as sole or as combination (radiosensitiser) therapy (NCT03921021 and NCT04402541). This thesis aims to generate tools to answer the vital biological question of, <strong>how is p97 directed to disassemble the MRN complex from DNA after DSBs</strong>. To achieve this aim, it is intended to elucidate both the binding partners and post-translational modification (PTMs) of the MRN complex, using APEX2 proximity ligation assays in combination with mass spectrometry analysis. To this end, molecular cloning techniques were used to synthesise plasmids expressing fusion proteins of MRE11, RAD50 and NBS1 tagged with either APEX2 or SSH. The expression and functionality of these fusion proteins were validated using western blotting and co-immunoprecipitation, respectively. Subsequent stable cell lines were generated using these plasmids via the Flp-In system.</p> <p>Additional studies identified post-translational modifications of the MRN complex using co-immunoprecipitation where ubiquitination of MRN complex was observed.</p> |
| spellingShingle | Koukouravas, SAR MRE11-RAD50-NBS1 complex disassembly from DNA double-strand breaks after ionising radiation |
| title | MRE11-RAD50-NBS1 complex disassembly from DNA double-strand breaks after ionising radiation |
| title_full | MRE11-RAD50-NBS1 complex disassembly from DNA double-strand breaks after ionising radiation |
| title_fullStr | MRE11-RAD50-NBS1 complex disassembly from DNA double-strand breaks after ionising radiation |
| title_full_unstemmed | MRE11-RAD50-NBS1 complex disassembly from DNA double-strand breaks after ionising radiation |
| title_short | MRE11-RAD50-NBS1 complex disassembly from DNA double-strand breaks after ionising radiation |
| title_sort | mre11 rad50 nbs1 complex disassembly from dna double strand breaks after ionising radiation |
| work_keys_str_mv | AT koukouravassar mre11rad50nbs1complexdisassemblyfromdnadoublestrandbreaksafterionisingradiation |