Characterisation of KEAP1 as a novel factor in DNA base damage repair and investigation of its relationship with PARPs
<p>Genomes are constantly subjected to various genotoxic stresses that can cause DNA damage, which, if unrepaired, can lead to cancer or cell death. To counteract this threat, cells have evolved a network of pathways that resolve DNA damage, termed DNA damage response (DDR). These crucial path...
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | English |
| Published: |
2023
|
| Subjects: |
| _version_ | 1797112492258754560 |
|---|---|
| author | Rokicka, JJ |
| author2 | Lakin, N |
| author_facet | Lakin, N Rokicka, JJ |
| author_sort | Rokicka, JJ |
| collection | OXFORD |
| description | <p>Genomes are constantly subjected to various genotoxic stresses that can cause DNA damage, which, if unrepaired, can lead to cancer or cell death. To counteract this threat, cells have evolved a network of pathways that resolve DNA damage, termed DNA damage response (DDR). These crucial pathways and the interactions between them are regulated by post-translational modifications, including poly(ADP- ribosyl)ation, which is carried out by poly(ADP-ribose) polymerases (PARPs). PARP1 and PARP2 play important roles in the regulation of base damage repair. BER, one of the key base damage repair pathways, has been extensively studied, however, its regulation and crosstalk with alternative pathways resolving DNA base lesions is not fully understood. This work aims to identify novel factors contributing to base damage repair and assess their relationship with PARPs.</p>
<p>Using a genome-wide CRISPR-Cas9 screen, we identified KEAP1 as a potential novel factor in alkylation base damage repair. KEAP1 acts as a substrate adaptor for the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex and had not been previously implicated in base damage repair. To validate the screen, we generated KEAP1- deficient cell lines using genome editing. We found that loss of KEAP1 increases cellular sensitivity to the alkylating agent MMS. We determined that all of the KEAP1 domains necessary for its function in the E3 ubiquitin ligase complex are also required for it to convey tolerance to MMS-induced damage. Disruption of KEAP1 is not epistatic with inhibition of repair through BER or direct repair, suggesting that KEAP1 acts in an alternative base damage repair pathway. Following damage, KEAP1 is recruited to chromatin in a PARP1-dependent manner. Interestingly, loss of KEAP1 results in chromatin enrichment of PARP1 and PARP2 when exposed to MMS. We also identify a novel synthetic lethal interaction between KEAP1-deficiency and PARP inhibition, which could have therapeutic potential in the treatment of KEAP1- deficient tumours.</p> |
| first_indexed | 2024-03-07T08:24:56Z |
| format | Thesis |
| id | oxford-uuid:97d3ef72-df49-42ad-94f8-cfe232bc91dc |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T08:24:56Z |
| publishDate | 2023 |
| record_format | dspace |
| spelling | oxford-uuid:97d3ef72-df49-42ad-94f8-cfe232bc91dc2024-02-12T13:24:43ZCharacterisation of KEAP1 as a novel factor in DNA base damage repair and investigation of its relationship with PARPsThesishttp://purl.org/coar/resource_type/c_db06uuid:97d3ef72-df49-42ad-94f8-cfe232bc91dcDNA damageEnglishHyrax Deposit2023Rokicka, JJLakin, NMcHugh, PHiggs, MRonson, G<p>Genomes are constantly subjected to various genotoxic stresses that can cause DNA damage, which, if unrepaired, can lead to cancer or cell death. To counteract this threat, cells have evolved a network of pathways that resolve DNA damage, termed DNA damage response (DDR). These crucial pathways and the interactions between them are regulated by post-translational modifications, including poly(ADP- ribosyl)ation, which is carried out by poly(ADP-ribose) polymerases (PARPs). PARP1 and PARP2 play important roles in the regulation of base damage repair. BER, one of the key base damage repair pathways, has been extensively studied, however, its regulation and crosstalk with alternative pathways resolving DNA base lesions is not fully understood. This work aims to identify novel factors contributing to base damage repair and assess their relationship with PARPs.</p> <p>Using a genome-wide CRISPR-Cas9 screen, we identified KEAP1 as a potential novel factor in alkylation base damage repair. KEAP1 acts as a substrate adaptor for the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex and had not been previously implicated in base damage repair. To validate the screen, we generated KEAP1- deficient cell lines using genome editing. We found that loss of KEAP1 increases cellular sensitivity to the alkylating agent MMS. We determined that all of the KEAP1 domains necessary for its function in the E3 ubiquitin ligase complex are also required for it to convey tolerance to MMS-induced damage. Disruption of KEAP1 is not epistatic with inhibition of repair through BER or direct repair, suggesting that KEAP1 acts in an alternative base damage repair pathway. Following damage, KEAP1 is recruited to chromatin in a PARP1-dependent manner. Interestingly, loss of KEAP1 results in chromatin enrichment of PARP1 and PARP2 when exposed to MMS. We also identify a novel synthetic lethal interaction between KEAP1-deficiency and PARP inhibition, which could have therapeutic potential in the treatment of KEAP1- deficient tumours.</p> |
| spellingShingle | DNA damage Rokicka, JJ Characterisation of KEAP1 as a novel factor in DNA base damage repair and investigation of its relationship with PARPs |
| title | Characterisation of KEAP1 as a novel factor in DNA base damage repair and investigation of its relationship with PARPs |
| title_full | Characterisation of KEAP1 as a novel factor in DNA base damage repair and investigation of its relationship with PARPs |
| title_fullStr | Characterisation of KEAP1 as a novel factor in DNA base damage repair and investigation of its relationship with PARPs |
| title_full_unstemmed | Characterisation of KEAP1 as a novel factor in DNA base damage repair and investigation of its relationship with PARPs |
| title_short | Characterisation of KEAP1 as a novel factor in DNA base damage repair and investigation of its relationship with PARPs |
| title_sort | characterisation of keap1 as a novel factor in dna base damage repair and investigation of its relationship with parps |
| topic | DNA damage |
| work_keys_str_mv | AT rokickajj characterisationofkeap1asanovelfactorindnabasedamagerepairandinvestigationofitsrelationshipwithparps |