Investigating the role of homologous recombination in the maintenance of chromosome stability
<p>Homologous recombination (HR) is an essential DNA break repair pathway that maintains genome stability. BRCA2, PALB2, and RAD51 are key modulators of this pathway, mutations within which are associated with tumourigenesis. Centromeres are large repetitive DNA regions in the genome, defined...
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| Format: | Thèse |
| Langue: | English |
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2023
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| _version_ | 1826314622407278592 |
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| author | Graham, E |
| author2 | Esashi, F |
| author_facet | Esashi, F Graham, E |
| author_sort | Graham, E |
| collection | OXFORD |
| description | <p>Homologous recombination (HR) is an essential DNA break repair pathway that maintains genome stability. BRCA2, PALB2, and RAD51 are key modulators of this pathway, mutations within which are associated with tumourigenesis. Centromeres are large repetitive DNA regions in the genome, defined through the presence of CENP-A. There is increasing evidence that centromeres experience instability across the cell cycle, impeding their function.</p>
<p>In this thesis, I first demonstrate a role for de novo RAD51 loading in mitosis after mild replication stress to promote mitotic DNA synthesis (MiDAS). Utilising RAD51 separation-of-function mutant cell lines, I present evidence suggesting that RAD51’s single stranded DNA binding activity is important to promote MiDAS at undamaged loci. Failure to complete MiDAS in RAD51 deficient cells leads to mitotic defects and increased aberrant centromere recombination and fragility. I next explored the role of HR factors: BRCA2, PALB2, and RAD51 at centromeres RPE1 cells. Here, I show evidence of a role for BRCA2 and RAD51, but not PALB2, in maintaining centromeric CENP-A levels and, using Exo-FISH, show that BRCA2 may have a distinct role in minimising centromeric single stranded DNA accumulation in a cell cycle-dependent manner. Finally, I generated and validated an auxin-inducible degron system to rapidly degrade BRCA2 and PALB2 in the mismatch repair defective, colorectal cancer cell line, HCT116. Using these tools, I highlight the impact of short-term and long-term BRCA2 and PALB2 depletion on chromosome stability, providing an insight into a potential mechanism for tumourigenesis. Investigations into the role of BRCA2 and PALB2 in centromere identity and stability further identified BRCA2 as important for maintaining centromere stability, different from RPE1 cells, highlighting their different genetic backgrounds. Hence, I propose a role for BRCA2 and RAD51 in preserving centromere stability and function, collectively increasing our comprehension of the importance of HR factors at centromeres and how different cellular and chromatin contexts may influence repair at this region to limit tumourigenesis.</p> |
| first_indexed | 2024-03-07T08:02:21Z |
| format | Thesis |
| id | oxford-uuid:96df32c1-f344-40c3-a66c-05175c2e88c4 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-09-25T04:36:20Z |
| publishDate | 2023 |
| record_format | dspace |
| spelling | oxford-uuid:96df32c1-f344-40c3-a66c-05175c2e88c42024-09-23T11:46:57ZInvestigating the role of homologous recombination in the maintenance of chromosome stabilityThesishttp://purl.org/coar/resource_type/c_db06uuid:96df32c1-f344-40c3-a66c-05175c2e88c4EnglishHyrax Deposit2023Graham, EEsashi, F<p>Homologous recombination (HR) is an essential DNA break repair pathway that maintains genome stability. BRCA2, PALB2, and RAD51 are key modulators of this pathway, mutations within which are associated with tumourigenesis. Centromeres are large repetitive DNA regions in the genome, defined through the presence of CENP-A. There is increasing evidence that centromeres experience instability across the cell cycle, impeding their function.</p> <p>In this thesis, I first demonstrate a role for de novo RAD51 loading in mitosis after mild replication stress to promote mitotic DNA synthesis (MiDAS). Utilising RAD51 separation-of-function mutant cell lines, I present evidence suggesting that RAD51’s single stranded DNA binding activity is important to promote MiDAS at undamaged loci. Failure to complete MiDAS in RAD51 deficient cells leads to mitotic defects and increased aberrant centromere recombination and fragility. I next explored the role of HR factors: BRCA2, PALB2, and RAD51 at centromeres RPE1 cells. Here, I show evidence of a role for BRCA2 and RAD51, but not PALB2, in maintaining centromeric CENP-A levels and, using Exo-FISH, show that BRCA2 may have a distinct role in minimising centromeric single stranded DNA accumulation in a cell cycle-dependent manner. Finally, I generated and validated an auxin-inducible degron system to rapidly degrade BRCA2 and PALB2 in the mismatch repair defective, colorectal cancer cell line, HCT116. Using these tools, I highlight the impact of short-term and long-term BRCA2 and PALB2 depletion on chromosome stability, providing an insight into a potential mechanism for tumourigenesis. Investigations into the role of BRCA2 and PALB2 in centromere identity and stability further identified BRCA2 as important for maintaining centromere stability, different from RPE1 cells, highlighting their different genetic backgrounds. Hence, I propose a role for BRCA2 and RAD51 in preserving centromere stability and function, collectively increasing our comprehension of the importance of HR factors at centromeres and how different cellular and chromatin contexts may influence repair at this region to limit tumourigenesis.</p> |
| spellingShingle | Graham, E Investigating the role of homologous recombination in the maintenance of chromosome stability |
| title | Investigating the role of homologous recombination in the maintenance of chromosome stability |
| title_full | Investigating the role of homologous recombination in the maintenance of chromosome stability |
| title_fullStr | Investigating the role of homologous recombination in the maintenance of chromosome stability |
| title_full_unstemmed | Investigating the role of homologous recombination in the maintenance of chromosome stability |
| title_short | Investigating the role of homologous recombination in the maintenance of chromosome stability |
| title_sort | investigating the role of homologous recombination in the maintenance of chromosome stability |
| work_keys_str_mv | AT grahame investigatingtheroleofhomologousrecombinationinthemaintenanceofchromosomestability |