AbstractThe DNA within a cell is damaged on a continuous basis by both endogenous and exogenous agents. One of the most dangerous types of damage a cell can incur are DNA Double stranded breaks (DSBs) as failure to repair these types of lesions can lead to mutations, cell death and cancer. In order to maintain genome integrity, cells have several different mechanisms to repair various types of DNA damage. BRCA1 is a tumour suppressor gene commonly mutated in many inherited breast and ovarian cancers. BRCA1 is fundamental to the repair to DNA damage especially the faithful repair of DSB by homologous recombination (HR) as loss of BRCA1 results in aberrant HR, chromosomal translocation and cancer. BRCA1 is almost always found in the nucleus bound to BARD1. BARD1 retains BRCA1 in the nucleus and enhances the E3 ubiquitin ligase function of BRCA1. The importance of BRCA1 in the repair of DNA DSBs has been extensively researched, however the role of BARD1 in repair remains unknown.
Similar to BRCA1, BARD1 contains tandem BRCT domains at its C-terminus which are important for binding phosphorylated proteins involved in DNA repair. Mutations in the BARD1 BRCT domains have been discovered in a few breast and ovarian cancers which suggested that BARD1 is important in the DNA damage response (DDR) independent of its function in stabilising BRCA1. Loss of the BARD1 BRCT domains also results in a decrease in the ability of cells to carry out HR. This thesis examines the contribution of the BARD1 BRCT domains to the repair of DNA DSBs. Using homologous recombination assays and colony survival assays, Bard1 DT40 cells reconstituted with BARD1?BRCT showed a significant decrease in their ability to carry out HR and were also extremely sensitive to PARP inhibitors suggesting that the BARD1 BRCT domains do indeed play an important role in HR. Work carried out in this thesis suggests that BARD1 contributes to the stabilisation of the BRCA1-C complex which is important in the repair of DSBs. The BARD1 BRCT domains also showed defects in localisation and delayed recruitment to sites of damage. The defects in HR and localisation seen in the absence of the BARD1 BRCT domains potentially implicate a functional role for the BARD1 BRCT domains in DSB repair. Potential BARD1 BRCT domain interacting proteins were also identified my Mass spectrometry, which may shed some light onto the functional role of BARD1 in DNA repair.
|Date of Award||2014|
|Supervisor||Kevin Hiom (Supervisor)|