Role of the BRCA2 Protein in the Maintenance of Genome Stability
Predisposition to breast cancer may be inherited in an autosomal dominant manner as a result of germline mutation in the BRCA2 gene. The BRCA2 gene and its mouse homologue Brca2 have been cloned and sequenced. BRCA2 is nuclear protein that is expressed in a cell cycle dependent manner. Early experiments aimed at determining the function of BRCA2 suggested a role in DNA repair and the maintenance of genome stability. The aim of this thesis was to characterise the role of BRCA2 in maintenance of genome stability and DNA double-strand break (DSB) repair using mouse and cellular models carrying targeted mutations in Brca2. Initially the chromosome instability phenotype of mouse embryo fibroblasts (MEFs) homozygous for a hypomorphic Brca2 mutation was characterised identifying a role for Brca2 in the suppression of spontaneous chromosome breaks, aberrations and aneuploidy. Loss of wild-type Brca2 was found to lead to abnormalities in centrosome function and chromosome segregation in MEFs. To investigate the role of Brca2 in DNA DSB repair, the effect of Brca2 mutation on ionising radiation sensitivity and DNA DSB rejoining was characterised in both actively replicating and quiescent MEFs. BRCA2 interacts with the homologous recombination repair protein RAD51. To specifically examine homology directed repair mechanisms a conditional targeted mutation in Brca2 was generated in mouse embryonic stem (ES) cells and an homologous recombination substrate, was created and integrated at a single chromosomal locus. This allowed the examination of the affect of Brca2 mutation on mechanisrTI of repair of a single chromosomal DSB. Finally the role of Brca2 in the in vivo response to both endogenous and ionising radiation induced DNA damage was investigated by generating mice with both a targeted Brca2 mutation and a transgenic mutation reporter gene array. This allowed characterisation of the effect of Brca2 mutation on both ill vivo somatic mutation frequency and mutation type.