Volume 8 Supplement 2
Regulation of recombinational repair by the familial breast cancer susceptibility protein BRCA2
© BioMed Central Ltd 2006
Published: 01 November 2006
Inherited mutations in BRCA2 are associated with a predisposition to early-onset breast cancers. The underlying basis of tumourigenesis is thought to be linked to defects in DNA double-strand break repair by homologous recombination (HR), as indicated by the spontaneous chromosomal instability phenotype of BRCA2-defective cell lines. The BRCA2 protein interacts with ssDNA and the RAD51 recombination protein, and is proposed to recruit RAD51 to the damage site for the HR repair.
Recombinant BRCA2 fragments that cover the entire length of BRCA2 were tested for interaction with RAD51 and for their phosphorylation using cell free extracts. An antibody that specifically recognises BRCA2 phosphorylated at serine 3291 was generated and used to analyse the phosphorylation status of endogenous BRCA2 during the cell cycle and after DNA damaging treatment. A cell line that stably expresses a C-terminal BRCA2 fragment was generated, to allow the analysis of RAD51 interactions and ability to promote homologous recombinational repair (HRR).
We found that the C-terminal region of BRCA2, which directly interacts with RAD51, contains a site (S3291) that is phosphorylated by cyclin-dependent kinases. Phosphorylation of S3291 increases as cells progress towards mitosis, and was shown to block C-terminal interactions between BRCA2 and RAD51. However, DNA damage overcomes cell cycle regulation by reducing S3291 phosphorylation and stimulating interactions with RAD51. HRR is defective in cells overexpressing the C-terminal fragment of BRCA2, indicating that interactions between RAD51 and the C-terminal region of endogenous BRCA2 are important for repair.
We suggest that S3291 phosphorylation provides a molecular switch that can regulate RAD51-mediated HRR. Loss of phosphorylation in response to DNA damage allows interactions between RAD51 and the C-terminal region of BRCA2 and may facilitate the loading of RAD51 on damaged DNA . Importantly, a S3291 nonphosphorylatable mutation (P3292L) has been found in familial breast cancer patients, implicating a role of S3291 phosphorylation in the maintenance of genome integrity.
This research was supported by Breast Cancer Campaign (SCW), Cancer Research UK (SCW, FE), the Human Frontiers Science Program (FE) and the Japan Society for the Promotion of Science (FE).