Homologous recombination, also called homology-directed repair (HDR), is a major DNA repair pathway for lesions such as double-strand breaks (DSBs). Several proteins critical to the HDR pathway have been identified as tumor suppressors. Notable among these is the breast cancer suppressor BRCA2 which interacts with and promotes the function of the RAD51 recombinase, the critical protein for strand exchange between homologous sequence. BRCA2 mutations are also associated with the developmental disorder Fanconi anemia. The long-term objective is to understand the role of HDR proteins in tumor-relevant tissue types, as HDR deficiency is a major target for tumor therapies under development.
The specific aims are: 1. To investigate roles of HDR proteins in human mammary epithelial cells. Genetic loss of HDR proteins results in embryonic lethality in the mouse, but mammary tumors form in conditional mouse models as in patients with germline mutations. To understand the cellular roles of HDR proteins in the tumor-relevant cell type, we plan to construct isogenic human mammary epithelial cell lines with mutations in the HDR proteins BRCA2, RAD51, and select RAD51 paralogs. A major question to be addressed is whether loss of any one of the HDR proteins results in a cell lethal phenotype. For mutants that are inviable, rescue experiments will be attempted. Mutants that are viable will be interrogated for a number of properties, including HDR deficiency, chromosome instability, and sensitivity to DNA damaging agents. In limited cases, epistatic relationships between HDR proteins will be determined. 2. To determine the requirement for RAD51 and BRCA2 in mouse mammary epithelial cells. As a complement to Aim1 plan to delete BRCA2 from primary mouse mammary epithelial cell cultures in the presence or absence of p53 to determine whether BRCA2 is required for cellular viability. We will also develop a conditional RAD51 deletion model to address its requirement for somatic cell viability and tumor suppression. 3. To determine the role of the BRCA2 C terminus in HDR, replication fork protection, and genome integrity. BRCA2 interaction with RAD51 at a C terminal site has been implicated in stabilizing RAD51 filaments, although the BRCA2 C terminus may have additional functions which promote HDR. We plan to investigate HDR in cells and tissues from mice deleted for the BRCA2 C terminus using a novel transgenic mouse model. We will also address whether loss of heterozygosity is increased in these mice, and whether they are susceptible to endogenous genotoxins similar to Fanconi anemia mice. Germ cell development will also be examined as a model for stem cell maintenance and recombination. RAD51 filament stabilization by BRCA2 was recently found by our lab to be of critical importance for the protection of stalled replication forks from being degraded. A separation of function mutation in BRCA2 will be developed to determine the physiological effects of loss of replication fork protection.
Lesions that arise in the genome compromise its integrity and so must be repaired. Loss of repair or misrepair leads to chromosome loss and genomic rearrangements, which are associated with many tumor types, including breast cancer. This project addresses fundamental questions about tumor suppressors involved in repair between similar sequences in mouse and human mammary cells.
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