A role for BRCA2 in DNA repair was originally established based on its interaction with RAD51, a key player in HR. RAD51 plays a central role in recombination, assembling onto single-stranded DNA as a nucleoprotein filament, and catalyzing the invasion and exchange of homologous DNA sequences. A major hurdle in any biochemical study is to purify the protein of interest to homogeneity in an active state. At 384 kDa, BRCA2 is a large protein that has proven difficult to purify. Recently, we have successfully developed a method to purify the full-length protein from human cells. We initially characterized BRCA2’s ability to bind DNA substrates, protein partners involved in homologous recombination, and its capability to catalyze DNA strand exchange, an in vitro measure of recombination. Our findings revealed that BRCA2 binds RAD51 with a defined stoichiometry, prevents nucleation onto dsDNA, and enforces binding of RAD51 onto ssDNA thus stabilizing the nascent nucleoprotein filament.
A major focus of the lab is to understand the role BRCA2 plays in the DSB response as well as its role in homologous recombination (HR). Homologous recombination is a high fidelity form of DSB repair utilizing substrates such as the sister chromatid after DNA replication to repair a break with restoration of the original DNA sequence. Other pathways for DSB repair, such as non-homologous end joining (NHEJ), operate in the G1 phase of the cell cycle and can be deleterious if sequence information is deleted or altered at the break.
In order to better understand how BRCA2 and other players in HR signal and catalyze repair reactions, we are using a multi-disciplinary approach encompassing biochemistry, genetics, cell biology, structural biology, and proteomics. We are currently using human cells to both express and purify many of the proteins involved in HR to further understand detailed mechanisms of action. We can then look at enzymatic activities biochemically such as DNA binding preferences, direct protein interactions, and DNA strand exchange activity. Several proteins participate in the HR process and many of the players, such as the RAD51 paralogs, remain ill-defined as to what exact role they are playing. BRCA2 itself interacts with several proteins: PALB2, BRCA1, FANCD2, EMSY, DMC1, DSS1 and it is unclear how these interacting partners influence HR. Our goal is to understand the functional consequences of these interactions and how disruptions in the HR pathway lead to cancer.
Biochemical and genetic characterization of tumor associated mutations in the BRCA2 gene.
Genetic screens to further understand the molecular pathogenesis leading to tumor formation in BRCA2 carriers.
Proteomic strategies to identify novel proteins that interact with BRCA2 after DNA damage.
Identifying mechanisms and proteins that govern the regulatory decision between HR and NHEJ after a DSB is generated in mammalian cells.