The Functions of BRCA2 in Repairing DNA Damage

Lio,Yi-Ching
Lawrence Berkeley National Laboratory

Breast cancer is the most common malignancy among women worldwide, and its incidence is increasing. The discovery of the breast cancer genes, BRCA1 and BRCA2 about six years ago promised to open new doors to understanding the disease. However, exactly how loss of the functions of these two genes causes predisposition to breast cancer has remained elusive. The work proposed here is directed toward answering this question for BRCA2.

Living cells possess sophisticated mechanisms for repairing damage to their DNA. Recent evidence has indicated that the BRCA2 protein plays a role in a specific type of DNA repair mechanism known as "homologous recombinational repair" (HRR). This repair process utilizes the information provided by the complimentary DNA strand to restore a break and is thought to be an error-free repair. The Rad51 protein is a central player, which with the help of other proteins, such as BRCA2, promotes the key reaction required for HRR. Thus, when the BRCA2’s functions become impaired, the HRR mechanism becomes defective. DNA damage begins to accumulate in the cell's genes and can finally result in uncontrolled cell growth. Therefore, dysfunction of the DNA repair pathway may be a general phenomenon in the major cases of hereditary breast cancer. We will provide direct experimental evidence for the functions of BRCA2 in HRR.

Recent evidence supports a concept that a multiprotein complex involving Rad51 and five Rad51-related proteins may operate as a functional unit in regulating HRR. We hypothesize that the BRCA2 protein participates directly in HRR through physical interaction with the key protein Rad51, such that loss of the BRCA2-Rad51 interaction suppresses HRR. We further hypothesize that the BRCA2 protein is an essential component of this HRR complex functioning to directly regulate the biochemical activities of Rad51. The aims of this project are:

1. Using cellular approaches, we will test whether the BRCA2 protein directly regulates HRR by modifying a human cell line so that they carry an artificial 'reporter' gene. The reporter gene will allow us to measure recombination activity with and without disruption of the BRCA2-Rad51 interaction.

2. Using protein biochemistry approaches, including a novel baculovirus co-expresssion strategy, enzyme activity assays, protein binding experiments, and special chromatography, we propose to demonstrate the functions of BRCA2 in controlling the actions of Rad51, and the formation of a stable HRR complex involving BRCA2 and Rad51.

The demonstration of the functions of BRCA2 in HRR would be an important step toward elucidating the mechanism underlying the pathogenesis of BRCA2-mediated breast cancer. Also, we would get important clues to the role of HRR in sporadic breast cancer. Such an understanding would reveal new targets for prognostic interpretation and therapeutic intervention in breast cancer.

Progress Report, Year 1 (2002)

The BRCA2 breast cancer gene was found to be a major contributor to a dominantly inherited predisposition to the disease. A critical function of BRCA2 has been recently implicated in recombinational DNA repair and chromosome stability. This information came by the discovery of a repair deficiency of Brca2-/- mice and by physical interaction of the BRCA2 protein with Rad51, the essential protein for homologous recombinational repair (HRR). Exactly how BRCA2 functions in DNA repair is yet a mystery. Recent studies have shown that Rad51 and five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2 and XRCC3) form multiprotein complexes and may operate as functional units in HRR. We propose to investigate the involvement of BRCA2 in the Rad51-dependent DNA repair complexes and to provide direct in vivo and in vitro evidence for the functions of BRCA2 in DNA homologous recombinational repair.

We hypothesize that the BRCA2 protein participates directly in HRR through the interaction with Rad51 and regulates this repair process in some critical way, such that disruption of the BRCA2-Rad51 interaction with dominant-negative domains results in a reduced capacity for repair of chromosome break by homologous recombination. We hypothesize further that the BRCA2 protein is an essential component of the Rad51-dependent DNA repair complex, functioning to mediate the biochemical activities of Rad51 and forming a stable complex with Rad51 and five Rad51 paralogs. Our specific aims are: (1) To test the hypothesis that disruption of the BRCA2-Rad51 interaction affects homologous recombinational repair in vivo. (2) To test the hypothesis that the BRCA2 protein functions to mediate the biochemical activities of Rad51. (3) To test the hypothesis that the BRCA2 protein forms a stable complex with Rad51 and Rad51 paralogs.

For in vivo study, three fragments of the BRC repeats of BRCA2, including BRC1-4, BRC5-8 and BRC1-8, have been individually constructed into a mammalian vector, pIREShyg2. An artificial HRR reporter locus system in HT1080 cells has been used to measure the repair capacity for HRR. We found that expression of the BRC1 domain of BRCA2 greatly inhibits HRR, suggesting that the BRCA2-Rad51 interaction via BRC1 is important for regulating HRR. The effects of the BRC1-4, BRC5-8 and BRC1-8 fragments on HRR will further be examined. For in vitro study, these three BRC fragments of BRCA2 have also been constructed into a His-tagged baculoviral vector.
We have successfully expressed these three proteins in insect cells, Sf9. The expressions were confirmed by Western blotting using either alpha-BRC4 or alpha-BRC5 antibody as well as alpha-His antibody. The purification of the individual fragments is currently undertaking. Three in vitro biochemical assays of Rad51 have been established in our laboratory, including DNA binding, ATPase, and DNA strand pairing and transfer. Using these three assays, the biochemical activities of the Rad51B and Rad51C proteins have been determined. The effects of the BRCA2 fragments on these activities of Rad51 will be examined. In addition, various combinations of BRCA2 fragments and Rad51 paralogs have been co-expressed in Sf9 cells and the Ni-NTA pull-down experiments are currently carried out to determine the complex formation between these proteins.

The work is to provide direct evidence for the functions and the mechanism of actions of BRCA2 in DNA repair. It will direct toward elucidating the mechanism underlying the pathogenesis of BRCA2-mediated breast cancer as well as toward providing a new approach for understanding the role of homologous recombination repair in sporadic breast cancer. Such an understanding would reveal new targets for prognostic interpretation and therapeutic intervention in breast cancer.