The Functions of BRCA2 in Repairing DNA Damage

Institution: Lawrence Berkeley National Laboratory
Investigator(s): Yi-Ching Lio, Ph.D. -
Award Cycle: 2001 (Cycle VII) Grant #: 7KB-0019 Award: $485,757
Award Type: New Investigator Awards
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2001)
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.


Final Report (2004)
Background information: “Seven percent of breast cancers and ten percent of ovarian cancers are related to susceptibility genes, mainly BRCA1 and BRCA2. Obviously not all susceptibility genes have yet been identified. It is suspected that as many as 1,000,000 (about 0.5% - 0.6%) United States women are carriers of the altered BRCA1 or BRCA2 gene. Ashkenazi Jewish women are at particularly high risk with over 1% carrying the gene. Women that carry the mutation have an 82% risk of breast cancer and a 44% risk for ovarian cancer (BRCA1) by the age of 70. Interestingly, these women are often afflicted at a younger age with a 59% risk of breast cancer before the age of fifty, and often these patients will be afflicted with cancer before the age of 40.” [from, Woman’s Cancer Information Center]

Loss of a functioning copy of BRCA2 gene has been demonstrated in a large proportion of familial breast cancer, but there is no well-defined explanation for how defective BRCA2 predisposes to breast cancer. Current evidence indicates that the critical functions of BRCA2 are associated with an error-free DNA repair mechanism called "homologous recombination" (HR). In nature, homologous recombination is a DNA maintenance pathway that protects chromosomes against damage affecting both DNA strands, such as double strand breaks (DSBs). Homologous recombinational repair (HRR) provides high fidelity in repairing DNA damage and is therefore essential and critical for the maintenance of genome stability and tumor avoidance. It was found that the BRCA2 protein physically interacts with the key HR protein, called Rad51, and controls the actions of Rad51. There are a number of isoforms (closely related proteins) of Rad51, including 51B, Rad51C and XRCC3. It become apparent several years ago that understanding how BRCA2 functions in DNA repair would be the key to elucidate the pathogenesis of BRCA2-mediated breast cancer. Our research goals were to investigate the functions of BRCA2 in DNA HR repair using both cellular and biochemical approaches. The aims of this project are: (1) To test the hypothesis that disruption of the BRCA2-Rad51 interaction affects HRR in human HT1080 cells. (2) To test the hypothesis that the BRCA2 protein mediates the biochemical activities of Rad51, including DNA binding, ATPase and DNA strand exchange activities. (3) To test the hypothesis that the BRCA2 protein forms a stable complex with Rad51 and two Rad51 "paralogs", Rad51 B and Rad51 C.

Using an in vivo (i.e., “test tube) HR assay, we demonstrated that individual expression of several small BRCA2 fragments in human HT1080 cells (human fibrosarcoma-derived) results in a reduced frequency in HR, suggesting that disruption of the normal BRCA2-Rad51 interaction in cells by the “dominant-negative” (i.e., made genetically inactive) BRCA2 fragments causes a defect in HR. Our data show the direct cellular evidence that the BRCA2-Rad51 interaction is crucial for HRR and multiple regions of BRCA2 protein, including BRC repeats and C-terminal region, are involved in regulating HRR. In further experiments, we used the baculovirus (i.e., a technique of producing proteins in insect cells) co-expression and Ni-NTA "pull-down" strategies to show that BRCA2 forms a multi-protein complex with Rad51, Rad51B and Rad51C DNA repair proteins. A strong interaction was found between BRCA2 and Rad51, as well as between Rad51 B and Rad51 C, while the interaction between Rad51 and Rad51 C in this system was found to be weak. We also found that the BRC repeats of BRCA2 do not directly interact with Rad51B or Rad51C. Using an in vitro biochemical assay, we found that the BRC1 domain of BRCA2 inhibits the ATPase activity of Rad51, indicating of a role for the BRC1 domain in modulating the ATP binding and/or hydrolysis activity of Rad51. Finally, we had proposed to purify the Rad51-binding domain (BRC repeats) of BRCA2 and investigate its effects on Rad51 activities. We have successfully expressed three BRC regions using baculovirus system, including BRC1-4, BRC5-8 and BRC1-8, however; the purification of these proteins was found to be difficult because these proteins were extremely unstable and tended to be degraded during the purification process. Although the BRC repeats can not be stably purified for biochemical assay, we instead found the first evidence indicating that BRCA2 is phosphorylated in a DNA damage-dependent manner. It is very possible that ionizing radiation-induced BRCA2 phosphorylation may play a key role in modulating the interaction between BRCA2 and Rad51. Further work remains to confirm this observation.

In addition, to the specific aims in the CBCRP initial grant, we also performed studies on the Rad51 family by using recently-introduced RNA interference (RNAi) technology (i.e., 21–25-nucleotide small interfering RNA (siRNA) that can induce specific gene silencing). These studies, “…..demonstrated the following: (i) RNA interference effectively depletes Rad51C in human cells; (ii) depletion of Rad51C destabilizes the XRCC3 protein; (iii) inhibition of Rad51C impairs HRR of chromosomal DSBs; (iv) Rad51C-deficient HeLa cells are sensitive to MMC and IR; and (v) radiosensitivity of Rad51C-deficient HeLa cells is associated with the S/G2 phase of the cell cycle. These findings are the first in vivo evidence for the functions of human Rad51C in repairing DNA DSBs through homologous recombination. “ [from Lio et al., J. Biol. Chem., Vol. 279, Issue 40, 42313-42320, October 1, 2004]

In conclusion, our results provide direct cellular and biochemical evidence of the functions of BRCA2 + the Rad51 protein family in DNA repair. The information may also prove new approaches for understanding the role of HR repair in sporadic breast cancer.


Symposium Abstract (2003)
The BRCA2 breast cancer protein is a major contributor to a dominantly inherited predisposition to this disease. Exactly how the mutations of BRCA2 cause breast cancer is not yet understood. Recent evidence has indicated that the BRCA2 protein functions in DNA repair though a specific mechanism known as “homologous recombinational repair (HRR)”. This HRR repair process utilizes the information provided by the complementary DNA strand to restore a break and is thought to be an error-free repair. It has been shown that BRCA2 physically interacts with Rad51, a key protein in HRR. Our research aims to provide direct experimental evidence for the functions and mechanism of actions of BRCA2 in the HRR pathway.

We are using an experimental system in human cells, which measures the capability for HRR with and without disruption of the BRCA2-Rad51 interaction. Our approach is to use various fragments of BRCA2 to define the process. Expression of a BRC repeat or a C-terminal region of BRCA2 was found to suppress HRR, suggesting that the BRCA2-Rad51 interaction is important for regulating HRR. The effect of larger fragments of BRCA2 will be further determined. To understand how BRCA2 regulates HRR, we have expressed and purified three fragments of BRCA2. We found that a small fragment of BRCA2 displays an inhibitory effect on the ATP hydrolysis activity of Rad51. Five similar human Rad51 proteins, called “paralogs”, have been recently identified as possible players in HRR. Using a novel biochemical “pull-down” strategy, we have demonstrated that BRCA2 interacts simultaneously with Rad51 and two Rad51 paralogs, Rad51B and Rad51C, to form a multi-protein complex. Whether BRCA2 regulates the complex formation of Rad51 paralogs is yet to be examined.

The success of the work will provide a new fundamental concept for elucidating the role of BRCA2 in HRR, and this provides insights into understanding the pathogenesis of BRCA2-mediated breast cancer.

Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro.
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Lio YC, Mazin AV, Kowalczykowski SC, and Chen DJ
Yr: 2003 Vol: 278 Nbr: Abs: Pg:2469-2478

Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Lio YC, Mazin AV, Kowalczykowski SC, Chen DJ.
Yr: 2003 Vol: 278 Nbr: 4 Abs: Pg:2469-78

Human Rad51C deficiency destabilizes XRCC3, impairs recombination, and radiosensitizes S/G2-phase cells.
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Lio YC, Schild D, Brenneman MA, Redpath JL, Chen DJ.
Yr: 2004 Vol: 279 Nbr: 40 Abs: Pg:42313-20