It is well established that faults in the BRCA2 gene (and the BRCA1 gene that prompted actress Angelina Jolie to undergo a mastectomy) increase the risk of breast, ovarian, prostate and other cancers. And since its discovery 20 years ago, this gene and its protein product, also called BRCA2, have been under intensive investigation.
The importance of the BRCA2 protein lies in the central roles it plays in DNA damage repair – but we’ve never actually seen it before now. Using electron microscopy, we’ve been able to get 3D pictures of the BRCA2 protein for the first time. These pictures, published in Nature Structural and Molecular Biology, not only reveal its structure and how it interacts with other proteins and DNA but will help in further understanding its role in DNA repair and cancer risk.
When repair goes wrong
The risk of breast cancer with BRCA1 and BRCA2 is around 50-80% over a lifetime and around 10-40% for ovarian cancer.
Our genomic DNA contains the information required for replication as well as instructions for making proteins, which then carry out the majority of cellular activities. So the integrity and fidelity of DNA are extremely important for the function and proliferation of the cells. However, due to toxic chemicals, UV irradiation and other natural metabolic by-products, our DNA suffers tens of thousands of events each day that cause damage.
Among the various forms of DNA damage, the most severe type is DNA double-strand breaks, as un-repaired or incorrectly repaired breaks can lead to mutations, chromosomes translocation (an abnormality caused by rearrangement of parts between chromosomes with the same genes) and deletion, all of which can contribute to cell death or cancer development.
BRCA2 plays a central role in repair, which uses an undamaged sister chromatin (a family of macromolecules that consist of DNA, protein and RNA in cells) as a template for faithful repair. In this process, the damaged DNA is first processed to create a single stranded DNA tail. Molecules called RAD51 form well-ordered filaments on the single stranded DNA tail, aided by BRCA2, and this long filament is then used to search for matching strands in the sister chromatin.
But if there are mutations in BRCA2 this can cause defects in this repair process, making the repair inefficient or forcing cells to use alternative repair methods that are prone to mistakes, all of which contribute to mutations in the genomic DNA and so increase the risk of cancer developing.
If we could understand how intact BRCA2 protein repairs DNA, and the nature of the mutations, we could then develop methods to correct the defects in BRCA2 to ensure repair is carried out properly. Alternatively, we could develop ways to hamper repair in cancer cells so that they are left to die.
One of the most powerful methods to investigate how a protein works is knowing its 3D structure. These 3D structures not only tell us what a protein or a protein complex looks like, but also how they work. However, to study protein structures we need to find a way to extract the protein of interest from other proteins in the cell. With 3,418 amino acids as building blocks, BRCA2 is one of the largest proteins in the cell – and one of the most difficult.
In 2010, three independent groups finally accomplished the task of purifying the BRCA2 protein; among them was Stephen West of the Cancer Research UK London Research Institute, who is also joint lead author of our research. This enabled us to use electron microscopy to image thousands of purified BRCA2 molecules or BRCA2-RAD51 complexes that could subsequently be analysed using computers and algorithms. This allowed us to determine and align differently oriented molecules to generate 3D structures of BRCA2 as well as its complex with RAD51.
What we discovered
Our study reveals that BRCA2 proteins exist as pairs and a BRCA2 pair recruits two sets of RAD51 molecules. Our study also showed how a single-stranded DNA binds across the BRCA2. We also showed that BRCA2 increases the number of short RAD51 filaments on the DNA. These multiple filaments are then linked to form a longer filament, which is required for searching for the matching strand. These results not only define the precise roles of BRCA2 in helping RAD51 form filaments, but how it helps RAD51 load onto single-stranded DNA.
Our studies set the foundation for a greater understanding of BRCA2 protein’s structures and mechanisms. With increasing knowledge of the workings of this important protein, we will be a step closer to develop therapeutics to protect healthy cells and to combat cancer.