structures and mechanism of BRCA2 in meiotic recombination

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In reproducing organisms, two sets of genomes need to be exchanged and integrated. The perturbations to our DNA in the form of double stranded breaks are required for parental chromosomes to exchange genetic information and for genetic recombination to occur. Interestingly, the same form of break is also one of most severe types of damage to our DNA in non-sexually reproducing (mitotic) cells as failure to repair it can cause cell death while mis-repair could cause mutations in DNA that can lead to the development of cancer. Even more remarkable is that the two processes use similar processes to repair these breaks. In mitotic cells, this process is called homologous recombination where it utilizes the sister chromatid as templates for repair while in meiosis, the process is called meiotic recombination, which utilizes the homologous chromosome. In both processes, they use a recombinase protein that coats long stretches of single stranded DNA. These protein-DNA filaments are used to search for homologous DNA sequence in the partner chromosome. In homologous recombination, a single recombinase called RAD51 is required while in meiotic recombination, both RAD51 and another recombinase called DMC1, which is very similar to RAD51, are required. Both processes also use a key protein called BRCA2. BRCA2 is one of the breast cancer susceptibility proteins, and mutations in BRCA2 gene increase the risk of breast, ovarian and other cancers. It is partially understood why mutations in BRCA2 can increase the risk of cancer as it reduces the ability of cells to repair a DNA damage. Given the importance of BRCA2 in meiotic recombination, it is not surprising that, in mice, defects in BRCA2 cause male infertility. However, it is unclear if this is also true in human. It is thus intriguing and important to understand what the exact roles BRCA2 plays in meiotic recombination and how BRCA2 fulfills its roles. Furthermore, it is fascinating as why both RAD51 and DMC1 are required in this process and what the exact role of RAD51 is in this process. In this proposal, We plan to investigate these important questions. We plan to use the cutting edge electron microscopy single particle technique to study the 3-dimensional structures of BRCA2 and DMC1 and possibly BRCA2, RAD51 and DMC1 together. Furthermore, we will use a number of biochemical and biophysical techniques to visualize and quantify the length and number of DMC1 filaments under the influence of BRCA2. Our studies will provide a detailed mechanistic understanding of what and how BRCA2 plays these important roles in meiotic recombination, the essential process in generating genetic diversity and reproduction.