structures and mechanism of BRCA2 in meiotic recombination

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.

In reproducing organisms, two sets of genomes need to be exchanged and integrated. Double stranded breaks are required for genetic exchange to occur as they serve as recombination sites. Interestingly, a DSB is also one of the 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 can cause mutations in DNA that can lead to the development of cancer. Even more remarkable is that mitotic and meiotic DSBs use similar mechanisms for repair. In mitotic cells, homologous recombination utilizes the sister chromatid as templates for repair while meiotic recombination utilizes the homologous chromosome. In homologous recombination, RAD51 recombinase forms filaments on resected ssDNA and this nucleoprotein filament is used for homology search. In meiosis, DMC1, a RAD51 homologue, is the main recombinase although RAD51 is also required. The similarity between the two processes is further exemplified by the requirement of BRCA2. BRCA2 is one of the breast cancer susceptibility proteins, and mutations in BRCA2 gene increase the risk of breast, ovarian and other cancers due to the reduced ability to repair DNA damages. In mice, defects in BRCA2 cause male infertility. However, it is unclear if this is true in human. It is thus intriguing and important to understand what exact roles BRCA2 plays in meiotic recombination and how it 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 has in this process. In this proposal, We plan to use electron microscopy single particle technique to study the 3-dimensional structures of BRCA2 and DMC1 and possibly BRCA2, RAD51 and DMC1 and use biochemical and biophysical approaches to study the influence of BRCA2 on nucleoprotein filament formation to reveal the mechanistic action of BRCA2 and RAD51 in meiotic recombination.

The research will benefit scientists, postdocs, students, pharmaceutical and biotechnology industry, health care professionals and cancer patients through knowledge advance, staff and student training.

Advance in knowledge base
Meiotic recombination is a fundamental cellular process. A detailed understanding of the process will therefore have a profound effect in our understanding of how cells reproduce. In addition, this process shares high similarity with homologous recombination, which is the major repair pathway for DNA double-strand breaks, a fundamental course of cancer and aging. Furthermore, the key player BRCA2, a well known tumour suppressor, is shown to interact with many other tumor suppressors including p53. Structural information obtained here can therefore help us understand other cellular pathways. Due to the direct link of BRCA2 mutations to breast cancer and the demonstrated link of meiotic recombination to fertility in mice, the advanced knowledge in this system will have the added benefit of helping us to understand the basic mechanisms underlying breast cancer as well as infertility, which may then be exploited for novel therapeutic development.

Staff and student training
This project involves close collaborations between Prof. Xiaodong Zhang, Imperial College London and Dr. Steve West at the Crick institute (currently Cancer Research UK). We will utilize structural biology, biochemistry and functional studies. The interdisciplinary approach of the collaborating groups in this proposal will greatly enhance training of the associated RAs, especially with respect to their ability to work within large interdisciplinary teams. Quantitative approaches will feature highly, and the outcomes arising from the integration of the various data sets will demonstrate the power of the interdisciplinary approach. Such trained RAs (and associated PhD, masters and undergraduate students) are likely to benefit the biotechnology and pharmaceutical industries, as well as the academic base in the UK and abroad. Approaches to answering precise and penetrating questions of complex macromolecular systems will also feature in the training of staff associated with the project. We therefore anticipate medium term economic benefits arising from a well-trained UK and international research base, reflected in maintaining internationally competitive research intensive universities and associated industries.

Dissemination of results
Our work in this area has been widely presented and distributed, both at national and international meetings and in international high impact journals. In addition, our recent work on BRCA2-RAD51 has been featured at Imperial College London website as a press release and has attracted significant media attention. It was ranked 2nd by Altrimetric for papers published in Nat Struct. Mol. Biol. We plan to continue to present and disseminate our results in this way. In addition, this research will be available to larger community through our research websites.

Social and economic impact
Many important scientific advances are only found to be useful many years after the original discovery. The work here focuses on the mechanism of a fundamental cellular process and therefore we expect any commercial impacts will occur after the grant has finished. However due to its direct link to cancers, the work here will provide cancer patients with an improved knowledge about the cause of the disease and also offer opportunities for future novel therapeutic approaches that can interfere with DNA damage repair mechanisms. Furthermore, the link with infertility in mice suggests a possible link to human infertility, which is difficult to study and correlate. Our work might provide insights and potential novel treatment.