Structural and mechanistic studies of the breast cancer tumour suppressor BRCA2 in DNA double strand break repair

About 10% of breast cancers are due to genetic mutations that predispose these individuals to the cancer. Of these familial cases, 30-50% are associated with mutations in two breast cancer susceptibility genes, brca1 and brca2. For germ line brca2 mutations, the cumulative risk for breast cancer is as high as 50% and 10-15% for ovarian cancer. Since its discovery, brca2 and the 3418 amino acid protein it encodes (BRCA2) have been the subject of intense study. BRCA2 plays important roles in homologous recombination, a key mechanism in DNA damage repair. It carries out its function through interactions with other key components such as PALB2 and RAD51. It is therefore becoming clear that information on its interactions with these partners will be required in order to understand the mechanism and functions in the context of efficient DNA repair and tumourigenesis. Due to the large size and low abundance of BRCA2, it has been extremely challenging to purify the full length protein, which has hindered its biochemical, structural and mechanistic characterization. However, very recently, three groups have reported the purification and biochemical characterization of BRCA2, creating an exciting opportunity for structural studies. In this proposed research, we will employ electron microscopy single particle analysis to characterize the structures of BRCA2 alone and its complexes with RAD51 and PALB2 on ssDNA in order to provide a molecular basis for its roles in double-strand break repair and breast cancer. The information obtained will not only provide us with the first glimpse of the structures of this important protein and its interacting partners, but also reveal insights into how BRCA2 helps to load RAD51 onto single stranded DNA that eventually leads to strand invasion and homologous recombination. This will constitute a significant step forward in our understanding of this key cellular process that has a direct link to cancer and aging. It will improve our knowledge about the cause of the disease and also offer opportunities for novel therapeutic approaches for DNA damage repair mechanisms with direct consequences for cancer patients.

Since its discovery, the breast cancer tumor suppressor brca2 gene and the 3418 amino acid protein it encodes (BRCA2) have been the subject of intense study. BRCA2 plays important roles in homologous recombination, the main mechanism in error-free homology directed DNA double strand repair during the S and G2 phases. Human BRCA2 is one of the largest proteins in the cell. The large size and low abundance of BRCA2 made it extremely challenging to purify the full length protein, which has hindered its biochemical, structural and mechanistic characterization. However, very recently, three groups including that of Dr. Stephen West, have reported the purification and biochemical characterization of BRCA2, creating an exciting opportunity for structural studies. In this proposed research, we will employ biochemistry expertise in Dr. Stephen West's laboratory and electron microscopy single particle analysis technique in Prof. Xiaodong Zhang's laboratory in combination with protein homology modeling and algorithms development in Dr. Paul Bates group for fitting crystal structures into low resolution EM reconstructions. Together we will characterize the structures of BRCA2 alone and its complexes with RAD51 and PALB2 in order to provide a molecular basis for its roles in DNA double-strand break repair. The information obtained will not only provide us with the first glimpse of the structures of this important protein and its interacting partners, but also reveal insights into how BRCA2 helps to load RAD51 onto single stranded DNA that eventually leads to strand invasion and homologous recombination. This will constitute a significant step towards our understanding of this key cellular process that has a direct link to cancer and aging. It will improve our knowledge about the cause of the disease and also offer opportunities for novel therapeutic approaches for DNA damage repair mechanisms .

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

Knowledge advance
Homologous recombination and DNA double strand break repair are fundamental cellular processes. A detailed understanding of these processes will therefore have a profound impact in our understanding of how cells repair DNA damage and of the fundamental aspects of cancer biology. BRCA2 is also shown to interact with many other tumor suppressors including p53, therefore structural information obtained here can help us understand other cancer related pathways. Due to the direct link of brca2 mutations to breast cancer, the advanced knowledge in this system will have the added benefit of helping to understand the basic mechanisms underlying breast cancer which may then be exploited for novel drug development.

Staff and student training
This project involves close collaborations between Prof. Xiaodong Zhang, Imperial College London, Dr. Stephen West and Dr. Paul Bates, both at Cancer Research UK London Research Institutes. We will utilize structural biology, biochemistry and functional studies as well as computational modelling and molecular simulations to address the mechanism of homologous recombination. 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 interdisciplinary teams. 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. 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.

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 in the longer term. However due to its direct link to cancers, especially breast cancer, the work here will provide breast cancer patients with an improved knowledge about the cause of the disease and also offer opportunities for novel therapeutic approaches that can interfere with DNA damage repair mechanisms.