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

Lead Research Organisation: Imperial College London
Department Name: Life Sciences

Abstract

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.

Technical Summary

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 .

Planned Impact

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.

Publications

10 25 50
 
Description Mutations in the gene that encodes BRCA2 are well known for raising the risk of breast cancer and other cancers. Although the protein was known to be involved in DNA repair, its shape and mechanism have been unclear, making it impossible to target with therapies. We managed to obtain 3-dimensional images of BRCA2 and its partner protein RAD51. Our work show that BRCA2 protein works in pairs and binds to two sets of RAD51 that run in opposite directions to ensure that irrespective of how it binds to DNA, RAD51 will be able to form productive filaments
Exploitation Route This study improves our understanding of a fundamental cause of cancer. It's our first view of how the protein looks and how it works, and it gives us a platform to design new experiments to probe its mechanism in greater detail.

Once we have more details, it will help us to design ways to correct defects in BRCA2 and help cells repair DNA more effectively. We can also think about how to make the repair process less effective in cancer cells.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description A London Consortium for high resolution cryoEM
Amount £3,000,000 (GBP)
Funding ID 206175/Z/17/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2017 
End 02/2022
 
Description PhD studentship to Xiaodong Zhang
Amount £139,000 (GBP)
Organisation Breast Cancer Now 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2019 
End 09/2023
 
Description The Royal Society Newton International Fellowship to Yueru Sun
Amount £150,000 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2016 
End 12/2018
 
Description Wellcome Trust Investigator Award
Amount £2,000,000 (GBP)
Funding ID 210658/Z/18/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2019 
End 12/2023
 
Description Collaborations 
Organisation Curie Institute Paris (Institut Curie)
Country France 
Sector Academic/University 
PI Contribution intellectual input and structural studies which helped collaborator to secure tenure and grants
Collaborator Contribution advanced research including publications through materials and intellectual input
Impact Publications, further funding
Start Year 2016
 
Description New collaboration 
Organisation University of Birmingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration to combine structure and cell biology
Collaborator Contribution bringing cell biology expertise
Impact Resulted in joint PhD studentship
Start Year 2018