Recruitment of BRCA1-A complex to nucleosomes and sites of DNA damage

DNA is under constant attack from internal and external agents that cause DNA damage in the form of lesions and breaks. Double-strand breaks (DSBs) represent one of the most deleterious DNA lesions and needs to be resolved in a precise and timely manner. If this is not possible, errors in DNA coding sequences can persist leading to genome instability and uncontrolled cell growth, typically manifesting in different forms of cancer. Therefore, the cell dedicates a large machinery to repair DNA damage and a key player in this DNA repair process is the tumour suppressor breast cancer type 1 susceptibility protein (BRCA1). BRCA1 works by partnering with BARD1 (another tumour suppressor protein) and both need to be anchored near DNA double-strand breaks by a large complex called ARISC-RAP80. We refer to this supercomplex of seven proteins as the BRCA1-A complex and the molecular architecture of this large complex is currently unknown.

BRCA1-A recognises "flags", which the cell signalling machinery has produced near a damaged site in the form of a long poly-ubiquitin chain on chromatin, a mass of genetic material composed of DNA and proteins. The long poly-ubiquitin chains are presumed to be on histone protein H2A, which together with other histone proteins forms a core particle called nucleosome. How BRCA1-A and nucleosomes come together and what the exact roles of these ubiquitin chains are is currently unknown.

Throughout this project, we will uncover how BRCA1-A interacts with ubiquitylated nucleosome core particles (NCPs), and how BRCA1-A complex assembly correlates with its cellular functions. We will use a multidisciplinary approach and techniques varying from structural biology to determine protein structure, biophysical and biochemical experiments to assay enzyme activity and chemical biology to generate specific tools and reagents to probe BRCA1-A structure. These analyses will guide cell-based studies, where we will investigate how BRCA1-A assembly, localisation and activity are modulated in a biological context. Elucidating these mechanisms will transform our knowledge of how BRCA1-A functions, potentially opening new avenues for therapeutic targeting of DNA repair pathways.

BRCA1 forms three main complexes (A, B and C) in the cell, each having different roles in the DNA repair of double-strand breaks. The composition and recruitment of each complex to sites of DNA damage is well-known, however, it is difficult to delineate the exact function in vivo since there is overlap in sub-cellular localization, but also overlap of some molecular recognition modules that triggers the incorporation of BRCA1-BARD1 into the higher-order A- B- or C- supercomplexes. For example, all three supercomplexes require a phosphorylated pS-P-X-F motif that specifically interacts with the BRCT domains of BRCA1. The BRCA1-A complex is unique as it is selectively recruited after K63-linked ubiquitin chains have accumulated on chromatin. BRCA1-A also contains two sub-complexes with opposing activities: an E3 ligase module formed by BRCA1-BARD1, and a deubiquitylase module consisting of ARISC-RAP80. How these activities are coordinated in the context of the full BRCA1-A supercomplex and the precise function of each module is currently unknown. We have recently developed recombinant systems to produce active forms of each subcomplex as well as chemical biology methods to generate specific substrates for both. We aim to test each subcomplex in vitro for their enzymatic activity, either alone or together to delineate their precise function. We also have preliminary EM maps for both subcomplexes and our objective is to solve the structures of each subcomplex with and without substrates. We also aim to solve the structure of the fully assembled BRCA1-A supercomplex with and without ubiquitylated nuclear core particles after identifying the optimal nucleosomal substrate or nucleosomal binding partners. The molecular details and predictions revealed by our structural studies will be tested using in vitro and cell-based assays to understand the precise function of BRCA1-A in the DNA damage response pathway.

Who will benefit from this research?
Longer-term, the outputs of this research should benefit both the pharmaceutical industry and society as a whole. Shorter-term, this work will be of value to fundamental and translationally focused scientists in academia and industry.

How will they benefit from this research?
The BRCA1 and BARD1 tumour suppressor genes are frequently mutated in cancer. BRCA1 mutations lead to an increased risk of breast and ovarian cancers with hundreds of mutations discovered in the BRCA1 gene. Females with BRCA1 loss-of-function mutations have up to an 80% risk of developing breast cancer by age 90 and up to 55% risk of developing ovarian cancer. Therefore, elucidating the structure and precise function of the BRCA1-A supercomplex in DNA damage will be highly beneficial to better understand how disease-causing mutations lead to malignancy. In addition, cells are highly dependent on BRCA1 for repairing DNA damage, and any breakthrough in understanding how the BRCA1-A supercomplex functions will be beneficial to both academia, industry and charity organizations, whose mission is to identify and exploit new therapeutic targets for cancer.

What will be done to ensure that they benefit from this research?
We will disseminate the results of this project to the scientific community through publications and presentation at conferences and workshops. We will publish our data in Open Access journals and follow the MRC, University of Leeds and University of Edinburgh publications policy, which requires authors to upload the full text of all publications to the University Publications Databases. These will then be made open access via each institutional repository.
To ensure that basic discoveries, tools and reagents we generate during this project are developed to their full potential we will work with the Leeds and Edinburgh Research and Innovation teams to establish links with industry. We have regular links with industrial partners and we will present our work to special symposiums organized by our institutions to foster new links between academia and industry.
We will also communicate our findings to the public by taking part in annual Science festivals and Public events (e.g. Be curious: http://www.leeds.ac.uk/info/4000/around_campus/460/be_curious_festival-about_leeds_and_yorkshire). In addition, we will run BRCA1-focused sessions in partnership with Patient and Public Involvement (PPI) networks on cancer research.