Stephen West

 

Assembly of Macromolecular Complexes for DNA Repair

Individuals carrying mutations in their BRCA2 gene are predisposed to early onset breast or ovarian cancers. The cumulative incidence of breast cancer in germ-line BRCA2 mutation carriers approaches 80%, leading many women with familial history to make difficult choices relating to radical surgical approaches that can reduce the risk. Biallelic BRCA2 mutations have also been linked with the cancer-predisposition syndrome Fanconi anemia (FA) sub-type FA-D1. Recent research has shown that loss of BRCA2 leads to a hypersensitivity to DNA damaging agents and defects in the repair of DNA double-strand breaks (DSBs) by homologous recombinational repair (HR). BRCA2 protein interacts directly with RAD51 recombinase and facilitates the loading of RAD51 onto DNA where it forms an extended nucleoprotein filament that initiates recombination. The loss of BRCA2 activity through mutation affects the efficiency of RAD51 filament formation and results in reduced recombinational repair. These events are thought to underpin the tumourigenesis observed in individuals with BRCA2 mutations. 

In addition to its interaction with RAD51, BRCA2 interacts with a number of other proteins, including BRCA1 and PALB2 (Partner and Localiser of BRCA2), both of which are linked to breast cancer susceptibility. BRCA1 acquires E3 ubiquitin ligase activity through association with BARD1, and is involved in the initial recognition and signalling of the presence of a DSB, directing them into the HR pathway. PALB2 links BRCA1 with BRCA2, and is required for their association with chromatin. Accordingly, mutations in PALB2 destabilise the genome and, like BRCA2 mutations, increase the risk of breast cancer and diseases such as Fanconi anemia (PALB2 is defective in patients belonging to the FA-N sub-group). However, because PALB2 binds directly to DNA and also to RAD51, and stimulates RAD51-mediated recombination reactions in vitro, it is likely that the role of PALB2 goes beyond the simple localisation of BRCA2. Most likely, BRCA2 and PALB2 act as a heterodimeric complex that modulates RAD51 assembly.

In recent work, we determined a low-resolution structure for the BRCA2 tumour suppressor, and defined its role in the establishment of RAD51 filaments. However, little is known about the way that a BRCA2-PALB2 co-complex might direct RAD51 nucleoprotein filament formation. We are currently developing methodologies to purify homogeneous samples of BRCA2-PALB2 complex from human cells for detailed biochemical study. The successful applicant will take on one aspect of this project which involves collaboration with physical and structural biologists expert in the development of time-resolved cryo-electron microscopic analyses of macromolecular complexes. Specifically, we will use microfluidic techniques to spray BRCA2-PALB2-RAD51 assemblies, formed over millisecond time ranges, onto carbon-coated grids suitable for single particle analysis using the Francis Crick Institute's in-house Titan Krios transmission electron microscopes. It is anticipated that we will be able to visualise the assembly of RAD51 filaments by BRCA2-PALB2, followed by their dissociation. The way that BRCA2-PALB2 acts as a molecular chaperone for filament formation will be determined. 

1. Davies, A. A., Masson, J. Y., McIlwraith, M. J., Stasiak, A. Z., Stasiak, A., Venkitaraman, A. R. and West, S. C. (2001)
Role of BRCA2 in control of the RAD51 recombination and DNA repair protein.
Molecular Cell 7: 273-282. PubMed abstract

2. Esashi, F., Christ, N., Gannon, J., Liu, Y., Hunt, T., Jasin, M. and West, S. C. (2005)
CDK-dependent phosphorylation of BRCA2 as a regulatory mechanism for recombinational repair.
Nature 434: 598-604. PubMed abstract

3. Esashi, F., Galkin, V. E., Yu, X., Egelman, E. H. and West, S. C. (2007)
Stabilization of RAD51 nucleoprotein filaments by the C-terminal region of BRCA2.
Nature Structural & Molecular Biology 14: 468-474. PubMed abstract

4. Shahid, T., Soroka, J., Kong, E. H., Malivert, L., McIlwraith, M. J., Pape, T., West, S. C. and Zhang, X. (2014)
Structure and mechanism of action of the BRCA2 breast cancer tumor suppressor.
Nature Structural & Molecular Biology21: 962-968. PubMed abstract

5. Thorslund, T., McIlwraith, M. J., Compton, S. A., Lekomtsev, S., Petronczki, M., Griffith, J. D. and West, S. C. (2010)
The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA.
Nature Structural & Molecular Biology 17: 1263-1265. PubMed abstract