BRCA2Interact · Structural and biochemical characterization of pre-recombination complexes

Structural and biochemical characterization of pre-recombination complexes

(The fellowship described in this report was terminated pre-maturely after 18 months due to additional external funding being awarded to researcher Dr. Peuscher. Future directions of the work described are continueing under this newly awarded fellowship) Our genetic material, the DNA, is continuously subjected to damage. As maintaining genome stability is crucial for cell survival, broken DNA needs to be efficiently repaired. One essential mechanism to repair damaged DNA is homologous recombination (HR), and defects in different HR factors are linked with human disease and cancer pre-disposition. The RAD51 recombinase protein plays a central role in HR, forming filaments at sites of DNA damage and promoting homologous pairing and DNA strand exchange. RAD51 filament formation is mediated by the BRCA2 tumour suppressor protein, mutations in which lead to a high incidence of developing breast cancer. BRCA2 interacts with other HR factors, such as PALB2 and members of the RAD51 paralog family. Many of these proteins also function as tumour suppressors. To understand HR in more detail and gain new insights into the molecular mechanisms underlying genome instability, we have addressed the question how PALB2 and the RAD51 paralogs coordinate their activities with BRCA2 to promote the assembly of RAD51 filaments. Specifically, we have implemented a new protocol for the purification of RAD51, which has allowed us to purify RAD51 protein with improved solubility and activity. This protein is now being used in biochemical analyses and electron microscopy studies. In addition, the BRCA2-PALB2 complex has been purified and preliminary biochemical and structural analyses have been performed using this complex. To extend these studies by inclusion of the RAD51 paralogs, we have tested and optimized cloning and protein purification strategies for the two main paralog complexes. In conclusion, our studies have resulted in the successful purification of different critical HR proteins and complexes which can now be used to characterize the biochemical and structural properties of pre-recombination assemblies in more detail. This will help us to define the functional contribution of each individual protein within those complexes in promoting efficient HR, and teach us more about their role in genome maintenance and tumour avoidance.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз