Periodic Reporting for period 1 - BRCA2Interact (Structural and biochemical characterization of pre-recombination complexes)
Reporting period: 2015-06-01 to 2017-05-31
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.
To define the interplay between PALB2 and BRCA2 in promoting the formation of higher order HR complexes and RAD51 filament formation, an important aim of our studies is to determine the structure of BRCA2-PALB2 and BRCA2-PALB2-RAD51 protein complexes using cryo-electron microscopy and 3D reconstruction. We can report that BRCA2-PALB2 complexes have been successfully purified from baculo virus-infected insect cells. These complexes are being subjected to electron microscopy analysis and single particle selection for three-dimensional image reconstruction. To obtain a high-resolution structure for BRCA2-PALB2, a first large dataset is being collected for analysis. In order to reach the highest possible quality of structural and functional data, we continue to work to further improve the yield and solubility of the purified BRCA2-PALB2 complexes. To extend our structural determinations and reconstitution experiments, we will compare the purified BRCA2-PALB2 complexes with BRCA2-PALB2-RAD51. Purification of these RAD51-containing complexes is ongoing.
* Reconstitution of homologous pairing and strand exchange reactions using BRCA2-PALB2-RAD51
To investigate the interplay between BRCA2, PALB2, RAD51 and other HR factors on a functional level, we aimed to reconstitute homologous pairing and DNA strand exchange reactions using a series of purified BRCA2-containing protein complexes. We have successfully utilized an improved method for the purification of RAD51 from E.Coli that has been described recently (Subramanyam S, 2016), which increases both the yield and solubility of RAD51 by co-expression of the chaperone protein GroE. More importantly, strand exchange reactions have shown that RAD51 purified in this way is significantly more active when compared to RAD51 purified via other methods. We are now comparing this more active RAD51 in strand exchange assays with addition of newly purified BRCA2-PALB2 protein complexes as well as with BRCA2 alone. To carry out more controlled reconstitution experiments using both biochemical as well as EM approaches, we are purifying PALB2 from baculo virus-infected insect cells. Codon-optimized gene expression cassettes have been obtained to this order, and PALB2 expression has been optimized. When successful in purifying PALB2, we will extend our functional studies by generating PALB2 proteins disrupted in BRCA2 and RAD51 interaction domains. This will help us to define the contribution of each individual protein within the pre-nucleation complex.
To further develop the biochemical reconstitution experiments and EM visualization of pre-recombinational protein assembly, we will extend our experiments by inclusion of RAD51AP1, another critical HR factor interacting with PALB2, and the RAD51 paralogs. We have aimed to optimize the protein purification of the BCDX2 and CX3 paralog complexes by using newly created codon-optimized MultiBac expression vectors. However, obtaining sufficient expression of the RAD51C paralog has proven to be a challenge and we have tested different approaches both in the cloning and virus production stage to improve this. In addition, we are using CRISPR and other technologies to obtain a clean cell biological system in which to functionally study the effects of RAD51 paralog deficiency.