* Structural characterization of BRCA2-containing pre-recombination complexes (Collaboration with Prof Xiaodong Zhang, Imperial College, London)
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.
