3. BREAST CANCER-ASSOCIATED MISSENSE MUTANTS OF THE PALB2 WD40 DOMAIN, WHICH DIRECTLY BINDS RAD51C, RAD51 AND BRCA2, DISRUPT DNA REPAIR

WD40 domain - 7 blades beta-propeller fold 

Park J.Y., et al., 2014. Oncogene.


BRCA2 mediates the oligomerization of the Rad51 recombinase that is needed to form nucleoprotein filament with ssDNA. PALB2 associates with BRCA1 through its N-terminal coiled coil domain and with BRCA2 through its C-terminal WD40 domain. WD40 domains have ring structure of beta-propeller with seven blades that regulate protein interactions.

3 missense mutations of PALB2 WD40 domain have been found to disturb PALB2 protein more than its truncated mutations do. The disruption of PALB2, RAD51C, and BRAC2 interactions leads to breast cancer, overian cancer, and Fanconi anemia.

HF-RAD51C is purified using His6-FLAG-RAD51C based on which interactions with BRCA2 and PALB2 were detected via Co-IP. RAD51C was found to bind to WD4 domain of PALB2. However, Rad 51D does not interact with PALP2-BRCA2-RAD51C.

They created three missense mutations in the WD40 of PALP2 protein, including L939W, T103I, and L1143P at three different binding surfaces of PALB2 DW40 beta-propeller structure. The result suggested that PALB2 WD40 may scaffold the complex of HR and FA protein, and each HR protein is thought to bind to DW40 domain in independent manner at each distinct surface. XRCC3 is directly bound to PALB2 that resembles that of BRCA2, but completely distinct from that of RAD51C. These results may mean that RAD51D and XRCC3 interact with PALB2 in independent manner.   Therefore, formation of PALB2 - RAD51C complex or PALB2 - XRCC3 complex is doubted to compete with formation of RAD51C-XRCC3 complex, which is promoted in the absence of PALB2. [Does it mean RAD51C directly bound to XRCC3 in RAD51 paralogs, but they are in the same subcomplex CX3 in RAD51 paralogs??? I think M is doing experiments on this] 
Interestingly, RAD51C binding to PALB2 seemed to release BRCA2, leading to an increased amount of BRCA2 protein in mutant forms, and promotes RAD51 recruitment.
Since PALB2 is needed for RAD51 foci formation and CX3 only affects downstream of RAD51 foci formation. Therefore, it can be hypothesized that BRCA2-PALB2 was established first then RAD51C binds to PALB2, kicking BRCA2 out to promote RAD51 nucleoprotein formation, then PALB2 leaves after RAD51 foci formation to let RAD51C interacts with XRCC3 to form CX3 subcomplex for downstream of RAD51 foci formation in HR activity. But when does PALB2 interact with XRCC3 when PALB2 is busy in BRCA2-PALB2 complex, then in PALB2-RAD51C complex before RAD51 foci and needs to leave after RAD51 foci to allow RAD51C-XRCC3 complex formation? So...what is the purpose of PALB2 to bind to BRCA2 ????? Based on Olivia, 2009, PALB2-BRCA1 recognizes DSB sites, but how can they give a signal to recruit RAD51C or RAD51 paralogs to come to the damage sites when we know BRCA2 is a scaffolding protein? And what distinguishes the roles of BRCA1 and BRCA2?

L1143P mutant significantly reduced RAD51 filament formation, which is consistent with a significant decrease in DSB-HR associated with this mutant. L939W and L1143P also significantly increase in sensitivity to the IR.

In the opposite side, mutants of RAD51C were also tested, including L138F and D159N (breast and ovarian cancers) and R258H (FA), for interaction with PALP2. These mutants showed a decrease in binding of RAD51C to BRCA2, especially to PALB2.

RAD51 seemed not to be necessary for RAD51C or BRCA2 to bind to PALB2.

Nonfunctional truncated (complete pr partial removal) of DW40 in PALB2 protein are found have completely no DSB-initiated HR activities, but PALB2 missense mutations mentioned above only partially disrupt DSB-initiated HR pathway.

As a result, L939W, T103I, and L1143P mutations in PALB2 WD40 lead to genomic stability by at least two pathways. First, T103 is unstable in its expression in human cells, decreases in T103 WD40 PALB2 in cells may diminish its function in HR. Second, L939W and L1143P WD40 PALB2 suggested that they may alter the binding of PALB2 to HR proteins in vitro and human cells.

PALB2, BRCA2, and RAD51 are related to inherited breast cancer, while upstream FA pathway proteins are not.