Amplification of centrosomes in BRCA2 null MEF cells suggests that BRCA2 plays an important role in the regulation of centrosome duplication. However, how BRCA2 mediates the regulation of centrosome duplication remains undefined. Using two C-terminal fragments (aa 2393-3418 and aa 12393-2952) of BRCA2 as bait, we found BRCA2 interact with DSS1 and a novel BRCA2 binding protein, referred to as centrobin (centrosomal BRCA2 interacting protein). Extensive immunofluorescence studies demonstrated that centrobin is localized to the centrosome. We found that BRCA2 is also a centrosomal protein. Using a centrobin siRNA, we concluded that BRCA2 localized to the centrosomes via its interaction with centrobin. Disruption of BRCA2-centrobin interaction a three experimental approaches (dominant negative BRCA2 and centrobin mutants, antibody blocking and silencing of centrobin gene by siRNA) caused centrosome amplification while did not affect the normal centrosome duplication, indicating BRCA2 function as a checkpoint control for centrosome duplication. Further analysis of the interaction of centrobin and BRCA2 indicated that DSS1 and centrobin bind to an overlapping region of BRCA2 (aa 2393-2952). These data lead us to hypothesize that DSS1 may also be a centrosomal protein. Using a HA tagged version of DSS1, we found DSS1 localizes to the centrosomes during metaphase. Furthermore, overexpression of DSS1 suppresses the centrosome amplification in Cos cells, T47 cells, and U2OS cells. And silencing DSS1 expression by a siRNA leads to centrosome amplification in 76Tert cells and HeLa cells. Here we propose to characterize the DSS1 protein, determine the interactions between centrobin, BRCA2 and DSS1, and examine the function of DSS1 in the microtubule organization, spindle formation, cell ploidy; cytokinesis, centrosome duplication, and cell cycle control. DSS1 is one of the major BRCA2 binding protein with approximately half of the endogenous BRCA2 co-immunoprecipitates with DSS1 in HeLa cells. Elucidation of the function of DSS1 will provide important insight into the mechanism of BRCA2 mediated tumorigenesis.
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