Elucidating the Molecular Architecture of the FA-Core Complex Utilizing Biochemical Purification Approach.

FA is a rare, recessive disorder characterized by progressive bone marrow failure, developmental abnormalities, chromosome instability, cellular hypersensitivity to DNA cross-linking agents, and predisposition to cancer, mainly leukemias and squamous cell carcinomas of the head and neck. FA is genetically heterogeneous, represented by at least thirteen different complementation groups. The genes corresponding to groups A, B, C, D1 (BRCA2), D2, E, F, G, I, J, L, M and N have been cloned. The Fanconi proteins FANC -A, -B, -C, -E, -F, -G, -L, -M along with another newly discovered protein FAAP100 forms a core complex (CC) in the nucleus that is required for the monoubiquitination of two other Fanconi proteins FANCD2 and FANCI. In addition to their presence in nucleus, most of the Fanconi proteins are also found in the cytosol however their role in cytosol is not clear. Also, whether the FA-core complex proteins exist as a single complex or sub-complexes in the cytosol is not clear. The goals of this study are to

1. develop a method of purification of FA core complex from nuclear and cytosol

2. identify individual components of cytosolic and nuclear complex(s) of the FA proteins.

We have established a two-step purification method using 6XHis and FLAG tags for the biochemical and functional characterization of the FA core complex proteins. In an attempt to isolate interacting partners of FANCC, FANCG, FANCL and FAAP100 proteins; we have established four different HeLa cell lines; HeLa-HF-FANCC, HeLa-HF-FANCG, HeLa-HF-FANCL and HeLa-HF-FAAP100, stably expressing HF-FANCC, HF-FANCG, HF-FANCL and HF-FAAP100 recombinant proteins respectively. Affinity purification was carried out to isolate the complexes from the cytosol and nuclear extracts prepared from stable cell lines. The polypeptides purified from the complexes were identified by mass spectrometry. The results suggest that, in addition to the existence of the FA-core complex containing FANC -A, -B, -C, -E, -F, -G, -L and 100, the FA proteins also exists as sub-complexes in the cytosol. FANCL was found to form a sub-complex with FANCB and FAAP100 in cytosol. Also FANCC, a predominantly cytosolic protein was found to form a sub-complex with FANCE in cytosol. We found several novel proteins that interact with FA proteins.