Fanconi anemia is an inherited disorder characterized by chromosomal instability, cancer susceptibility, and a presumed defect in DNA repair. The long-term objectives of this proposal are to understand the biochemical function and cellular interactions of FACC, a protein that is deficient in Fanconi anemia complement group C patients. The cloning of the FACC gene by its ability to rescue lymphoblastoid cell lines derived from Fanconi anemia complement group C patients from the toxicity of bi- functional DNA cross-linking reagents (i.e., mitomycin-C and diepoxybutane [DEB]) has clearly established the role of this protein in the pathobiology of Fanconi anemia. However, fundamental questions concerning its mechanism of action, regulation, and biological function remain. The Principal Investigator has generated a polyclonal antibody directed against FACC and has been able to localize this polypeptide to the cytoplasm and perinuclear structures in a large number of mammalian cells, both under steady state conditions, with induction of cell cycle, and after exposure to DNA-damaging agents. He has also demonstrated that a chimeric FACC molecule that contains the constant region of human IgG1 heavy chain at its carboxy-terminus (FACC gamma 1) is able to correct the hypersensitivity of Fanconi anemia complement group C lymphoblastoid cells to mitomycin-C induced cytotoxicity and yet remains exclusively in the cytoplasm. Interestingly, when the nuclear localization signal of SV40 large T-antigen is subcloned upstream of FACC and this construct transfected into patient-derived lymphoblastoid cells, FACC localized to the nucleus, but was unable to correct mitomycin-C induced cytotoxicity. Taken together, these data (recently published in PNAS) clearly demonstrate the cytoplasmic localization of FACC, and thus suggest that its role in DNA repair must be indirect. The PI has also developed an in vitro assay for protein-protein interactions and demonstrated that FACC binds specifically to at least three ubiquitous cytoplasmic proteins of molecular weights 65 kD, 50 kD, and 35 kD (called the FACC-binding proteins, or FABPs). This has led him to propose that FACC forms a multimeric complex which could play a role in genomic stability, perhaps as a """"""""gatekeeper"""""""", regulating the access of genotoxic agents to the nucleus, or recruiting DNA repair proteins. He further hypothesizes that other complementation group abnormalities in Fanconi anemia could result from structural defects in other components of this multimeric complex.
Specific Aim 1 of this proposal is to study the subcellular targeting of FACC to cytoplasmic and perinuclear structures and its metabolic fate during stress states and passage through the cell cycle.
In Specific Aim 2, the Principal Investigator will elucidate the functional domains of FACC and its association with internal membranes, and will attempt to identify potential targeting signals and phosphorylation sites that may regulate its localization and function.
In Specific Aim 3, using a sensitive Southern blotting assay, the relationship of FACC to DNA cross-linking will be analyzed in an effort to identify specific molecular targets. Finally, in Specific Aim 4, structure of the FACC macromolecular complex will be elucidated by isolation of cDNA clones for the FABPs, development of assays to detect in vivo interactions of FACC and FABPs, and the analysis of the structure and expression of FABPs in both Fanconi anemia and non-Fanconi anemia cells. It is anticipated that these studies will provide fundamental insights into the regulation of genomic stability by cytoplasmic activities, and should also provide insight into the molecular pathobiology of Fanconi anemia.
Showing the most recent 10 out of 22 publications
