Fanconi anemia (FA) is a human autosomal recessive disorder distinguished by cellular hypersensitivity to DNA crosslinking agents, skeletal abnormalities, bone marrow failure, and greatly increased incidence of leukemia. At least five distinct FA complementation groups (A-E) have been identified. The cDNA for group C [FA(C)] has been cloned and sequenced, yet the predicted polypeptide [FA(C)] does not contain consensus motifs that reveal a biological function, although loss-of-function and gain-of-function studies have determined that human hematopoietic progenitor cells require FA(C) protein for optimal growth and differentiation. The existence of different FA complementation groups might be explained if the biological action of FA(C) is mediated through interaction with FA(C)-binding proteins as part of a concatenated signaling pathway, a protein complex, or a combination of these mechanisms. Affinity capture, immunoprecipitation experiments, and functional assays have suggested that FA(C) protein associates with several other cellular proteins. The long-term objectives of this proposal are to elucidate the role that the FA(C) protein plays in the development of malignancies and bone marrow failure. The objective of this study is to test the hypothesis that FA(C) protein functions to regulate growth and/or differentiation in hematopoietic stem cells and to facilitate DNA damage repair in all somatic cells, functioning at least in part by serving as a co-factor for other proteins to which it binds. The P.I. proposes the following specific aims: 1) To fully characterize FA(C)-binding candidate proteins, isolated in yeast two-hybrid screens, for functional activity in vitro and in vivo. Two clones obtained in two-hybrid screens encode proteins that specifically interact with domains of the FA(C) protein. Information about human inactivating mutations, cell lines representing different complementation groups, and assays based on co-immunoprecipitation, in vitro binding, and cellular resistance to crosslinking damage will be used to confirm biologically meaningful interactions. 2) To map functional domains in the FA(C) protein.
