The goal of this proposal is to delineate the relationship between the FAA and FAC gene products and the DNA repair defect in Fanconi anemia, complementation groups A (FA-A) and C (FA-C). It has been hypothesized that an underlying mechanism for this disorder may involve a DNA repair defect. We have isolated a DNA endonuclease complex from the nuclei of FA-A and FA-C cells and shown that it is defective in ability to incise DNA at sites of interstrand cross- links. Levels of a 230 kDa protein, associated with this complex and which binds to cross-linked DNA, are decreased in FA-A and FA-C cells. This protein has recently been identified as nonerythroid alpha spectrinllsigma* (alphaSpIIsigma*). The deficiency in alphaSpIIsigma* is corrected in FA-A cells transduced with a retroviral vector expressing the FAA cDNA, indicating that the FAA gene plays a role in its expression or stability. alphaSpIIsigma* also forms a complex with the FAA and FAC proteins in the nucleus which suggests that this complex may play role in DNA repair. It is possible that alphaSpllsigma* acts as a scaffold to help align or enhance interaction between proteins involved in the repair of interstrand cross-links and proteins that interact with FAA and FAC. The present proposal will address this by first determining the isoform of the alphaSpllsigma* we have identified and producing a recombinant protein that can be used in further studies. Exactly what proteins are associated with the FAA-FAC-alphaSpllsigma* complex, whether any of these proteins have binding affinity for DNA containing interstrand cross-links, and whether there is a deficiency in any of these proteins in FA-A and FA-C cells will be determined. The role of the FAA and FAC proteins in regulating the expression or stability of alphaSpllsigma* will be assessed as will the role of each of these three proteins in the repair of DNA interstrand cross-links. If alphaSpllsigma* is acting as a scaffolding protein, to help align and allow interactions between these as well as other proteins, this could have far reaching implications in a number of different processes, in addition to DNA repair, which have been associated with this protein, such as signal transduction and cell growth and development. A deficiency in alphaSpllsigma* in FA cells could thus ultimately affect hematopoietic differentiation and development. Isolation and identification of proteins associated with the FAA-FAC- alphaSpllsigma* complex and determination of their interactions with each other, other nuclear proteins, and DNA repair should help elucidate the basis of bone marrow failure and the development of aplastic anemia and leukemia in FA.