The goals of the proposed work are to identify genetic elements involved in the inherited disease Fanconi anemia (FA) and to study the molecular action of the identified genes. Cells isolated from patients with Fanconi anemia show unusual sensitivity to DNA damage by cross-linking agents such as mitomycin C (MMC) or diepoxybutane (DEB). Fanconi anemia results from a defect in any of several genes as indicated by the existence of four complementation groups. Initial efforts will be towards identification of the genetic element affected in Fanconi anemia complementation group A. For the identification of additional genetic elements involved in Fanconi anemia a cDNA expression library will be used to screen for complementation in fibroblasts by restoration of resistance to DNA cross-linking agents. Selection conditions allowing differentiation between normal cells and Fanconi anemia cells with either of these agents have been determined. A second goal is to understand the chain of events resulting in Fanconi anemia at the molecular level. Identified FA genes will be used to investigate the restoration of normal cellular function following DNA cross-linking in fibroblasts and lymphoblasts, the biochemical function defective in Fanconi anemia and the DNA repair deficiencies using an in vitro system. FA genes will be introduced into FA cells in pCMV-4, an expression vector. Comparison for survival after MMC or DEB will be tested along with chromosome stability. Repair of cross-linked DNA plasmids in FA (+) or (-) cells will be measured for transient or long term deficiencies. An in vitro system for measuring DNA repair will be made from FA (+) or (-) cells to allow identification of protein functions defective in FA. In order to provide an alternate approach to identification of function of FA gene products, purification of FA proteins and interacting proteins will be done followed by testing for candidate activities.
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