Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, a predisposition to cancer, hypersensitivity to DNA interstrand cross-linking agents and a defect in ability to repair cross-links produced by these agents. We have demonstrated that FA cells have a deficiency in the structural protein nonerythroid α spectrin (αSpII), which we have shown is involved in repair of DNA interstrand cross-links. αSpII co-localizes in damage-induced nuclear foci with FANCA and the DNA repair protein, XPF, after normal human cells are damaged with a DNA interstrand cross-linking agent. All three proteins co-immunoprecipitate with each other from chromatin-associated protein extracts from normal cells. In order to get a better understanding of the relationship between these proteins and their importance in the repair of DNA interstrand cross-links, studies were undertaken to determine whether these proteins interact directly with each other. Yeast two-hybrid analysis was carried out in which cDNAs for full length FANCA, XPF, and a central region of αSpII were separately subcloned into the LexA (pEG202) and pJG4-5 (B42) vectors. Yeast strain EGY194 was transformed with combinations of the FANCA, XPF and αSpII vectors. The results show that FANCA and XPF directly interact with each other as do FANCA and αSpII. To confirm that this binding was direct, affinity chromatography was carried out using purified proteins. FANCA was expressed in Sf9 insect cells using a recombinant baculovirus expression system. The FANCA fusion protein, which contained a 6-His tag, was purified using a TalonTM resin column. GST-tagged αSpII was expressed in E. coli andpurified on a glutathione Sepharose 4B column. Purified FANCA was shown to bind to αSpII using affinity chromatography. These studies are ongoing with XPF. The present studies, which demonstrate a direct interaction between FANCA and XPF, a protein shown to be involved in repair of DNA interstrand cross-links, point to an involvement of FANCA in the cross-link repair process, as does the binding of FANCA to αSpII. Since αSpII has been shown to bind to DNA containing interstrand cross-links and to be involved in repair of this type of damage, these results, combined with our nuclear co-localization data, further support our model that αSpII acts as a scaffold recruiting repair/FA proteins to sites of damage and thus facilitating the repair process. In FA cells where this scaffolding is deficient, these interactions are defective, which in turn correlates with reduced repair of DNA interstrand cross-links in these cells.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA