Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, 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 cells from FA complementation groups (FA-A, FA-B, FA-C, FA-D1, FA-F, and FA-G) have a deficiency in the structural protein nonerythroid α spectrin (αSpIIΣ*), which we have shown binds to DNA interstrand cross-links and is involved in repair of this lesion. αSpIIΣ* co-localizes in damage-induced nuclear foci with FANCA and XPF, a protein involved in the repair of DNA interstrand cross-links. αSpIIΣ* also co-immunoprecipitates with FANCA, FANCC, FANCG and XPF from chromatin-associated protein extracts, indicating an interaction with these proteins. The present study was undertaken to determine whether there are direct interactions between αSpIIΣ* and proteins associated with the FA core complex. These interactions were examined using the LexA based yeast two-hybrid system and affinity chromatography. Full length FANCA, FANCC, FANCF and FANCG and a central region of αII spectrin were separately subcloned into the LexA and pJG4-5 vectors. Yeast strain EGY48 was transformed with combinations of these vectors. FANCA and FANCG were shown to interact with the αII spectrin construct. This binding was confirmed using purified proteins and affinity chromatography. Yeast two-hybrid analysis did not show any direct binding between αII spectrin and either FANCC or FANCF. Since FANCA, FANCC, FANCF and FANCG all co-immunoprecipitate with αII spectrin, these results indicate that only the binding of FANCA and FANCG to αII spectrin is direct. Further studies indicate that in addition to αSpIIΣ*, FANCA and FANCG also play a role in the repair of DNA interstrand cross-links. Using a 140 bp DNA substrate containing one site-specific 4,5’,8-trimethylpsoralen interstrand cross-link, our results show that antibodies against both FANCA and FANCG inhibit the incisions produced by XPF on the 3’ and 5’ sides of the cross-link. These results taken together demonstrate that there are direct interactions between αII spectrin and at least two of the proteins associated with the FA core complex, FANCA and FANCG, and that all of these proteins play a role in the repair of DNA interstrand cross-links. These results further support our model that αSpIIΣ* acts as a scaffold, recruiting FA/repair proteins to sites of damage, aiding in their alignment, and thus facilitating the repair process. αSpIIΣ* may thus play an integral role in both the repair of DNA interstrand cross-links and genomic stability.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]