Resistance to mitomycin C requires direct interaction between the fanconi anemia proteins FANCA and FANCG in the nucleus through an arginine-rich domain

Fanconi anemia (FA) is a genetically heterogeneous disorder characterized b y bone marrow failure, birth defects, and chromosomal instability. Because FA cells are sensitive to mitomycin C (MMC), FA gene products could be invo lved in cellular defense mechanisms. The FANCA and FANCG proteins deficient in FA groups A and G interact directly with each other. We have localized the mutual interaction domains of these proteins to amino acids 18-29 of FA NCA and to two noncontiguous carboxyl-terminal domains of FANCG encompassin g amino acids 400-475 and 585-622, Site-directed mutagenesis of FANCA resid ues 18-29 revealed a novel arginine-rich interaction domain (RRRAWAELLAG). By alanine mutagenesis, Arg(1), Arg(2), and Leu(8) but not Arg(3), Trp(5), and Glu(7) appeared to be critical for binding to FANCG, Similar immunoloca lization for FANCA and FANCG suggested that these proteins interact in vivo . Moreover, targeting of FANCA to the nucleus or the cytoplasm with nuclear localization and nuclear export signals, respectively, showed concordance between the localization patterns of FANCA and FANCG, The complementation f unction of FANCA was abolished by mutations in its FANCG-binding domain. Co nversely, stable expression of FANCA mutants encoding intact FANCG interact ion domains induced hypersensitivity to MMC in HeLa cells. These results de monstrate that FANCA-FANCG complexes are required for cellular resistance t o MMC. Because the FANCC protein deficient in FA group C works within the c ytoplasm, we suggest that FANCC and the FANCA-FANCG complexes suppress MMC cytotoxicity within distinct cellular compartments.