M55 mutation restores STAT1 DNA-binding activity but fails to complement MMC hypersensitivity of FA-C cells.

(A) A schematic diagram of full-length FANCC and truncated mutant M55 that lacks amino-terminal 54 amino acids. The 3 motifs described in Figure 1 are indicated as black boxes. (B,C) EMSA analyses of STAT1 activation in M55-transduced FA-C lymphocytes. Normal FANCC- or M55-transduced HSC536N and PD4 cells were treated with IFN-γ (10 ng/mL) for 30 minutes and EMSA was performed using 5 μg of nuclear extracts and [³²P]-labeled oligonucleotides carrying the GAS from the IRF1 gene (B) or from the p21 gene (C). Competitive binding was carried out by incubation with 25-fold excess molar unlabeled GAS (lanes 5 and 6) oligonuleotides. Lane 7 indicates that binding reactions were incubated with an STAT1 antibody (5 μg). Arrows denote STAT1-GAS complexes. (D) Effect of M55 mutation on MMC sensitivity in FA-C lymphoblast cell line HSC536N. Cells were treated with varying concentrations of MMC for 5 days before staining with trypan blue dye to quantify viable cells. Cell viability is expressed as percentages of viable cells relative to the untreated control culture. Each data point represents the mean ± SD of at least triplicate determinations.