FANCA S1449A fails to completely correct FA-associated phenotypes. (A) Results of 3 growth inhibition assays are shown for GM6914 (FA-A) cells expressing wild-type Flag-FANCA, Flag-FANCA (S1449A), or the vector control. (B) Nuclear extracts were prepared from the same cells as in panel A treated with 0.1 μM MMC for 18 hours. Extracts were separated by SDS-PAGE and immunoblotted for FANCD2. Ku86 serves as a loading control. L/S ratios were calculated using densitometric measurements of short and long band intensities. (Bottom panel) The same cells as in panel A were treated with 0.1 μM MMC for the indicated time and then lysed directly in SDS loading buffer, separated by SDS-PAGE, and immunoblotted for FANCD2 and Ku86 as a loading control. L/S ratios were calculated by densitometry and plotted against time of treatment. (C) Histogram plots are shown of chromosomal aberrations seen on metaphase spreads from GM6914 cells as in panels A and B. (D) The frequency of homologous recombination in GM6914 DR-GFP cells expressing wild-type Flag-FANCA, Flag-FANCA S1449A, or the vector control was measured using flow cytometry after HDR-mediated repair of a GFP reporter substrate. Frequency is expressed as a percentage of the level of recombination seen in wild-type FANCA-expressing cells. FANCD2-L indicates long (mono-ubiquitylated) form of FANCD2; FANCD2-S, short (nonubiquitylated) form of FANCD2; and L/S Ratio, long/short ratio.