Figure 3.

The C-terminal SLD2 of UAF1 binds to a conserved SIM sequence on FANCI. (A) Identification of a conserved SIM in the primary amino acid sequence of human FANCI, aligned with the homologous regions of FANCI from other species: Homo sapiens (hs), Mus musculus (mm), Bos taurus (bt), Gallus gallus (gg), and Danio rerio (dr). (B) Schematic representation of FANCI-WT and two deletion mutants. FANCI-WT and C700 both contain the SIM region, while the SIM was absent in the C500 mutant. The N-terminal Flag epitope was indicated. (C) GST-SLD2 pulled down FANCI-WT and C700, but not C500. Plasmids expressing the indicated deletion mutants of human FANCI were transiently transfected into HEK293T cells. Cell lysates were used for the pull-down experiment with the indicated GST or GST-SLD2 fusion protein. Western blot was then performed using anti-Flag antibody. (D) FANCI interacted with full-length UAF1 in vivo. Flag-tagged wild-type FANCI and a mutant form of full-length FANCI (ΔVIPL) were transfected into HEK293T cells and immunoprecipitated using anti-Flag agarose beads. Wild-type FANCI specifically interacted with UAF1 after MMC treatment, and this interaction was impaired in the SIM mutant (ΔVIPL). (E) GST fusion proteins containing the indicated SUMO or SLD protein sequences were prepared and used to pull down wild-type and the ΔVIPL mutant form of full-length FANCI overexpressed in HEK293T cells. Only SLD2, but not SLD1 or SUMO1/2/3, pulled down wild-type FANCI, and this interaction was dependent on the SIM. By densitometry, the relative band intensities of lanes 6 and 7 were 1.00 and 0.57, respectively. (F) GST fusion proteins containing either SLD2 or SUMO1 were prepared and used to pull down wild-type Flag-FANCI or wild-type Flag-Pias1 protein, which were overexpressed in HEK293T cells. GST-SLD2 specifically pulled down FANCI and GST-SUMO1 specifically pulled down Pias1. (G) GST-SLD2 pull-down of wild-type FANCI but not of FANCI-ΔSIM (another FANCI mutant with a deletion of the entire SIM sequence).