Research Paper Volume 12, Issue 20 pp 20268—20284

TFG-maintaining stability of overlooked FANCD2 confers early DNA-damage response

Figure 3. AA 5-100 of TFG contributes to the interaction between TFG and FANCD2-V2. (A) Aminol terminal of TFG confers its association with FANCD2-V2. HEK293T cells were transfected with Stag-Flag-wtTFG, Stag-Flag-mtTFGΔaa5-100, Stag-Flag-mtTFGΔaa101-325, Stag-Flag-mtTFGΔaa326-400 or empty vector together with GFP-wtFANCD2V2. Both GFP and Stag antibodies’ IPs were performed and the pulldowns were detected by GFP and Flag antibodies. Red line-squares indicate the reduced interaction between GFP-wtFANCD2 and Stag-Flag-mtTFGΔaa5-100. (B) Docking of C-terminal of FANCD2-V2 and N-terminal of TFG supports their interaction. The predicted structure of human TFG (green) was adapted well to that of partial FANCD2-V1 protein from 1200-1441 (red). (C) Both the aa1437-1442 of FANCD2-V2 and the aa5-100 of TFG are important for the earlier action of FANCD2-V2 upon DNA damage. Live imaging was performed on TFG-normally expressed U2OS cells co-transfected with GFP-wtFANCD2-V2 and RFP-wtTFG or GFP-mtFANCD2-V2(Δaa1437-1442) and RFP-wtTFG; together with TFG expression-compromised U2OS cells co-transfected with GFP-wtFANCD2-V2 and RFP-wtTFG or GFP-wtFANCD2-V2 and RFP-mtTFG(Δaa5-100) (Moving images in Supplementary Videos 25 respectively). Photos were taken every 30min. Green focus duration and intensity was reduced in cells carrying mtFANCD2-V2Δaa1437-1442 or mtTFGΔaa5-100 comparing to the corresponding controls.