Fig. 3: The interaction of ATAD5 with RAD51 is increased under replication stress.

a–e, g, h HEK293T cells were transfected with a cDNA expression vector or siRNA as indicated. After 48 h, cells were treated with 2 mM HU and whole-cell extracts were prepared for immunoprecipitation. a, b Cells were transfected with a cDNA expression vector expressing FLAG-tagged ATAD5 (FLAG-ATAD5) or FLAG-tagged UAF1 (FLAG-UAF1), or an empty vector (FLAG). After 48 h, cells were treated simultaneously with 2 mM HU or 2 μM ATR inhibitor (ATRi, ETP-46464) for 6 h. c HEK293T cells transfected with ATAD5 siRNA for 48 h were labeled with 10 μM EdU for 20 min prior to the addition of 2 mM HU or 2 μM ATRi as indicated. Samples were processed for iPOND, and captured proteins were separated by SDS-PAGE and immunoblotted. The right panel shows chromatin-bound proteins extracted from a portion of cells in c separated by SDS-PAGE and immunoblotted. d, e Cells were transfected with an expression vector expressing full-length (FL) or deletion mutants of FLAG-ATAD5. A schematic diagram of the ATAD5 deletion mutants is shown on the top. Boxes in red represent RAD51-interacting regions. + indicates that the interaction exists. f Purified GST, GST-RAD51, or GST-UAF1 proteins were mixed with purified ATAD5 protein fragments and pulled down for Coomassie staining. g Cells were transfected with DNA vectors expressing deletion mutants of FLAG-ATAD5. A schematic diagram of the ATAD5 deletion mutants is shown on the top. Boxes in red represent RAD51-interacting regions. The symbol + indicates that the interaction exists. h Cells were transfected with a DNA vector expressing FLAG-tagged ATAD5 C-terminal fragment (∆N693). After 6 h of transfection, cells were transfected with RFC4 siRNA. i U2OS cells expressing ATAD5^AID were transfected with a cDNA expression vector, treated with auxin, and fixed for a SIRF assay. Three independent experiments were performed and one representative result was displayed. Statistical analysis for i: t test; ****p < 0.0001, ***p < 0.001, *p < 0.05. j Chromatin-bound proteins extracted from a portion of cells in i were subjected for immunoblotting.