Fig. 2: ATAD5 promotes RAD51 recruitment to stalled replication forks, which depends on the PCNA unloading activity of ATAD5.

a, b, d, l, m U2OS cells were transfected with siRNA under the Noco-APH condition. a, b Cells were treated with 2 mM HU for 3 h before fixation for immunostaining. a Representative images of chromatin-bound RAD51. Scale bar: 20 μm. b The intensity of RAD51 staining was quantified. c After transfection, HEK293T cells were processed for iPOND immunoblotting. The right panel shows chromatin-bound proteins. d After transfection, cells were subjected for a DNA combing assay (n = 5). e U2OS cells expressing ATAD5^AID transfected with a cDNA expression vector were subjected for a SIRF assay (n = 3 unless indicated). f, g U2OS-TetOn-ATAD5 cell lines were treated with doxycycline and transfected under the Noco-APH condition. f The staining intensity of chromatin-bound RAD51 was quantified. g Cells were analyzed using a DNA combing assay. The length of I-dU-labeled DNA track was measured. h Representative time traces of Alexa488 (blue), Cy3 (green), and Cy5 (red) at Alexa488 excitation (top panel), at Cy3 excitation (second panel), and at Cy5 excitation (third panel). The time trace of Cy3-Cy5 FRET at Cy3 excitation is presented at the bottom panel. Three events: ATP-Mg²⁺ injection (blue), formation of a four-way junction (yellow), and dissociation of daughter strands (green) are indicated by color lines, respectively. Time delays between the events are defined as τ_i (initiation time) and τ_d (FRET dwell time, region with red shade). i Percentages of molecules that exhibit fork reversal activity (n > 5). j Average τ_i (n > 5). k Average τ_d (n > 4). l DNA combing assay (n = 4). m Native BrdU assay. n HEK293T cells transfected were processed for iPOND immunoblotting. b, f, g, m Boxes indicate median and interquartile ranges and whiskers indicate the 5th–95th percentile. Statistical analysis: Mann–Whitney U test. d, e, l Statistical analysis: t test; ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, n.s., not significant. d, i–k, l Error bars represent standard deviation of the mean.