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 ATAD5AID 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 ti (initiation time) and td (FRET dwell time, region with red shade). i Percentages of molecules that exhibit fork reversal activity (n > 5). j Average ti (n > 5). k Average td (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.

Fig 3: Equimolar concentration of F10 induces increased replication stress compared to 5-FU.(A) HCT-116 cells were treated with similar concentration of F10 and 5-FU followed by CldU and IdU incorporation. (B) DNA fiber images were captured and analyzed. (C) F10 (10 nM) reduces fork velocity significantly compared to 5-FU (10 nM). (D) F10 (10 nM) increases terminal forks significantly compared to 5-FU (10 nM). Statistical significance represented is in comparison with the control. (E) HCT-116 cells were exposed to F10 (10 µm) for 2 hours, and evaluation of replication stress–associated proteins shows increased expression of Rad51, increased phosphorylation of Chk1 and ?H2AX, and increased ubiquitination of FANCD2.

Fig 4: Survival analysis of F10 and Chk1 inhibition by PF-477736.Colony survival assay performed in (A) HCT-116 and (C) HT-29 cells shows decreased survival of Chk1i treated colonies co-treated with F10 compared to DMSO-treated cells co-treated with F10. Western blot analysis shows decreased pChk1-317 and increased ?H2AX in the combination in both (B) HCT-116 and (D) HT-29 cell lines. Additionally, decreased expression of Rad51 and ubiquitination of FANCD2 were observed in both the cell lines treated with PF-477736.

Fig 5: Immunofluorescence analysis of ?H2AX and western blot analysis of DNA repair protein with BI6727 treatment: reduction in DNA repair proteins with increased DNA damage and increased tumor suppressor protein levelsATRT cell lines, MAF-A737 and BT16, both were treated with their respective IC50 of BI6727 or DMSO for 72 h. Cells were fixed and stained for ?H2AX (green) and the nuclei stained with DAPI (blue). Representative immunofluorescent images were shown; MAF-A737 (A) and BT16 (C). Quantification of ?H2AX foci at 72 h after BI6727 treatment normalized to DAPI was shown for each of the two cell lines. Percent± SEM is shown (B and D). (E) Lysates from ATRT cell lines treated with either DMSO or with IC50 of BI6727 were analyzed by western blotting for DNA damage repair proteins, 53BP1, RAD51 and KU80 along with tumor suppressor protein p53.