DNA fiber assay

The DNA fiber assay was performed as described previously²⁵ with slight modifications. Briefly, CARD14 inducible cell lines were labeled with 25 μM 5-chlorodeoxyuridine (CldU) (Sigma-Aldrich), washed with PBS, and exposed to 250 μM 5-iododeoxyuridine (IdU) (Sigma-Aldrich). To measure the efficiency of replication restart, cells were treated with 2 mM HU after CldU labeling and exposed to IdU. Labeled cells were harvested and resuspended in cold PBS. The cell suspension was mixed 1:4 with cell lysis solution (0.5% SDS, 50 mM EDTA, 200 mM Tris-HCl [pH 8.0]), placed on glass slides and carefully tilted at a 15° angle causing DNA fibers to spread into single molecules via gravity. Next, the DNA fibers were denatured by fixing with 4% paraformaldehyde in PBS and immersing in 2.5 M HCl for 80 min at 27°C. Slides were neutralized and washed with PBS before blocking with 3% BSA in PBS for 30 min at 37°C. DNA fibers were then incubated with primary and secondary antibodies diluted in 3% BSA in PBS overnight at 4°C (primary antibodies) or for 90 min at 37°C (secondary antibodies). Anti-BrdU antibodies (BU1/75 [ICR1]) (abcam, ab6326) for CldU and anti-BrdU antibodies (clone B44) (BD Biosciences, 347580) for IdU were used as primary antibodies. Alexa Fluor 488-conjugated goat anti-rat IgG (H+L) antibodies and Alexa Fluor 594-conjugated goat anti-mouse IgG (H+L) antibodies (Thermo Fisher Scientific, A11005) were used as secondary antibodies. Finally, slides were mounted in ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). Images were obtained using a FV1000 confocal laser scanning microscope (Olympus) and all data were analyzed via Computer Assisted Scoring & Analysis (CASA) software purchased from Dr. Paul Chastain. Tracts containing CldU were pseudocolored in red, and tracts containing IdU in green. Replication fork speed was estimated using a conversion factor of 2.59 kb/μm.²⁶