DNA fiber assay

The DNA fiber assay was performed as previously described (Lee et al., 2014; Merrick et al., 2004). Briefly, cells were treated sequentially with IdU for 10 min and CldU for 30 min and then incubated in spreading buffer (0.5% SDS, 200 mM Tris-Cl pH 7.4, and 50 mM EDTA) on a Silane-Prep Slide (S4651; Sigma-Aldrich). DNA fibers were then extended by tilting the slide and were fixed with 3:1 methanol/acetic acid. After treatment with 2.5 N HCl for 30 min, each slide was incubated with mouse anti-BrdU (for IdU detection) and rat anti-BrdU (for CldU detection). After wash with PBS, the slide was incubated with a mixture of secondary antibodies (Alexa Fluor^® 568 rabbit anti-mouse IgG [A11061; Invitrogen] and Alexa Fluor^®488 chicken anti-rat IgG [A21470; Invitrogen]) at RT for 30 min followed by another mixture of secondary antibodies (Alexa Fluor^®568 goat anti-rabbit IgG [A11011; Invitrogen] and Alexa Fluor^®488 goat anti-chicken IgG [A11039; Invitrogen]) after 15 min of blocking. The slides were mounted using Vectashield mounting medium and fluorescence microscopy images were captured using a Carl Zeiss LSM880 scanning laser confocal microscope (Fluorescence Core Imaging Center at Ewha Womans University). Tract length of fork progression was determined and converted to fork speed on the basis of 1-μm DNA being equivalent to approximately 2.8 kb.