Fig. 2

Protection of reversed forks from degradation by RIF1. a Top panel: schematics of experimental conditions for fork progression in WT and Rif1^−/− MEFs. Cells were labeled with CldU (red) followed by IdU (green) as indicated. Representative DNA fibers for progression in WT and Rif1^−/− MEFs are shown below the schematic. Progression was measured by tract lengths of CldU (red) and IdU (green) in micrometers (μM). b Top panel: schematic for labeling cells in fork degradation assay. Representative pictures of normal and degraded fork are shown below the schematic. Cells were labeled with CldU followed by IdU and then subjected to replication stress with 4 mM HU for 3 h. Ratio of IdU to CldU tract length was plotted as readout for fork degradation. c, d Fork degradation assay in WT and RIF1-KO HAP1 cells (c) and between two different clones of WT, Rif1^−/−, and 53bp1^−/− MEF cell line (d). Experimental conditions were similar as in b. e Representative electron micrographs of normal fork (left) and reversed replication fork (right) observed on treatment with HU. The black arrow pointing to four-way junction at the replication fork indicates fork reversal (P, Parental, D, Daughter strand, R, Reversed arm). f Percentage of fork reversal in WT and Rif1^−/− MEFs treated with or without HU (4 mM) for 3 h. Numbers of analyzed molecules are indicated in parentheses. g WT and Rif1^−/− MEFs were transfected with siRad51 (100 nmols, 48 h) followed by labeling and treatment with 4 mM HU for 3 h. Fork degradation was determined in the presence and absence of RAD51. h Fork reversal frequencies observed with and without depletion of RAD51 in WT and Rif1^−/− MEFs under HU treatment. Numbers of analyzed molecules are indicated within parenthesis. Red bars in a, b, c, d, and g represent mean values from 125 fibers from each genotype under each condition. P-values were derived from Kruskal–Wallis ANOVA with Benjamini Hochberg (BH) post test except in c, where Mann–Whitney was used and in f and h, where unpaired t-test was done (ns, non-significant, ****P < 0.0001). All experiments were repeated three times with similar outcomes (Supplementary Data 2 and Supplementary Fig. 7a–e)