Figure 3. POLE3 and DSCC1 contribute to DNA replication fork progression in an additive manner.

(A) Relative viability of WT, DSCC1-KO, and two POLE3-KO clones treated with ATR inhibitor (ATRi, AZD6738) or topoisomerase I inhibitor camptothecin assessed by CTB 5 d after addition of drug. Dots represent three technical replicates from a representative of two independent experiments. (B) Replication fork speed of POLE3-KOs assessed by a DNA fiber assay. At least 80 fibers were scored per condition. P-values were determined by an ordinary one-way ANOVA. (C) Cells were assessed for γH2AX foci using immunofluorescence. At least 42 cells were scored per experiment per condition in three independent experiments, and the percentage of cells with more than five foci is shown. Scale bar represents 5 µm. (D) WT and DSCC1-KO cells were transfected with indicated crRNAs and treated with doxycycline to induce Cas9 expression. After 5 d, DNA replication fork speed was assessed by a fiber assay. At least 50 fibers were scored per experiment per condition in two independent experiments. Indicated P-values are determined by an ordinary one-way ANOVA. (E) Flow cytometry analysis of WT and DSCC1-KO cells 5 d after transfection with indicated crRNA:tracrRNAs. Actively replicating cells (S) are identified by EdU incorporation and G1 and G2 by DNA content stained by DAPI.

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