Fig. 5: C-terminal non-catalytic part of POLE1 is critical for replication initiation.

a Schematic representation of POLE1 constructs used in the study: wild-type protein, catalytically dead protein with mutations D860A, D862A, catalytic domain of POLE1 (aa1–1261), C-terminal half of POLE1 (without the catalytic domain, aa 1262–2305), C-terminal part of POLE1 without the zinc-finger domain (aa 1261–2151). b 293FT cells were transfected with an empty vector or the constructs described in panel a, fused to FLAG tags at their N termini. 48 h later cells were lysed and FLAG-tagged proteins were immunoprecipitated using M2 agarose beads, followed by elution with FLAG peptide. Western blots of the eluted protein and input samples are shown. c, d Clone 16 cells were transfected with indicated constructs. 32 h later dox/aux or DMSO was added to the cells for 16 h. 10 µM EdU was added for the last 30 min. Flow cytometry histograms of the EdU incorporation are shown (c). The first panel shows the transfected untreated samples, colors match the legend on 2D, the other panels represent samples treated with dox/aux for 16 h. Comparisons of samples transfected with indicated constructs (orange) to the sample transfected with an empty vector (black) are shown (c). d Quantification of the percent of cells in the dox/aux treated cells, corresponding to the indicated fractions, normalized to the empty vector control, are shown. Fractions were gated as indicated in Fig. S2. The quantification is based on n = 4 independent experimental repeats (means + SD and p values are shown where they are significant. One-way ANOVA was used for statistical analyses). Source data are provided as a Source Data file.