Fig. 5: Role of RecQL4-S89 phosphorylation in regulating MTBP association with replication origins.

A Nascent strand abundance (left, red) and MTBP association (right, green) at baseline and dormant replication origins in unperturbed RecQL4 CRISPR knockout HCT116 cells (Null) complemented with either WT-RecQL4 or S89A-RecQL4. Baseline and dormant origins were stratified as described in the legend to Fig. 1. B MTBP binding at baseline and dormant replication origins in the cell variants described in panel A, without APH or 1 h after release from APH exposure (10 μM for 1 h). C, D The recovery of HCT116 cells from exposure to APH. Representative immunofluorescence (C) and Quantitative immunofluorescence-based cytometry quantification (D) of DAPI (a DNA content indicator), EdU (a DNA synthesis marker), RPA2 (a replication stress marker), and γH2AX (a DNA damage marker). HCT116 cells harboring either WT-RecQL4 (WT) (D, top panel) or S89A-RecQL4 (D, bottom panel) were untreated (UT) or exposed to APH (10 μM for 1 h) and then released (rel.) into a fresh medium for the indicated time periods. For each condition, cells were pre-labeled with EdU (10 μM for 30 min) and then detergent-extracted and fixed (see methods). Scale bar = 2 μm. EdU versus DAPI levels were plotted, and high-, low-RPA positive and RPA negative (neg.) cells (for calibration, see Supplementary Fig. 4E) are shown in red and blue, respectively. A minimum of 4 replicates were analyzed and plotted together. E Quantification of RPA and γH2AX in untreated cells and in cells recovering from exposure to APH as described in Fig. 5C, D. (for QIBC profiles, see Supplementary Fig. 5G and source Data file). Total number of cells quantified across 4 biological replicates: for WT- UT (n = 5010), 1 h (n = 5648), 4 h (n = 11,951), 8 h (n = 8528), 24 h (n = 7498) and S89A-UT (n = 5024), 1 h (n = 6443), 4 h (n = 8601), 8 h (n = 9288), 24 h (n = 6186). Stacked bars show the mean, error bars indicate SD. F Left, Colony formation in HCT116 cells harboring WT (left) and S89A-RecQL4 (roight) exposed to 10 μM APH for the indicated times. Right, mean colony formation in three independent experiments. Error bars indicate SD. Source data are provided as a Source data file.