Fig. 4: MTBP and pRecQL4 association with baseline origins after perturbation of DNA replication.

A Recovery of HCT116 cells from APH-induced replication stress. Top, experimental procedure. Below, representative quantitative immunofluorescence-based cytometry (QIBC) of cells exposed to APH (10 μM for 1 h) and released for the indicated time frames. B EdU incorporation in cells treated as indicated in panel A, quantified across 4 biological replicates: Control (n = 5010), APH (n = 5706), 1 h (n = 5648), 4 h (n = 11,951), 8 h (n = 8528), 24 h (n = 7498), and 36 h (n = 5024). Stacked bars show the mean, error bars indicate standard deviation (SD). Statistical analysis was performed for the “high EdU” population compared to the control using a two-sided paired Tukey test, adjusted for multiple comparisons: ***p = 0.0008; ****p < 0.0001; ns not significant. C Binding of MTBP (left) and pRecQL4-S89 (right) to baseline and dormant origins in HCT116 cells exposed to aphidicolin (APH, 10 μM for 1 h) and released at the indicated time points. D Fractions of baseline (top) and dormant (bottom) origins binding to MTBP, pRecQL4, or both, at the indicated timepoint post-APH removal. Vehicle (DMSO) treated (veh.) and untreated control (cont.). The line plot represents the average of two biological replicates. E Replication stress facilitates an interaction between MTBP and pRecQL4. Left, experimental strategy. Right, Chromatin extracts from asynchronous cells untreated or treated with APH (10 μM for 1 h) were first immunoprecipitated (IP) with PCNA. PCNA-depleted fractions were then immunoprecipitated (pooled five reactions) with pMCM2 antibodies to isolate pre-replication complexes. MTBP or pRecQL4 antibodies were used for further IPs (with three pooled reactions), and the presence or absence of MTBP or pRecQL4 was detected by immunoblotting. Representative immunoblots of 3 independent replicates. Source data are provided as a Source Data file.