ATR-Mediated Global Fork Slowing and Reversal

Cell Reports, Volume 24

Supplemental Information

ATR-Mediated Global Fork Slowing and Reversal Assist Fork Traverse and Prevent Chromosomal Breakage at DNA Interstrand Cross-Links Karun Mutreja, Jana Krietsch, Jeannine Hess, Sebastian Ursich, Matteo Berti, Fabienne K. Roessler, Ralph Zellweger, Malay Patra, Gilles Gasser, and Massimo Lopes

ATR-mediated global fork slowing and reversal assist fork traverse and prevent chromosomal breakage at DNA interstrand crosslinks

Figure S1, related to Figure 1. Refined synthesis of DIG-TMP and its validation in cellular assays for ICL-induced replication stress. (A) Simplified schematic display of the synthesis of DIG-TMP from TMP and DIG-NHS ester (detailed procedure in STAR methods). (B) FACS analysis of cell cycle profile (using propidium iodide stain) in U2OS cells that were left untreated (control) or treated with DIG-TMP (5 μM), TMP (30 nM) in combination with UVA (0.3 J/cm2 or 3 J/cm2). Cells were collected at the indicated time post irradiation. (C) Cell viability of U2OS cells in the absence or presence of the indicated doses of UVA, DIG-TMP and TMP. Cell proliferation was assessed using Cell titer blue assay 72h after UVA irradiation. Red stars, doses selected for functional assays in this study. (D) U2OS cells that were left untreated (control) or treated with DIG-TMP (5μM), TMP (30nM) in combination with UVA (0.3J/cm2; 3J/cm2). Cells were collected at the indicated time post irradiation and counted using a hemocytometer. (E) U2OS cells were left untreated (control) or treated with DIG-TMP (5μM), TMP (30nM) or UVA (0.3J/cm2; 3J/cm2) in indicated combinations. Following irradiation cells 6000 cells were plated in 60 mm dishes for each condition. 7-10 days later colonies were fixed, stained and counted. (F) U2OS cells were left untreated (control) or treated with MMC at mentioned doses for 1h. 8 hours post treatment cells were plated for clonogenic assays, as in (E).

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Figure S2, related to Figure 1. Replication impairment and DDR signalling upon ICL induction. (A-B) Assessment of DNA synthesis rate (EdU incorporation) and DNA damage signalling (γH2AX intensity) in cells that are in S-phase. Where indicated, asynchronously growing U2OS cells were incubated with either DIG-TMP (5μM) or TMP (30nM), and UVA (3J/cm2 or 0.3J/cm2). Then, all samples were pulsed with EdU (10μM) for 30min, stained for γH2AX and analyzed by FACS for EdU incorporation (A) and γH2AX-intensity (B). A minimum of 200 random cells was analyzed per sample. Kurskal-Wallis test (**** = p