The ATR-Chk1 pathway is critical for DNA damage responses and cell-cycle progression. Chk1 inhibition is more deleterious to cycling cells than ATR inhibition, raising questions about ATR and Chk1 functions in the absence of extrinsic replication stress. Here we show that a key role of ATR in S phase is to coordinate RRM2 accumulation and origin firing. ATR inhibitor (ATRi) induces massive ssDNA accumulation and replication catastrophe in a fraction of early S-phase cells. In other S-phase cells, however, ATRi induces moderate ssDNA and triggers a DNA-PK and Chk1-mediated backup pathway to suppress origin firing. The backup pathway creates a threshold such that ATRi selectively kills cells under high replication stress, whereas Chk1 inhibitor induces cell death at a lower threshold. The levels of ATRi-induced ssDNA correlate with ATRi sensitivity in a panel of cell lines, suggesting that ATRi-induced ssDNA could be predictive of ATRi sensitivity in cancer cells.
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