SRA

The ATR kinase, which coordinates cellular responses to DNA replication stress, is also essential for the proliferation of normal unstressed cells. Although its role in the replication stress response is well defined, the mechanisms by which ATR supports normal cell proliferation remain elusive. Here, we show that Atr is dispensable for the viability of G0-arrested naïve B cells. However, upon cytokine-induced proliferation, Atr-deficient B cells initiate DNA replication efficiently in early S phase, but by mid-S phase they display dNTP depletion, fork stalling, and replication failure. Nonetheless, productive DNA replication can be restored in Atr-deficient cells by pathways that suppress origin firing, such as downregulation of CDC7 and CDK1 kinase activities. Together, these findings indicate that ATR supports the proliferation of normal unstressed cells by tempering the pace of origin firing during the early S phase to avoid exhaustion of dNTPs and other replication factors. Overall design: We set up a genome-wide CRISPR-Cas9 screen targeting 18,424 different genes with 4-5 gRNAs each in v-abl transformed wild-type mouse B cells treated with the IC90 dose of 1 of 2 ATR inhibitors (VE821 or AZD6738) or a Chk1 inhibitor (LY2603618), in triplicates, for 6 days and compared gRNA representation to DMSO-treated controls.