ATR kinase supports normal proliferation in the early S phase by preventing replication resource exhaustion [HTGTS]

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. To determine how ATR protein affects the repair phase of CSR, we analyzed thousands of CSR and internal deletion junctions from activated control, CD21-Cre AtrC/- and CD21-Cre AtrC/KD B cells using the high-throughput genome-wide translocation sequencing (HTGTS) technology. Cells were cultured in RPMI medium plus IL-4 and anti-CD40 cytokines for 4 days and total genomic DNA was collected for HTGTS.