Nucleotide metabolism supports RNA synthesis and DNA replication to enable cell growth and division. Nucleotide depletion can inhibit cell growth and proliferation, but how cells sense and respond to changes in the relative levels of individual nucleotides is unclear. Moreover, the nucleotide requirement for biomass production changes over the course of the cell cycle, and how cells coordinate differential nucleotide demands with cell cycle progression is not well understood. Here we find that excess levels of individual nucleotides can inhibit proliferation by disrupting the relative levels of nucleotide bases needed for DNA replication and impeding DNA replication. The resulting purine and pyrimidine imbalances are not sensed by canonical growth regulatory pathways like mTORC1, Akt and AMPK signalling cascades, causing excessive cell growth despite inhibited proliferation. Instead, cells rely on replication stress signalling to survive during, and recover from, nucleotide imbalance during S phase. We find that ATR-dependent replication stress signalling is activated during unperturbed S phases and promotes nucleotide availability to support DNA replication. Together, these data reveal that imbalanced nucleotide levels are not detected until S phase, rendering cells reliant on replication stress signalling to cope with this metabolic problem and disrupting the coordination of cell growth and division.

核苷酸代谢支持 RNA 合成和 DNA 复制，从而促进细胞生长和分裂。核苷酸消耗可以抑制细胞生长和增殖，但细胞如何感知和响应个体核苷酸相对水平的变化尚不清楚。此外，生物质生产的核苷酸需求在细胞周期过程中发生变化，并且细胞如何协调不同的核苷酸需求与细胞周期进展尚不清楚。在这里，我们发现过量的单个核苷酸可以通过破坏 DNA 复制所需的核苷酸碱基的相对水平并阻碍 DNA 复制来抑制增殖。由此产生的嘌呤和嘧啶失衡无法被 mTORC1、Akt 和 AMPK 信号级联等典型生长调节途径感知到，从而导致细胞过度生长，尽管增殖受到抑制。相反，细胞依靠复制应激信号在 S 期核苷酸失衡期间生存并从中恢复。我们发现 ATR 依赖性复制应激信号在不受干扰的 S 期被激活，并促进核苷酸可用性以支持 DNA 复制。总之，这些数据表明，直到S期才检测到不平衡的核苷酸水平，使得细胞依赖复制应激信号来应对这种代谢问题，并破坏细胞生长和分裂的协调。