Alterations of bases in DNA constitute a major source of genomic instability. It is believed that base alterations trigger base excision repair (BER), generating DNA repair intermediates interfering with DNA replication. Here, we show that genomic uracil, a common type of base alteration, induces DNA replication stress (RS) without being processed by BER. In the absence of uracil DNA glycosylase (UNG), genomic uracil accumulates to high levels, DNA replication forks slow down, and PrimPol-mediated repriming is enhanced, generating single-stranded gaps in nascent DNA. ATR inhibition in UNG-deficient cells blocks the repair of uracil-induced gaps, increasing replication fork collapse and cell death. Notably, a subset of cancer cells upregulates UNG2 to suppress genomic uracil and limit RS, and these cancer cells are hypersensitive to co-treatment with ATR inhibitors and drugs increasing genomic uracil. These results reveal unprocessed genomic uracil as an unexpected source of RS and a targetable vulnerability of cancer cells.

DNA 碱基的改变是基因组不稳定的一个主要来源。据信，碱基改变会触发碱基切除修复（BER），产生干扰 DNA 复制的 DNA 修复中间体。在这里，我们证明基因组尿嘧啶（一种常见的碱基改变类型）会诱导 DNA 复制应激 (RS)，而无需经过 BER 处理。在缺乏尿嘧啶 DNA 糖基化酶 (UNG) 的情况下，基因组尿嘧啶积累到高水平，DNA 复制叉减慢，PrimPol 介导的重新启动增强，在新生 DNA 中产生单链缺口。 UNG 缺陷细胞中的 ATR 抑制会阻止尿嘧啶诱导的间隙修复，从而增加复制叉崩溃和细胞死亡。值得注意的是，一部分癌细胞上调 UNG2 以抑制基因组尿嘧啶并限制 RS，并且这些癌细胞对 ATR 抑制剂和增加基因组尿嘧啶的药物的联合治疗高度敏感。这些结果揭示了未加工的基因组尿嘧啶是 RS 的意外来源和癌细胞的可靶向脆弱性。