Saxena, S.; Patel, P. S.; Nabel, C. S.; Kawale, A. S.; Crosby, C. R.; Vander Heiden, M. G.; Hata, A. N.; Zou, L.

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 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 repair of uracil-induced gaps, increasing replication fork collapse and cell death. Notably, a subset of cancer cells harboring high levels of genomic uracil upregulate UNG2 to 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.