ABSTRACT Most chemotherapies target DNA replication, and their efficacy depends on the DNA damage response that integrates DNA repair with the cell cycle. Widely used standard-of-care antimetabolites inhibit thymidylate synthase and increase the incorporation of deoxyuridine (dU) into DNA by polymerases. dU contamination in DNA is limited by the DDR kinase ATR that induces cell cycle checkpoints and reduces the rate of DNA replication. This project investigates the impact of dU contamination on innate immune responses. ATR inhibitors (ATRi) induce origin firing across active replicons and cause ribonuclease reductase degradation in otherwise unperturbed cells. This increases both the amount of DNA replication and the amount of free dUTP in cells. Thus, ATRi increase the incorporation of dU into DNA by polymerases. Since ATRi also inhibit cell cycle checkpoints, ATRi induce more dU contamination than antimetabolites. Our preliminary data show that ATRi-induced IFN-β is dependent on uracil DNA glycosylase (UNG) which removes dU from DNA. Our data are consistent with a model in which UNG-dependent base excision repair (BER) removes dU from DNA and generates cytoplasmic dsDNA that induces IFN-α/β. Unexpectedly, however, we did not observe a difference in the amount of cytoplasmic dsDNA in wild-type (WT) and UNG knockout (∆UNG) cells after treatment with ATRi. We reasoned that cytoplasmic DNA in ∆UNG cells may not be recognized by pattern recognition receptors because it is dA:dU-rich. Indeed, replacing just 20 thymidine bases (T) with dU in a 200 bp dsDNA fragment significantly decreased IFN-β induction in cells. Our data suggest that the integrity of dA:T base pairs is critical for the IFN-α/β response in cells treated with chemotherapy and ionizing radiation (XRT). cGAS recognizes DNA in a sequence-independent manner. However, dsDNA fragments are transcribed in the cytoplasm by RNA Polymerase III (Pol III) to generate RNA that is recognized by RIG1. It was recently sh