Uracil incorporation into DNA is implicated in several deleterious health outcomes including megaloblastic anemia, neural tube defects (NTDs), and neurodegeneration. Uracil incorporation can induce DNA strand breaks and apoptosis. There is emerging evidence that uracil incorporation also leads to transcriptional stalling, gene activation, and necrosis. Accumulation of uracil into DNA is influenced by cytosine deamination, de novo thymidylate biosynthesis, salvage thymidylate biosynthesis, dUTPase, and DNA repair initiated by uracil DNA glycosylases (UDGs). Secondary roles of UDGs in DNA demethylation and transcriptional repression suggest that genomic uracil may impact gene expression. 5-fluorouracil (5-FU) is a chemotherapeutic which targets the de novo thymidylate biosynthesis enzyme thymidylate synthase (TYMS). The mechanisms by which 5-FU inhibits cell growth, and the mechanisms of resistance, are reviewed. Nuclear localization of de novo thymidylate biosynthesis enzymes is predictive of chemoresistance. Because SUMOylation is critical for nuclear localization of the de novo thymidylate biosynthesis enzymes, targeting of SUMO may augment antifolate-based treatments. These studies examined the effects of SHMT1 ablation on uracil incorporation, salvage thymidylate biosynthesis, dUTPase, and uracil repair in HeLa and A549 cells. Notably, SHMT1 KO in HeLa cells is associated with unchanged levels of uracil in DNA, increased salvage thymidylate synthesis, and increased DNA repair. Conversely, treatment of A549 cells with SHMT1 siRNA was associated with elevated uracil in DNA, decreased thymidylate salvage synthesis and decreased dUTPase expression. A novel assay was developed to measure uracil excision activity of cell lysates. Treatment of A549 cells with the apoptotic drugs, etoposide and cisplatin, induced loss of thymidine kinase 1 (TK1) and dUTPase protein levels; this suggests that the loss of these enzymes seen in SHMT1 siRNA treatment is a result of apoptosis. Drug treatments led to increased uracil in DNA and apoptosis in A549 cells, both rescued by dT supplementation. Conversely, treatment of HeLa cells with etoposide or cisplatin induced apoptosis, without increasing uracil levels in DNA, or decreasing TK1 or dUTPase expression. This indicates uracil incorporation into DNA is critical to drug-mediated apoptosis in A549, but not HeLa cells. Loss of TK1 and dUTPase, and increased uracil incorporation induced by cisplatin treatment may explain the synergistic mechanism by which the combination chemotherapy of 5-fluorouracil and cisplatin occurs.