FIGURE 3.
Deoxyuridine is incorporated into DNA as dT in HeLa and mouse embryonic fibroblast nuclear DNA and does not increase uracil in DNA. (A) HeLa (black bars, n = 3) or mouse embryonic fibroblast (gray bars, Shmt1+/+, n = 3; hatched bars, Shmt1+/−, n = 3) cells were grown for 4 doublings in DMEM supplemented with 2.35 nM [2,8-3H]-2′-dA and either 33 nM [5-3H]-U or 33 nM [5-3H]-2′-dU. DNA was digested to nucleosides, which were then separated via HPLC. Radioactivity from dT and dA fractions were quantified by using a Beckman LS6500 scintillation counter and shown as the ratio of [3H]-dT to [3H]-dA for each treatment. Data are shown as means ± SDs of n = 3 biological replicates, with significance determined by using a Student’s t test with a Bonferroni correction. (B) HeLa cells (n = 3 per condition) were grown for 4 doublings in folate-deficient DMEM supplemented with either 50 μM U or 50 μM dU both with and without 1 mg/L folic acid. DNA was isolated with a DNeasy kit (Qiagen), uracil was excised by using uracil glycosylase, and uracil was measured with gas chromatography–mass spectrometry. Data are shown as means ± SDs of n = 3 biological replicates, with significance determined by using a Student’s t test with a Bonferroni correction. There were no significant differences between the diets. CPM, counts per minute; dA, deoxyadenosine; DMEM, defined minimal essential medium; dT, thymidine; dU, deoxyuridine; U, uridine.