De novo Thymidylate Biosynthesis and Uracil Misincorporation in DNA as Related to Neural Tube Defect and Cancer Outcomes in Mouse Models

Abstract

In the absence of adequate thymidylate (dTMP) synthesis, uracil is misincorporated into DNA during replication and/or repair, as DNA polymerases do not distinguish dTTP from dUTP. Current dogma holds that the dUTP/dTTP ratio determines the rate at which dU is incorporated into DNA. Here we show that the dTMP precursors uridine (U) and deoxyuridine (dU) have distinct fates in DNA. Radioactive tracer studies in HeLa and mouse embryonic fibroblast (MEF) cells demonstrate that exogenous dU is incorporated into DNA as the base dT, and not misincorporated as dU. Exogenous U is incorporated primarily as the pyrmidine base dC. In HeLa cells both exogenous U and dU modestly stimulate proliferation, but U in culture media also increase caspase 3/7 activity, a marker of apoptosis. Exogenous U and dU also have distinct and opposing effects on cytotoxicity of 5‐flourouracil (5‐FU) in HeLa cells.The ApcMin/+ and Shmt1‐/‐ mouse models were used to investigate whether supplementation of folate‐deficient diets with U, dU, or thymidine (T) affected colorectal cancer or NTD incidence, respectively. Unexpectedly, exencephaly in offspring from Shmt1‐/‐ mice was rescued by dietary dU but exacerbated by dietary U. Conversely, U supplementation reduced intestinal tumor number in ApcMin/+ mice by more than 30% relative to the unsupplemented diet, whereas dietary dU did not affect tumor number. Supplemental thymidine did not affect the phenotype of either mouse model. Taken together, these data challenge the assumption that uracil accumulation in DNA is driven simply by the dUMP/dTMP ratio.