De novo Thymidylate Biosynthesis and dUTPase Interact in DNA Synthesis

Abstract

Impaired folate mediated one‐carbon metabolism is implicated in several deleterious health outcomes including neural tube defects, neurodegeneration, and colon cancer. Folate is a cofactor for de novo thymidylate biosynthesis. The de novo thymidylate biosynthesis enzymes serine hydroxymethyl‐transferase (SHMT1), dihydrofolate reductase (DHFR), thymidylate synthase (TYMS), and methylene tetrahydrofolate dehydrogenase (MTHFD1) are SUMOylated, translocate to the nucleus, and form a complex at sites of DNA replication and repair. The nuclear de novo thymidylate biosynthesis complex is unique to mammalian cells and does not form in yeast. Impaired de novo thymidylate biosynthesis leads to increased uracil incorporation into DNA in cell culture models and in mice, subsequently leading to repair‐mediated DNA damage and cell death. In this study, we demonstrate that disruption of de novo thymidylate biosynthesis via SHMT1 CRISPR knock‐out in HeLa cells does not increase uracil levels in DNA. This suggests de novo thymidylate biosynthesis may not be a major determinant of uracil incorporation into HeLa cell nuclear DNA. We also show that dUTPase is a key regulator of uracil incorporation in HeLa cells, and that dUTPase physically interacts with SHMT1 and is a major source of dUMP substrate for de novo thymidylate biosynthesis. These data indicate that dUTPase‐catalyzed dUTP degradation and de novo thymidylate biosynthesis complex interact during DNA synthesis and repair to limit uracil accumulation in DNA.Support or Funding InformationNational Institutes of Health Grants DK58144 and HD059120