Identifying the role of genomic uracil in folate deficiency
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Folate deficiency is implicated in deleterious health outcomes including neural tube defects (NTDs), neurodegeneration and colon cancer. Folate is a cofactor for the de novo synthesis of thymidylate (dTMP), and folate deficiency increases DNA damage, alters histone methylation, and increases uracil misincorporation into the genome during DNA synthesis. Excision of uracil in DNA is shown to cause DNA damage, but paradoxically, knockdown of uracil excision enzyme uracil DNA glycosylase (UNG) increases rates of DNA damage. The biological significance of uracil incorporation into DNA in folate deficiency is unknown, but shows tissuespecific differences. It is not known whether uracil in DNA has functional or regulatory consequences, but uracil incorporation in DNA interferes with transcription factor binding in vitro. Uracil levels in nuclear DNA are postulated to be governed by the cellular dTTP/dUTP ratio. Cellular dUTP levels are regulated by the enzyme dUTP diphosphatase (dUTPase), which cleaves two phosphate groups from dUTP, yielding dUMP, which can be used as the substrate for synthesis of dTMP. Cellular dTTP levels are determined by rates of de novo dTMP biosynthesis, which requires folate cofactors and functions as a multienzyme complex in the nucleus. In this study, we sought to determine the contribution of these two activities to uracil accumulation in DNA.The multienzyme complex that synthesizes dTMP is comprised of serine hydroxymethyltransferase (SHMT1 and SHMT2), thymidylate synthase (TYMS), dihydrofolate reductase (DHFR), and methylenetetrahydrofolate dehydrogenase (MTHFD1). Both SHMT and MTHFD1 synthesize 5,10methylenetetrahydrofolate, the cofactor required to synthesize dTMP. SHMT also acts as a scaffold for the multienzyme complex and is critical for recruitment of these proteins at sites of DNA replication and repair. CRISPR knockout of SHMT1 in HeLa cells leads to a 2fold increase in uracil in DNA, compared to control cells. Treatment of HeLa cells with dUTPase siRNA leads to a 2.5fold increase in uracil in DNA and a 9fold increase in gammaH2A.X, a marker of DNA damage.These data suggest that dUTPase prevents genomic uracil incorporation by its enzymatic activity, keeping dUTP concentrations low, and that SHMT1 prevents uracil incorporation by its scaffolding activity, indicating that the uracil incorporation into DNA is regulated beyond the nuclear dTTP/dUTP ratio.Support or Funding InformationNational Institutes of Health Grants DK58144 and HD059120
