Nuclear Localization of de Novo Thymidylate Biosynthesis Pathway Is Required to Prevent Uracil Accumulation in DNA

• Published in: The Journal of biological chemistry 2011-12, Vol.286 (51), p.44015-44022
• Main Authors: MacFarlane, Amanda J., Anderson, Donald D., Flodby, Per, Perry, Cheryll A., Allen, Robert H., Stabler, Sally P., Stover, Patrick J.
• Format: Article
• Language: English
• Subjects: Active Transport, Cell Nucleus; Animals; Cell Nucleus - metabolism; DNA - genetics; DNA - metabolism; DNA Synthesis; Folate; Glycine Hydroxymethyltransferase - genetics; Glycine Hydroxymethyltransferase - physiology; Metabolism; Mice; Mice, Transgenic; Nucleoside Nucleotide Biosynthesis; Oligonucleotide Array Sequence Analysis; One Carbon Pool; Pyridoxal Phosphate; Pyridoxal Phosphate - metabolism; Pyrimidine; Pyrimidines - chemistry; S Phase; SHMT1; Thymidylate Synthase - chemistry; Tissue Distribution; Transgenes; TYMS; Uracil - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.307629

Uracil accumulates in DNA as a result of impaired folate-dependent de novo thymidylate biosynthesis, a pathway composed of the enzymes serine hydroxymethyltransferase (SHMT), thymidylate synthase (TYMS), and dihydrofolate reductase. In G1, this pathway is present in the cytoplasm and at S phase undergoes small ubiquitin-like modifier-dependent translocation to the nucleus. It is not known whether this pathway functions in the cytoplasm, nucleus, or both in vivo. SHMT1 generates 5,10-methylenetetrahydrofolate for de novo thymidylate biosynthesis, a limiting step in the pathway, but also tightly binds 5-methyltetrahydrofolate in the cytoplasm, a required cofactor for homocysteine remethylation. Overexpression of SHMT1 in cell cultures inhibits folate-dependent homocysteine remethylation and enhances thymidylate biosynthesis. In this study, the impact of increased Shmt1 expression on folate-mediated one-carbon metabolism was determined in mice that overexpress the Shmt1 cDNA (Shmt1tg+ mice). Compared with wild type mice, Shmt1tg+ mice exhibited elevated SHMT1 and TYMS protein levels in tissues and evidence for impaired homocysteine remethylation but surprisingly exhibited depressed levels of nuclear SHMT1 and TYMS, lower rates of nuclear de novo thymidylate biosynthesis, and a nearly 10-fold increase in uracil content in hepatic nuclear DNA when fed a folate- and choline-deficient diet. These results demonstrate that SHMT1 and TYMS localization to the nucleus is essential to prevent uracil accumulation in nuclear DNA and indicate that SHMT1-mediated nuclear de novo thymidylate synthesis is critical for maintaining DNA integrity. Background: S phase nuclei contain the thymidylate synthesis pathway. Results: Mice overexpressing a Shmt1 transgene exhibit elevated expression of SHMT1 and TYMS, impaired nuclear localization of the thymidylate biosynthesis pathway, and elevated uracil in DNA. Conclusion: SHMT1 and TYMS localization to the nucleus is essential to prevent uracil accumulation in DNA. Significance: SHMT1-mediated nuclear de novo thymidylate synthesis is critical for maintaining DNA integrity.