title: Nuclear localization of de novo thymidylate biosynthesis pathway is required to prevent uracil accumulation in DNA.
abstract: 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.
authors: Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853.
authors: Amanda J
authors: AJ
authors: MacFarlane
authors: Graduate Field of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
authors: Donald D
authors: DD
authors: Anderson
authors: Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853.
authors: Per
authors: P
authors: Flodby
authors: Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853.
authors: Cheryll A
authors: CA
authors: Perry
authors: Department of Medicine and Division of Hematology, University of Colorado School of Medicine, Aurora, Colorado 80045.
authors: Robert H
authors: RH
authors: Allen
authors: Department of Medicine and Division of Hematology, University of Colorado School of Medicine, Aurora, Colorado 80045.
authors: Sally P
authors: SP
authors: Stabler
authors: Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853; Graduate Field of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853. Electronic address: pjs13@cornell.edu.
authors: Patrick J
authors: PJ
authors: Stover
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics* / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D020411:Oligonucleotide Array Sequence Analysis; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011743:Pyrimidines / Q000737:chemistry; D016196:S Phase; D013940:Thymidylate Synthase / Q000737:chemistry*; D014018:Tissue Distribution; D019076:Transgenes; D014498:Uracil / Q000737:chemistry*
chemical_list: D011743:Pyrimidines; D014498:Uracil; D011732:Pyridoxal Phosphate; D004247:DNA; D013940:Thymidylate Synthase; D012696:Glycine Hydroxymethyltransferase; C559685:Shmt1 protein, mouse; C030986:pyrimidine