Folate rescues vitamin B^sub 12^ depletion-induced inhibition of nuclear thymidylate biosynthesis and genome instability

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 114; no. 20; p. E4095
• Main Authors: Palmer, Ashley M, Kamynina, Elena, Field, Martha S, Stover, Patrick J
• Format: Journal Article
• Language: English
• Published: Washington National Academy of Sciences 16.05.2017
• Subjects: 5-Methyltetrahydrofolate-homocysteine S-methyltransferase; Accumulation; Anemia; Binding; Biosynthesis; Cell culture; Cofactors; Cytosol; Damage; Deoxyribonucleic acid; Depletion; DNA; DNA biosynthesis; DNA damage; Enrichment; Folic acid; Genomes; Genomic instability; Histone H2A; Homocysteine; Inhibition; Inhibitors; Mathematical models; Methionine; Nitrous oxide; Nuclei; Nuclei (cytology); Robustness; Serine; Stability; Trapping; Vitamin B; Vitamin deficiency
• ISSN: 0027-8424; 1091-6490

Clinical vitamin B^sub 12^ deficiency can result in megaloblastic anemia, which results from the inhibition of DNA synthesis by trapping folate cofactors in the form of 5-methyltetrahydrofolate (5-methylTHF) and subsequent inhibition of de novo thymidylate (dTMP) biosynthesis. In the cytosol, vitamin B^sub 12^ functions in the remethylation of homocysteine to methionine, which regenerates THF from 5-methylTHF. In the nucleus, THF is required for de novo dTMP biosynthesis, but it is not understood how 5-methylTHF accumulation in the cytosol impairs nuclear dTMP biosynthesis. The impact of vitamin B^sub 12^ depletion on nuclear de novo dTMP biosynthesis was investigated in methionine synthase-null human fibroblast and nitrous oxide-treated HeLa cell models. The nucleus was the most sensitive cellular compartment to 5-methylTHF accumulation, with levels increasing greater than fourfold. Vitamin B^sub 12^ depletion decreased de novo dTMP biosynthesis capacity by 5-35%, whereas de novo purine synthesis, which occurs in the cytosol, was not affected. Phosphorylated histone H2AX (γH2AX), a marker of DNA double-strand breaks, was increased in vitamin B^sub 12^ depletion, and this effect was exacerbated by folate depletion. These studies also revealed that 5-formylTHF, a slow, tight-binding inhibitor of serine hydroxymethyltransferase (SHMT), was enriched in nuclei, accounting for 35% of folate cofactors, explaining previous observations that nuclear SHMT is not a robust source of one-carbons for de novo dTMP biosynthesis. These findings indicate that a nuclear 5-methylTHF trap occurs in vitamin B^sub 12^ depletion, which suppresses de novo dTMP biosynthesis and causes DNA damage, accounting for the pathophysiology of megaloblastic anemia observed in vitamin B^sub 12^ and folate deficiency.