Journal Article

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

Palmer, Ashley M., Kamynina, Elena, Stover, Patrick J., Field, Martha S.

2017

Overview

Vitamin B 12 deficiency causes hematological and neurological pathologies by impairing DNA synthesis. The nucleus is shown to be highly sensitive to 5-methyltetrahydrofolate (5-methylTHF) accumulation induced by vitamin B 12 depletion in the cytosol, leading to impaired nuclear de novo thymidylate synthesis and genome instability. These effects of the 5-methylTHF trap are exacerbated by folate depletion in nitrous oxide-treated HeLa cells and methionine synthase loss-of-function fibroblasts. These results further our understanding of mechanisms underlying vitamin B 12 –folate interrelationships in pathologies, including megaloblastic anemia and potentially neural tube defects. Clinical vitamin B 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 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 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 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 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 12 depletion, which suppresses de novo dTMP biosynthesis and causes DNA damage, accounting for the pathophysiology of megaloblastic anemia observed in vitamin B 12 and folate deficiency.

• Subject: DNA Damage / Female / Fibroblasts - metabolism / Genomic Instability / Glycine Hydroxymethyltransferase - metabolism / HeLa Cells / Humans / Infant / Tetrahydrofolates - metabolism / Thymidine Monophosphate - biosynthesis / Vitamin B 12 Deficiency - genetics / Vitamin B 12 Deficiency - metabolism