Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria.

Article Details

Citation

Anderson DD, Quintero CM, Stover PJ

Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria.

Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):15163-8. doi: 10.1073/pnas.1103623108. Epub 2011 Aug 26.

PubMed ID
21876188 [ View in PubMed
]
Abstract

The de novo and salvage dTTP pathways are essential for maintaining cellular dTTP pools to ensure the faithful replication of both mitochondrial and nuclear DNA. Disregulation of dTTP pools results in mitochondrial dysfunction and nuclear genome instability due to an increase in uracil misincorporation. In this study, we identified a de novo dTMP synthesis pathway in mammalian mitochondria. Mitochondria purified from wild-type Chinese hamster ovary (CHO) cells and HepG2 cells converted dUMP to dTMP in the presence of NADPH and serine, through the activities of mitochondrial serine hydroxymethyltransferase (SHMT2), thymidylate synthase (TYMS), and a novel human mitochondrial dihydrofolate reductase (DHFR) previously thought to be a pseudogene known as dihydrofolate reductase-like protein 1 (DHFRL1). Human DHFRL1, SHMT2, and TYMS were localized to mitochondrial matrix and inner membrane, confirming the presence of this pathway in mitochondria. Knockdown of DHFRL1 using siRNA eliminated DHFR activity in mitochondria. DHFRL1 expression in CHO glyC, a previously uncharacterized mutant glycine auxotrophic cell line, rescued the glycine auxotrophy. De novo thymidylate synthesis activity was diminished in mitochondria isolated from glyA CHO cells that lack SHMT2 activity, as well as mitochondria isolated from wild-type CHO cells treated with methotrexate, a DHFR inhibitor. De novo thymidylate synthesis in mitochondria prevents uracil accumulation in mitochondrial DNA (mtDNA), as uracil levels in mtDNA isolated from glyA CHO cells was 40% higher than observed in mtDNA isolated from wild-type CHO cells. These data indicate that unlike other nucleotides, de novo dTMP synthesis occurs within mitochondria and is essential for mtDNA integrity.

DrugBank Data that Cites this Article

Polypeptides
NameUniProt ID
Serine hydroxymethyltransferase, mitochondrialP34897Details
Thymidylate synthaseP04818Details
Dihydrofolate reductaseP00374Details
Dihydrofolate reductase 2, mitochondrialQ86XF0Details