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Mitochondrial de novo thymidylate biosynthesis
Author(s) -
Anderson Donald Dean,
Quintero Cynthia Marcela,
Stover Patrick J
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.892.1
Subject(s) - thymidylate synthase , dihydrofolate reductase , dna synthesis , biology , uracil , biochemistry , dna replication , mitochondrion , biosynthesis , de novo synthesis , serine hydroxymethyltransferase , microbiology and biotechnology , dna , serine , phosphorylation , enzyme , genetics , fluorouracil , chemotherapy
The de novo thymidylate (dTMP) pathway in mammals consists of serine hydroxymethyltransferase (SHMT1 and SHMT2α ), thymidylate synthase (TYMS) and dihydrofolate reductase (DHFR). Recent work in our lab has shown that the de novo dTMP pathway is localized to the nucleus in S and G2/M phases and that nuclear de novo dTMP biosynthesis occurs in purified mouse liver nuclei. We have observed that decreasing levels of SHMT1 in the nucleus results in a depletion of nuclear dTMP synthesis and increases in uracil misincorporation. Increased uracil misincorporation occurred although cytosolic SHMT1 levels were increased six‐fold. This suggests an important role for nuclear de novo dTMP biosynthesis in inhibiting uracil misincorporation. These data suggest that de novo dTMP synthesis for nuclear DNA replication may occur exclusively in the nucleus. In this study, the ability of mitochondria to conduct de novo dTMP synthesis for DNA synthesis in that compartment was investigated. We have now shown the localization of TYMS, DHFR, and SHMT2 in the mitochondria of HepG2 cells using confocal microscopy and IPs of purified HepG2 mitochondrial fractions. DHFR activity was also observed in mitochondrial lysates suggesting that de novo dTMP biosynthesis can also occur in mitochondria. Collectively, these studies demonstrate that cytoplasmic dTMP synthesis is not necessary to support DNA replication.