Document Detail


Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria.
MedLine Citation:
PMID:  21876188     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Donald D Anderson; Cynthia M Quintero; Patrick J Stover
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2011-08-26
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  108     ISSN:  1091-6490     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  2011 Sep 
Date Detail:
Created Date:  2011-09-14     Completed Date:  2011-11-09     Revised Date:  2013-06-27    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  United States    
Other Details:
Languages:  eng     Pagination:  15163-8     Citation Subset:  IM    
Affiliation:
Division of Nutritional Sciences, Cornell University, 127 Savage Hall, Ithaca, NY 14853, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
Biosynthetic Pathways*
CHO Cells
Cricetinae
Cricetulus
DNA, Mitochondrial / metabolism
Gene Expression Regulation
Glycine / metabolism
Humans
Mammals / metabolism*
Mitochondria / enzymology,  metabolism*
Molecular Sequence Data
Protein Transport
Sequence Alignment
Tetrahydrofolate Dehydrogenase / chemistry,  genetics,  metabolism
Thymidine Monophosphate / biosynthesis
Thymidylate Synthase / metabolism
Thymine Nucleotides / biosynthesis*
Uracil / metabolism
Grant Support
ID/Acronym/Agency:
DK58144/DK/NIDDK NIH HHS; R37 DK058144/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
0/DNA, Mitochondrial; 0/Thymine Nucleotides; 365-07-1/Thymidine Monophosphate; 365-08-2/thymidine 5'-triphosphate; 56-40-6/Glycine; 66-22-8/Uracil; EC 1.5.1.3/Tetrahydrofolate Dehydrogenase; EC 2.1.1.45/Thymidylate Synthase
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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