Abstract
Because deoxyribonucleoside triphosphates (dNTPs) are the critical substrates for DNA replication and repair, dNTP pools have been studied in context of multiple basic biochemical processes. Over the last 12 years, interest in dNTPs, and specifically the mitochondrial dNTP pools, has expanded to biomedical science because several mitochondrial diseases have been found to be caused by dysfunctions of several enzymes involved in dNTP catabolism or anabolism. Techniques to reliably measure mitochondrial dNTPs should be sensitive and specific to avoid interference caused by the abundant ribonucleotides. Here, we describe detailed protocols to measure mitochondrial dNTPs from two specific samples, cultured skin fibroblasts and mouse liver. The methods can be easily adapted to other types of samples. The protocol follows a polymerase-based method, which is the most widely used approach to measure dNTP pools. Our description is based on the latest update of the technique, which minimizes the potential interference from ribonucleotides.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bourdon A, Minai L, Serre V, Jais JP, Sarzi E, Aubert S, et al. (2007). Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion. Nat Genet 39:776–780.
Mandel H, Szargel R, Labay V, Elpeleg O, Saada A, Shalata A, et al. (2001). The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA. Nat Genet 29:337–341.
Nishino I, Spinazzola A, Hirano M (1999). Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder. Science 283:689–692.
Saada A, Shaag A, Mandel H, Nevo Y, Eriksson S, Elpeleg O (2001). Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy. Nat Genet 29:342–344.
Pohjoismaki JL, Holmes JB, Wood SR, Yang MY, Yasukawa T, Reyes A, et al. (2010). Mammalian mitochondrial DNA replication intermediates are essentially duplex but contain extensive tracts of RNA/DNA hybrid. J Mol Biol 397:1144–1155.
Yasukawa T, Reyes A, Cluett TJ, Yang MY, Bowmaker M, Jacobs HT, et al. (2006). Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand. Embo J 25:5358–5371.
Kaukonen J, Juselius JK, Tiranti V, Kyttala A, Zeviani M, Comi GP, et al. (2000). Role of adenine nucleotide translocator 1 in mtDNA maintenance. Science 289:782–785.
Chen P, Liu Z, Liu S, Xie Z, Aimiuwu J, Pang J, et al. (2009). A LC-MS/MS method for the analysis of intracellular nucleoside triphosphate levels. Pharm Res 26:1504–1515.
Decosterd LA, Cottin E, Chen X, Lejeune F, Mirimanoff RO, Biollaz J, et al. (1999). Simultaneous determination of deoxyribonucleoside in the presence of ribonucleoside triphosphates in human carcinoma cells by high-performance liquid chromatography. Anal Biochem 270:59–68.
Di Pierro D, Tavazzi B, Perno CF, Bartolini M, Balestra E, Calio R, et al. (1995). An ion-pairing high-performance liquid chromatographic method for the direct simultaneous determination of nucleotides, deoxynucleotides, nicotinic coenzymes, oxypurines, nucleosides, and bases in perchloric acid cell extracts. Anal Biochem 231:407–412.
Hennere G, Becher F, Pruvost A, Goujard C, Grassi J, Benech H (2003). Liquid chromatography-tandem mass spectrometry assays for intracellular deoxyribonucleotide triphosphate competitors of nucleoside antiretrovirals. J Chromatogr B Analyt Technol Biomed Life Sci 789:273–281.
Shewach DS (1992). Quantitation of deoxyribonucleoside 5′-triphosphates by a sequential boronate and anion-exchange high-pressure liquid chromatographic procedure. Anal Biochem 206:178–182.
Solter AW, Handschumacher RE (1969). A rapid quantitative determination of deoxyribonucleoside triphosphates based on the enzymatic synthesis of DNA. Biochim Biophys Acta 174:585–590.
Sherman PA, Fyfe JA (1989). Enzymatic assay for deoxyribonucleoside triphosphates using synthetic oligonucleotides as template primers. Anal Biochem 180:222–226.
Bianchi V, Borella S, Rampazzo C, Ferraro P, Calderazzo F, Bianchi LC, et al. (1997). Cell cycle-dependent metabolism of pyrimidine deoxynucleoside triphosphates in CEM cells. J Biol Chem 272:16118–16124.
Dorado B, Area E, Akman HO, Hirano M Onset and organ specificity of Tk2 deficiency depends on Tk1 down-regulation and transcriptional compensation. Hum Mol Genet 20:155–164.
Lopez LC, Akman HO, Garcia-Cazorla A, Dorado B, Marti R, Nishino I, et al. (2009). Unbalanced deoxynucleotide pools cause mitochondrial DNA instability in thymidine phosphorylase-deficient mice. Hum Mol Genet 18:714–722.
Saada A, Ben-Shalom E, Zyslin R, Miller C, Mandel H, Elpeleg O (2003). Mitochondrial deoxyribonucleoside triphosphate pools in thymidine kinase 2 deficiency. Biochem Biophys Res Commun 310:963–966.
Song S, Wheeler LJ, Mathews CK (2003). Deoxyribonucleotide pool imbalance stimulates deletions in HeLa cell mitochondrial DNA. J Biol Chem 278:43893–43896.
Ferraro P, Franzolin E, Pontarin G, Reichard P, Bianchi V (2010). Quantitation of cellular deoxynucleoside triphosphates. Nucleic Acids Res 38:e85.
Fernandez-Vizarra E, Ferrin G, Perez-Martos A, Fernandez-Silva P, Zeviani M, Enriquez JA Isolation of mitochondria for biogenetical studies: An update. Mitochondrion 10:253–262.
Frezza C, Cipolat S, Scorrano L (2007). Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat Protoc 2:287–295.
Pontarin G, Gallinaro L, Ferraro P, Reichard P, Bianchi V (2003). Origins of mitochondrial thymidine triphosphate: dynamic relations to cytosolic pools. Proc Natl Acad Sci USA 100:12159–12164.
Ferraro P, Pontarin G, Crocco L, Fabris S, Reichard P, Bianchi V (2005). Mitochondrial deoxynucleotide pools in quiescent fibroblasts: a possible model for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). J Biol Chem 280:24472–24480.
Ferraro P, Nicolosi L, Bernardi P, Reichard P, Bianchi V (2006). Mitochondrial deoxynucleotide pool sizes in mouse liver and evidence for a transport mechanism for thymidine monophosphate. Proc Natl Acad Sci USA 103:18586–18591.
Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254.
Acknowledgment
Dr. Martí is supported by a grant from the Spanish Instituto de Salud Carlos III (PS09/01591). Dr. Hirano is supported by NIH grants R01 HD056103 (cofunded by NICHD and the NIH Office of Dietary Supplements), R01 HD057543, and RC1 NS070232; MDA grant 115567; and the Marriott Mitochondrial Disorder Clinical Research Fund.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Martí, R., Dorado, B., Hirano, M. (2012). Measurement of Mitochondrial dNTP Pools. In: Wong, Ph.D., LJ. (eds) Mitochondrial Disorders. Methods in Molecular Biology, vol 837. Humana Press. https://doi.org/10.1007/978-1-61779-504-6_9
Download citation
DOI: https://doi.org/10.1007/978-1-61779-504-6_9
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-503-9
Online ISBN: 978-1-61779-504-6
eBook Packages: Springer Protocols