Abstract
Regulation of dNTP pools in an intracellular environment is not only vital for DNA replication but also plays a major role in maintaining genomic stability. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP synthesis and altered regulation of RNR leads to imbalanced dNTP pools. Increased dNTP levels are mutagenic and have the potential to interfere with pathways that are involved in DNA replication, repair and DNA damage control. However, the mechanisms through which altered dNTP pools affect these pathways are poorly understood. Nonetheless, altered dNTP pools have been identified in a number of cellular contexts, including cancer. In order to interpret and analyze the effects of altered dNTP pools, we need quantitative information about dNTP pools in different genetic and environmental contexts in vivo. Here we describe a high-throughput fluorescence-based assay that uses a qPCR-based approach to quantify dNTP levels for use with Saccharomyces cerevisiae extracts.
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
Mathews CK (2006) DNA precursor metabolism and genomic stability. FASEB J 20:1300–1314
Mathews CK (2014) Deoxyribonucleotides as genetic and metabolic regulators. FASEB J 28:3832–3840
Mathews CK (2015) Deoxyribonucleotide metabolism, mutagenesis and cancer. Nat Rev Cancer 15:528
Johansson R, Jonna VR, Kumar R, Nayeri N, Lundin D, Sjöberg B-M, Hofer A, Logan DT (2016) Structural mechanism of allosteric activity regulation in a ribonucleotide reductase with double ATP cones. Structure 24:906–917
Pai CC, Kearsey S (2017) A critical balance: dNTPs and the maintenance of genome stability. Genes 8:57
Aye Y, Li M, Long MJC, Weiss RS (2015) Ribonucleotide reductase and cancer: biological mechanisms and targeted therapies. Oncogene 34:2011–2021
Schmidt TT, Reyes G, Gries K, Ceylan CÜ, Sharma S, Meurer M, Knop M, Chabes A, Hombauer H (2017) Alterations in cellular metabolism triggered by URA7 or GLN3 inactivation cause imbalanced dNTP pools and increased mutagenesis. Proc Natl Acad Sci 114:E4442–E4451
Xu X, Page JL, Surtees JA, Liu H, Lagedrost S, Lu Y, Bronson R, Alani E, Nikitin AY, Weiss RS (2008) Broad overexpression of ribonucleotide reductase genes in mice specifically induces lung neoplasms. Cancer Res 68:2652–2660
Chabes A, Georgieva B, Domkin V, Zhao X, Rothstein R, Thelander L (2003) Survival of DNA damage in yeast directly depends on increased dNTP levels allowed by relaxed feedback inhibition of ribonucleotide reductase. Cell 112:391–401
Watt DL, Buckland RJ, Lujan SA, Kunkel TA, Chabes A (2016) Genome-wide analysis of the specificity and mechanisms of replication infidelity driven by imbalanced dNTP pools. Nucleic Acids Res 44:1669–1680
Buckland RJ, Watt DL, Chittoor B, Nilsson AK, Kunkel TA, Chabes A (2014) Increased and imbalanced dNTP pools symmetrically promote both leading and lagging strand replication infidelity. PLoS Genet 10:e1004846
Wilson PM, LaBonte MJ, Russell J, Louie S, Ghobrial AA, Ladner RD (2011) A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates. Nucleic Acids Res 39:e112
Koc A, Mathews CK, Wheeler LJ, Gross MK, Merrill GF (2006) Thioredoxin is required for deoxyribonucleotide pool maintenance during S phase. J Biol Chem 281:15058–15063. https://doi.org/10.1074/jbc.M601968200
Wheeler LJ, Mathews CK (2012) Effects of a mitochondrial mutator mutation in yeast POS5 NADH kinase on mitochondrial nucleotides. J Biol Chem 287:31218–31222
Soriano-Carot M, Bañó MC, Igual JC (2012) The yeast mitogen-activated protein kinase Slt2 is involved in the cellular response to genotoxic stress. Cell Div 7:1
Prasad Maharjan R, Ferenci T (2003) Global metabolite analysis: the influence of extraction methodology on metabolome profiles of Escherichia coli. Anal Biochem 313:145–154
Villas-Bôas SG, Højer-Pedersen J, Akesson M, Smedsgaard J, Nielsen J (2005) Global metabolite analysis of yeast: evaluation of sample preparation methods. Yeast 22:1155–1169
Eriksson S, Gudas LJ, Ullman B, Clift SM, Martin DW (1981) DeoxyATP-resistant ribonucleotide reductase of mutant mouse lymphoma cells. Evidence for heterozygosity for the protein M1 subunits. J Biol Chem 256:10184–10188
Reichard P, Eliasson R, Ingemarson R, Thelander L (2000) Cross-talk between the allosteric effector-binding sites in mouse ribonucleotide reductase. J Biol Chem 275:33021–33026
Acknowledgments
We are grateful to members of the Surtees lab (past and present) for helpful discussions and input. We thank Jumana Badar for her help in working out calibration curves. N.A.L. is a University at Buffalo Presidential Scholar. This work was supported by the American Cancer Society (RSG-14-235-01 to J.A.S.). J.A.S. is an ACS Research Scholar.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Subramaniam, R., Lamb, N.A., Hwang, Y., Johengen, L., Surtees, J.A. (2019). Extracting and Measuring dNTP Pools in Saccharomyces cerevisiae. In: Balakrishnan, L., Stewart, J. (eds) DNA Repair. Methods in Molecular Biology, vol 1999. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9500-4_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9500-4_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9499-1
Online ISBN: 978-1-4939-9500-4
eBook Packages: Springer Protocols