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.
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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.
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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
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DOI: https://doi.org/10.1007/978-1-4939-9500-4_6
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