Subramaniam Radha, Lamb Natalie A, Hwang Yoonchan, Johengen Lauren, Surtees Jennifer A
Genetics, Genomics and Bioinformatics Program, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY, USA.
Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY, USA.
Methods Mol Biol. 2019;1999:103-127. doi: 10.1007/978-1-4939-9500-4_6.
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.
在细胞内环境中，脱氧核苷三磷酸（dNTP）库的调节不仅对DNA复制至关重要，而且在维持基因组稳定性方面也起着主要作用。核糖核苷酸还原酶（RNR）催化dNTP合成中的限速步骤，RNR调节的改变会导致dNTP库失衡。dNTP水平升高具有致突变性，并有可能干扰参与DNA复制、修复和DNA损伤控制的途径。然而，dNTP库改变影响这些途径的机制尚不清楚。尽管如此，在包括癌症在内的许多细胞环境中都已发现dNTP库发生了改变。为了解释和分析dNTP库改变的影响，我们需要关于体内不同遗传和环境背景下dNTP库的定量信息。在此，我们描述了一种基于荧光的高通量检测方法，该方法使用基于定量聚合酶链反应（qPCR）的方法来定量酿酒酵母提取物中的dNTP水平。
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