Deoxynucleotide-interconverting enzymes and the quantification of deoxynucleoside triphosphates in mammalian cells

Abstract

We have demonstrated that methanol extracts of human cells are heterogeneous with regard to content of dNDP (deoxynucleoside diphosphate) and dNMP (deoxynucleoside monophosphate) kinases. The presence of these enzymes can affect the reliability of techniques used to measure intracellular pools of deoxynucleotides. An optimized extraction procedure and enzymic assay for dNTP species in haematopoietic cells are described which provide sensitivity to measure 0.1-40pmol of dATP, dTTP and dGTP, and 1.0-40pmol of dCTP. The extraction and assay give linear results with (2.5-15)x10(6) nucleated cells and (0.1-1.5)x10(9) red blood cells. Under these conditions, extracts equivalent to ~0.5x10(6) nucleated haematopoietic cells catalyse the phosphorylation of 0-8% of dNDP and dNMP standards to dNTP and incorporate them into deoxynucleotide polymer under circumstances where 100% of an equimolar dNTP standard would be incorporated. By contrast, extracts of 0.4x10(6) HeLa cells totally converted dADP, dTDP and dGDP into dNTP with subsequent polymerization. Conversion of dCDP was somewhat less efficient. The results demonstrate conclusively that the activities of deoxynucleotide interconverting enzymes differ in different types of human cells. They can interfere with assay of nucleotides, but may not do so in many types of cell extracts. In particular, dNTP concentrations can be measured in human haematopoietic cells after extraction with 60% (v/v) methanol and are not artificially elevated by deoxynucleotide interconversions. It is apparent that extraction and assay procedures for measurement of dNTP species should be analysed for each cell type in order to minimize contaminating enzyme activities and ensure accuracy of dNTP quantification.

Macrophage Tropism of HIV-1 Depends on Efficient Cellular dNTP Utilization by Reverse Transcriptase

“…1). Consistent with this function, numerous studies have reported that dNTP contents in cancer cells and transformed cell lines are elevated 3-10-fold compared with normal cells and that dividing cells have higher dNTP contents than non-dividing cells (1)(2)(3)(4)(5)(6). Cellular dNTP levels also fluctuate during the cell cycle and upon DNA damage, when replication and repair DNA synthesis levels are altered (6 -10).…”

HIV-1 Transmission, Replication Fitness and Disease Progression

“…Several studies have identifi ed available dNTP levels as a factor that can affect RT fi delity (Back and Berkhout, 1997;Vartanian et al 1997), yet very little research has been performed with HIV-1 RT fi delity mutations that occur naturally within infected hosts in the absence of HAART, despite the fact that cell types known to be targets for HIV-1 infection (e.g. macrophages, resting T cells, activated T cells) have variable dNTP pools (Traut, 1994;Diamond et al 2004;Hauschka, 1973;Fuller et al 1982;Skoog and Bjursell, 1974;Yao et al 2003). A recent study with SIV RT variants isolated from infected macaques demonstrated rapid selection against an RT variant with higher replication fi delity (Biesinger et al 2008).…”

Genetic Studies on the Role of the Nucleoside Transport Function in Nucleoside Efflux, the Inosine Cycle, and Purine Biosynthesis

“…Measurement of intraceflular nucleotide concentrations. Cells were extracted either by the perchloric acid precipitation method, followed by neutralization with 1 N KOH (34), or with 60% methanol for 24 h in the presence of 50 ,uM sodium tripolyphosphate (8). Nucleotides were separated and quantitated by highpressure liquid chromatography on a Partisil-Strong Anion Exchange (Whatman) column with ammonium phosphate buffers (pH 3.4) at a flow rate of 2.5 ml/min as the mobile phase, 10 mM for the nucleoside monophosphates, and 400 mM for the nucleoside triphosphates.…”