Quantitation of cellular deoxynucleoside triphosphates

Abstract

Eukaryotic cells contain a delicate balance of minute amounts of the four deoxyribonucleoside triphosphates (dNTPs), sufficient only for a few minutes of DNA replication. Both a deficiency and a surplus of a single dNTP may result in increased mutation rates, faulty DNA repair or mitochondrial DNA depletion. dNTPs are usually quantified by an enzymatic assay in which incorporation of radioactive dATP (or radioactive dTTP in the assay for dATP) into specific synthetic oligonucleotides by a DNA polymerase is proportional to the concentration of the unknown dNTP. We find that the commonly used Klenow DNA polymerase may substitute the corresponding ribonucleotide for the unknown dNTP leading in some instances to a large overestimation of dNTPs. We now describe assay conditions for each dNTP that avoid ribonucleotide incorporation. For the dTTP and dATP assays it suffices to minimize the concentrations of the Klenow enzyme and of labeled dATP (or dTTP); for dCTP and dGTP we had to replace the Klenow enzyme with either the Taq DNA polymerase or Thermo Sequenase. We suggest that in some earlier reports ribonucleotide incorporation may have caused too high values for dGTP and dCTP.

Figure 1.

Enzymatic assay for dCTP. During incubation with a specific oligonucleotide template a DNA polymerase incorporates three molecules of labeled dATP for each dCTP converting them into an acid-insoluble form. The amount of dCTP is calculated from the incorporated radioactivitiy in a standard curve with known amounts of dCTP.

Figure 2.

CTP replaces dCTP during assay with the Klenow DNA polymerase. In the absence of dCTP we incubated CTP (50 or 1000 pmol) with the Klenow enzyme (0.2 or 1 unit) together with labeled dATP (0.25 or 2.5 μM) as described in ‘Materials and Methods’ section and determined the incorporation of radioactivity into the oligonucleotide shown in Figure 1. The recovered acid-insoluble radioactivity was transformed into apparent pmol dCTP (ordinate) as calculated from a parallel standard curve with known amounts of dCTP.

Figure 3.

Effect of rNTPs on the recovery of dNTPs in assays with the Klenow polymerase. (A) CTP (at 100- or 1000-fold higher concentration than dCTP) in dCTP (0.5 or 1 pmol) assay with 0.2 units of enzyme and either 0.25 or 2.5 μM labeled dATP. The ordinate shows the increased apparent recovery of dCTP in the assay, due to misincorporation of CTP in the polymer. (B) GTP in dGTP assay. Conditions as in A. (C) UTP in dTTP assay. We tested two enzyme concentrations (0.2 and 1 unit) at 0.25 μM labeled dATP. Other conditions as in A. Very similar values for dTTP recovery were obtained also with 200- and 2000-fold excess UTP. (D) ATP in dATP assay. Quantitation of dATP (0.5 or 1 pmol) in the presence of 5000 pmol ATP and 0.25 μM labeled dTTP at various enzyme concentrations (0.025, 0.05 or 0.2 units).

Figure 4.

Assays of dCTP and dGTP with Taq DNA polymerase or Thermo Sequenase. (A) Effect of CTP (100- or 1000-fold excess) in dCTP (0.5 or 1 pmol) assay at 0.25 μM dATP with 2 units Taq DNA polymerase. (B) GTP in dGTP assay. Conditions as in A. (C) Thermo Sequenase in dGTP assay. dGTP (0.2–1 pmol) was assayed with 2 units Thermo Sequenase in the presence of 200 pmol GTP. General conditions as in Figure 2.