ATP-Linked Chimeric Nucleotide as a Specific Luminescence Reporter of Deoxyuridine Triphosphatase

Abstract

Nucleotide surveillance enzymes play important roles in human health, by monitoring damaged monomers in the nucleotide pool and deactivating them before they are incorporated into chromosomal DNA or disrupt nucleotide metabolism. In particular, deamination of cytosine, leading to uracil in DNA and in the nucleotide pool, can be deleterious, causing DNA damage. The enzyme deoxyuridine triphosphatase (dUTPase) is currently under study as a therapeutic and prognostic target for cancer. Measuring the activity of this enzyme is important both in basic research and in clinical applications involving this pathway, but current methods are nonselective, detecting pyrophosphate, which is produced by many enzymes. Here we describe the design and synthesis of a dUTPase enzyme-specific chimeric dinucleotide (DUAL) that replaces the pyrophosphate leaving group of the native substrate with ATP, enabling sensitive detection via luciferase luminescence signaling. The DUAL probe functions sensitively and selectively to quantify enzyme activities in vitro and in cell lysates. We further report the first measurements of dUTPase activities in eight different cell lines, which are found to vary by a factor of 7-fold. We expect that the new probe can be of considerable utility in research involving this clinically significant enzyme.

Figure 1

Structure of the DUAL probe and mechanism for signaling dUTPase activity. Cleavage between α and β phosphate groups nearest deoxyuridine (deaminated base shown in blue) releases ATP, which generates a luminescence signal in the presence of luciferase and luciferin.

Figure 2

Substrate capabilities of DUAL and other ATP-linked nucleotides. A. DUAL is a very poor direct substrate for firefly luciferase, as shown by relative signals of DUAL nucleotide and ATP in equimolar amounts. B. dUTPase enzyme shows high selectivity for the DUAL probe over ATP-linked versions of the four canonical deoxynucleotides (dG, dC, dA, dT).

Figure 3

Enzyme substrate parameters of DUAL. At left is a saturation plot, showing the reaction rate as a function of DUAL concentration; at right are Michaelis–Menten parameters as compared with values reported for the native dUTP substrate.

Figure 4

Performance of the DUAL probe. A. Selectivity of DUAL for the dUTPase enzyme relative to two other nucleotide sanitation enzymes (MTH1, ITPA). B. Sensitivity of DUAL as measured by linearity of luminescence response at increasingly lower enzyme concentrations.

Figure 5

Measuring siRNA-mediated suppression of dUTPase activity in HEK293T cell lysate. A. Raw luminescence intensity measurements of untreated cells vs siRNA-treated cells (24 h). B. dUTPase protein quantity by Western blot, normalized to β-actin.

Figure 6

Relative dUTPase activity in eight cell lines measured by the DUAL assay. A. Relative dUTPase activity as luminescence signals from DUAL activity assay. B. Western blot data showing relative protein quantities. C. Correlation plot showing relatively low/downregulated dUTPase activity (relative to protein quantity) in HEK293T cells and high/upregulated activity in H1299 and A549 cell lines.