Quantification of uracil in DNA by LC/MS/MS

Genomic uracil was quantified by a method described previously (29), but with some modifications. Cells (5–10 mill) were suspended in a buffer containing 10 mM Tris–HCl (pH 8.0), 10 mM NaCl, 1% SDS, 25 mM DTT, 0.1 mg/ml proteinase K (Worthington Biochemical), 0.1 mg/ml RNase A (Sigma-Aldrich), 50 μM deaminase inhibitor tetrahydrouridine (THU, Merck Millipore) and lysed by passing through 21G and 23G syringe needles followed by incubation at 37 °C for 1 h with 1000 RPM shaking. Proteins and lipids were subsequently extracted from the lysates with phenol:chloroform:isoamyl alcohol (25:24:1, Sigma), followed by two rounds with chloroform:isoamyl alcohol (24:1, Sigma). DNA (nuclear and mitochondrial) in the aqueous phase was precipitated by adding 0.3 volumes 10 M ammonium acetate (pH 7.9) and 1 volume 100% isopropanol. Pellets were washed twice in 70% ethanol. Residual RNA and free nucleotides were removed from the DNA samples by treatment with 50 μg RNaseA in 10 mM ammonium bicarbonate (pH 7.0)/10 mM MgCl₂ for 30 min at 37 °C, followed by a subsequent isopropanol/ammonium acetate precipitation. The DNA pellets were washed twice in 70% ethanol, dissolved in water and the outputs were quantified. UNG-treated genomic DNA was used as a negative control. For these samples, 15 μg pooled genomic DNA from various mouse and human cells was treated with 77 ng of a truncated form of human UNG containing only the catalytic domain (Δ84UNG (21)) in 50 mM tris-HCl (pH 8.0), 1 mM EDTA, 0.5 mg/ml BSA, 1 mM DTT, 200 μM THU, and 8 U HindIII for 1 h, followed by isopropanol/ammonium acetate precipitation.

DNA (5–10 μg) was hydrolyzed to nucleosides by treatment with 0.8 U Nuclease P1 (Sigma-Aldrich), 80 U Benzonase (Santa Cruz Biotechnology), and 7.5 U Antarctic Phosphatase (New England Biolabs) in 50 μl reactions, containing 10 mM ammonium acetate (pH 5.5), 1 mM MgCl₂, 0.1 mM ZnCl₂ and 240 μM THU, for 60 min at 37 °C. The hydrolysis reaction was spiked with a heavy isotope-labeled internal standard, ¹³C¹⁵N₂-dU. Enzymes were then removed from the reactions by adding 3 volumes of ice-cold acetonitrile. The tubes were incubated on ice for 10 min before centrifugation (16 000 g, 30 min, 4 °C) and supernatants were transferred to new tubes and lyophilized until dry.

To separate dU from dC, the samples were dissolved in water and fractionated on an Agilent 1100 HPLC system (with a UV detector set to 260 nm to identify the canonical nucleosides) and a mixed mode Primesep 200 column (2.1 mm × 150 mm, 5 μm, SieLC) kept at 30 °C using a flow rate of 0.4 ml/min and water and acetonitrile as mobile phase, each containing 0.1% formic acid, as the mobile phase. The 12-min-long HPLC gradient was as follows: 5% acetonitrile for 30 s, ramp to 35% acetonitrile by 1.5–2.5 min, and return to 5% acetonitrile by 2.51 min. The dU-containing fractions were collected from 1.6 to 1.7 min and vacuum centrifuged until dry.

Samples were dissolved in water and analyzed by LC/MS/MS using a reverse phase column (2.1 mm × 150 mm, 1.8 μm, EclipsePlusC18 RRHD, Agilent Technologies) kept at 25 °C with a flow rate of 0.3 ml/min on a 1290 Infinity II HPLC coupled to a 6495 Triple Quadrupole mass spectrometer with an electrospray ion source (Agilent Technologies). Water and methanol were used as the mobile phase, each containing 0.1% formic acid. The 13-min-long HPLC gradient was as follows: 5% methanol for 3 min, ramp to 13% methanol by 3.5 min, ramp to 17% methanol by 5.5 min to 7 min, and return to 5% methanol by 8 min. Analysis was performed in positive ionization multiple reaction monitoring mode, using the mass transitions (229.08 to 113.0 Da) for dU and (232.08 to 116.0 Da) for ¹³C¹⁵N₂-dU (internal standard).