Yeast were grown to logarithmic phase, diluted to 5 × 106 cells/ml into media +/− 150 µM 5-FU, and incubated for varying times (wt, 6 h, ung1, 6 h, apn1, 3 h) at 30°C with agitation in order to kill 70–90% of the wt and apn1 cells and achieve maximum cell killing with the ung1 yeast at this 5-FU concentration. Small aliquots were diluted and plated, and the remaining cells were spun down, lysed with lyticase (Sigma) and genomic DNA was isolated using Genomic-tips (Qiagen). Each sample (3 µg) was digested overnight at room temperature with 3 nM Escherichia coli uracil DNA glycosylase (Ung) and 3 nM human AP endonuclease (Ape1) in buffer containing 50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 50 mM NaCl and 10 mM MgCl2. Half of each reaction was run on a 0.8% agarose gel, which was stained with 1 µg/ml EtBr prior to imaging.
For GC-MS, yeast cells were grown to logarithmic phase and diluted to 5 × 106 cells/ml into 5-FU to achieve 70–90% cell killing across all samples (wt, 150 µM 5-FU for 6 h, ung1, 3 mM 5-FU for 4 h, and apn1, 150 µM 5-FU for 3 h). Aliquots were diluted and plated, and the genomic DNA was purified and digested with 10 nM Ung overnight at room temperature in buffer containing 10 mM Tris–HCl, pH 7.5, 2.5 mM MgCl2 and 25 mM NaCl. Following digestion, aliquots of uracil-13C4, 15N2 and 5-FU-13C4, 15N2 were added as internal standards (stable isotope labeled U and 5-FU were purchased from Cambridge Isotope Laboratories). Then the DNA was precipitated with 70% EtOH, centrifuged and the supernatant and pellet fractions were separated. Ethanol was removed from supernatant fractions under vacuum in a SpeedVac at room temperature. Aqueous supernatant fractions were frozen in liquid nitrogen, lyophilized to dryness for 18 h, and then trimethylsilylated and analyzed by GC-MS as described (32 (link)–34 (link)). For identification and quantification, selected-ion monitoring was used to monitor the characteristic ions of the trimethylsilyl derivatives of uracil (m/z 256 and m/z 241), uracil-14C4,15N2 (m/z 262 and m/z 247), 5-FU (m/z 274 and m/z 259) and 5-FU-13C4,15N2 (m/z 280 and m/z 265) during GC/MS analysis. (In each case, the first ion is the molecular ion and the second one is the ion that results from the loss of methyl radical from the molecular ion.)
For RNA 5-FU incorporation analysis, wt yeast were grown to logarithmic phase, diluted to 5 × 106 cells/ml into YEPD +/− one EC50 of 5-FU, and shaken at 30°C for 6 h. Cellular RNA was then isolated using the RNeasy Kit (Qiagen). RNA (10 µg) was digested to nucleosides using mung bean nuclease (10 U) and calf intestinal phosphatase (10 U) overnight at 37°C in buffer containing 10 mM Tris–HCl, pH 7.9, 10 mM MgCl2, 50 mM NaCl and 1 mM DTT (all reagents from New England Biolabs). High-performance liquid chromatography (HPLC) was carried out using an analytical Aqua reversed-phase C18 column (Phenomenex) and isocratic elution with 3% acetonitrile in aqueous 0.1 M TEAA, pH 7.0, at a flow rate of 1 ml/min.
For GC-MS, yeast cells were grown to logarithmic phase and diluted to 5 × 106 cells/ml into 5-FU to achieve 70–90% cell killing across all samples (wt, 150 µM 5-FU for 6 h, ung1, 3 mM 5-FU for 4 h, and apn1, 150 µM 5-FU for 3 h). Aliquots were diluted and plated, and the genomic DNA was purified and digested with 10 nM Ung overnight at room temperature in buffer containing 10 mM Tris–HCl, pH 7.5, 2.5 mM MgCl2 and 25 mM NaCl. Following digestion, aliquots of uracil-13C4, 15N2 and 5-FU-13C4, 15N2 were added as internal standards (stable isotope labeled U and 5-FU were purchased from Cambridge Isotope Laboratories). Then the DNA was precipitated with 70% EtOH, centrifuged and the supernatant and pellet fractions were separated. Ethanol was removed from supernatant fractions under vacuum in a SpeedVac at room temperature. Aqueous supernatant fractions were frozen in liquid nitrogen, lyophilized to dryness for 18 h, and then trimethylsilylated and analyzed by GC-MS as described (32 (link)–34 (link)). For identification and quantification, selected-ion monitoring was used to monitor the characteristic ions of the trimethylsilyl derivatives of uracil (m/z 256 and m/z 241), uracil-14C4,15N2 (m/z 262 and m/z 247), 5-FU (m/z 274 and m/z 259) and 5-FU-13C4,15N2 (m/z 280 and m/z 265) during GC/MS analysis. (In each case, the first ion is the molecular ion and the second one is the ion that results from the loss of methyl radical from the molecular ion.)
For RNA 5-FU incorporation analysis, wt yeast were grown to logarithmic phase, diluted to 5 × 106 cells/ml into YEPD +/− one EC50 of 5-FU, and shaken at 30°C for 6 h. Cellular RNA was then isolated using the RNeasy Kit (Qiagen). RNA (10 µg) was digested to nucleosides using mung bean nuclease (10 U) and calf intestinal phosphatase (10 U) overnight at 37°C in buffer containing 10 mM Tris–HCl, pH 7.9, 10 mM MgCl2, 50 mM NaCl and 1 mM DTT (all reagents from New England Biolabs). High-performance liquid chromatography (HPLC) was carried out using an analytical Aqua reversed-phase C18 column (Phenomenex) and isocratic elution with 3% acetonitrile in aqueous 0.1 M TEAA, pH 7.0, at a flow rate of 1 ml/min.
