Chapter 8 Determination of Deoxyribonucleoside Triphosphate Concentrations in Yeast Cells by Strong Anion-Exchange High-Performance Liquid Chromatography Coupled with Ultraviolet Detection Shaodong Jia, Lisette Marjavaara, Robert Buckland, Sushma Sharma, and Andrei Chabes Abstract DNA polymerase assays are commonly used for the detection of deoxyribonucleoside triphosphates (dNTPs) in biological samples. For better specificity and accuracy, high-performance liquid chromatography (HPLC) methods have been developed for the analysis of the four dNTPs in complex samples. Here we describe a simple method using isocratic strong anion-exchange (SAX) chromatographic separation coupled with ultraviolet detection (UV) for the analysis of the four dNTPs in budding yeast Saccharomyces cerevisiae. This method can be applied to other species of yeast or bacteria. Key words Boronate column separation, Deoxyribonucleoside triphosphates, Liquid chromatography, Strong anion exchange, Budding yeast

1  Introduction Optimal levels of dNTPs are important for accurate DNA replication and repair [1, 2], and insufficient or imbalanced dNTP pools can lead to genetic abnormalities and cell death [3]. Thus, there is a need for accurate and sensitive methods for the measurement of dNTPs in biological samples. Two significant challenges in the analysis of dNTP pools in cells are the relatively low levels of dNTPs and the difficulty in separating them from the more abundant ribonucleoside triphosphates (rNTPs) [4]. The concentrations of rNTPs in budding yeast can be up to 200-fold greater than their corresponding dNTPs [5], and DNA polymerase assays for measuring dNTPs can be hampered by the presence of rNTPs or other metabolites that can interfere with the enzymatic activity of DNA polymerase [6].

Sonya Vengrova and Jacob Dalgaard (eds.), DNA Replication: Methods and Protocols, Methods in Molecular Biology, vol. 1300, DOI 10.1007/978-1-4939-2596-4_8, © Springer Science+Business Media New York 2015

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To overcome this problem, a number of HPLC methods have been developed for analyzing dNTPs in complex sample matrices. A key difference to the DNA polymerase assay is that current HPLC methods commonly apply extraction and purification technologies for the pretreatment of the sample to ensure high accuracy, specificity, and sensitivity. In some cases, a mass spectrometer (MS) has been used with HPLC as the detector to provide even higher specificity and sensitivity [6, 7] but at significantly greater expense. Here, we describe a five-step protocol for dNTP pool measurement, including cell lysis, dNTP extraction with trichloroacetic acid (TCA), Freon/trioctylamine neutralization of the extracts, boronate column separation of dNTPs from rNTPs, and finally HPLC separation coupled with UV detection of dNTPs and rNTPs. This protocol, which is an adaptation of the previously published method [4], allows us to analyze dNTP pool levels from different yeast strains using a simple and economical system.

2  Materials All solutions are prepared in ultrapure water from a Milli-Q water system. No sodium azide is added to the solutions. All solutions are prepared and stored at room temperature unless indicated otherwise. 2.1  Culture and Harvest Yeast Cells

1. YPDA medium: 1 % yeast extract, 2 % peptone, 2 % dextrose, 0.002 % adenine sulfate. 2. Millipore 0.8 μm nitrocellulose filter. 3. Vacuum pump system and adapter.

2.2  Extraction of dNTPs and rNTPs from Yeast Cells

1. Cell lysis solution: 12 % TCA (w/v) in 15 mM MgCl2. To prepare 15 mM MgCl2 stock solution, weigh 0.305 g of MgCl2 ⋅ 6H2O in a weigh boat and dissolve it with 5 mL of water in the boat. Transfer the solution to a clean glass bottle and rinse the boat into the bottle with 10 mL water. Finally, add water to the bottle to make up the volume to 100 mL. Filter the solution into another clean glass bottle (see Note 1). To prepare 25 mL of cell lysis solutions, weigh 3.0 g of TCA in a clean 50 mL conical tube and add 25 mL of 15 mM MgCl2 to the tube (see Note 2). 2. Neutralization solution: 2.8 mL 98 % trioctylamine, 10 mL Freon (1,1,2-trichloro-1,2,2-trifluoroethane), mix well (see Note 3). 3. Liquid nitrogen.

Determination of dNTP concentrations in yeast cells

2.3  Separation of dNTPs from rNTPs with the Boronate Column

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1. Boronate column packing: The Bio-Rad Affi-Gel Boronate media is hydrated and packed into the Bio-Rad Glass Econo-­ column. Store the packed columns at 4 °C (see Note 4). 2. Boronate column washing buffer: 0.1 M sodium boronate solution, pH 8.9. Weigh 3.092 g of boric acid (H3BO3) into a 500 mL beaker with about 490 mL of water. Dissolve the boric acid well with a magnetic stirring bar and adjust the pH to 8.9 with 2 M sodium hydroxide. Transfer the solution to a graduated cylinder and make up to 500 mL with water (see Note 5). 3. Ambic buffer: 50 mM (NH4)2CO3, 15 mM MgCl2. For a 1.0 M ammonium carbonate stock solution, weigh 9.609 g of ammonium carbonate ((NH4)2CO3) into a weigh boat. Dissolve it with 5 mL of water in the boat. Transfer the solution to a clean glass bottle and rinse the boat twice with 5 mL of water into the bottle. Finally, add water to the bottle to make up the volume to 100 mL (see Note 6). For a 150 mM MgCl2 stock solution, weigh 15.25 g of MgCl2 ⋅ 6H2O and dissolve it with 5 mL water in the boat. Transfer the solution to a clean glass bottle and rinse the boat twice with 5 mL water into the bottle. Fill to ~400 mL with water; dissolve by stirring before finally adding water to the bottle to make up the volume to 500 mL. Filter the solution into another clean glass bottle. 4. Mobile phase HPLC buffer: 352.7 mM KH2PO4, pH 3.4, 2.5 % acetonitrile. Weigh 240 g of potassium dihydrogen phosphate (KH2PO4) into a 5 L beaker. Add 4.5 L of water and stir until all of the solid material is dissolved. Slowly add 125 mL of acetonitrile to the beaker in a fume hood, and then adjust the pH to 3.4 with ortho-phosphoric acid. Adjust the volume to 5 L with water and filter the solution into a clean 5 L glass bottle. 5. 1 M ammonium carbonate. 6. 10 mL plastic tubes.

2.4  HPLC Separation of the Four dNTPs and Four rNTPs

1. dNTP standard: 0.1 μM 2′-deoxycytidine-5′-triphosphate (dCTP), 0.1 μM thymidine-5′-triphosphate (dTTP), 0.1 μM 2′-deoxyadenosine-5′-triphosphate (dATP), 0.1 μM 2′-deoxyguanosine-5′-triphosphate (dGTP) in ambic buffer, pH 3–4. A 10 mM mixture of the four dNTPs is prepared from 100 mM stock solutions with sterile water and serially diluted in sterile water to make final stock solution of 10 μM (see Note 7). 12.5 μL from the 10 μM stock solution is mixed with 1237.5 μL of ambic buffer to make the final concentration of 0.1 μM. The sample is then adjusted to pH 3–4 with 6 M hydrochloride (HCl) solution for HPLC analysis (see Note 8).

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2. rNTP standards: cytidine-5′-triphosphate (CTP), uridine-5′triphosphate (UTP), adenosine-5′-triphosphate (ATP), and guanosine-5′-triphosphate (GTP) are prepared the same way as the dNTP standards. 3. Experimental sample for dNTP measurement: 1.25 mL of the eluted solution from the boronate column is adjusted to the pH 3–4 with 6 M HCl solution for HPLC analysis (see Note 9). 4. Experimental sample for rNTP measurement: 23.75 μL of the solution after extraction and neutralization is mixed with 1.226 mL of ambic buffer and adjusted to pH 3–4 with 6 M HCl (see Note 10). 5. A Hitachi HPLC system: Hitachi L-2130 pump, Hitachi L-2200 auto-sampler, Hitachi L-2400 UV detector. 6. A partisphere SAX column (125 mm  ×  4.6 mm, 5 μm, Hichrom, UK). 7. EZChrom Elite software. 8. 1.5 mL glass injection vials.

3  Method 3.1  Culture and Harvest Yeast Cells

1. Yeast cultures are grown in the media of choice, e.g., YPDA medium at 30 °C and 180 rpm. The cell number is determined by measuring the optical density of cell suspensions at 600 nm (OD600) and converting to cell number with a standard curve. 2. At a density from 0.4 to 0.8 × 107 cells/mL, ∼3.7 × 108 cells are collected onto a 0.8 μm nitrocellulose filter. Place the filter over a water vacuum pump adapter attached to a vacuum pump system. Wet the filter with water before screwing the top of the adapter on. Fill the whole adapter with water and then fit a syringe barrel over it (see Note 11). 3. Transfer the required volume of medium with cultured yeast cells into the syringe barrel (see Note 12). Turn on the water pump to start the filtration (see Note 13).

3.2  Extraction of dNTPs and rNTPs from Yeast Cells

1. After filtration, immediately transfer the filter with collected cells is into a 1.5 mL Eppendorf tube containing 0.7 mL cold cell lysis solution and freeze in liquid nitrogen. At this stage, the samples can be stored at −80 °C for a week. Thaw samples on ice (see Note 14). Briefly vortex the tubes for ~30 s and then shake for 15 min on a shaker with vibration in a 4 °C cold room. 2. Place the tubes from the shaker on ice. Take one of the tubes and knock the liquid to the top. Puncture a small hole on the bottom of the tube and insert the tube into an empty 2 mL prechilled Eppendorf tube (see Note 15). Do the same for all

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the remaining tubes. Briefly spin all the liquid down into the empty tubes. Remove the top tubes and centrifuge the lower tubes at 20,000 × g for 1 min to pellet the cell debris. 3. Add 0.8 mL neutralization solution to a new 2.0 mL Eppendorf tube and add 0.7 mL neutralization solution to a new 1.5 mL Eppendorf tube. Maintain the tubes on ice. Transfer the supernatant from step 2 to the first 2.0 mL tube containing 0.8 mL ice-cold neutralization solution and vortex for 20 s (see Note 16). Separate the layers by centrifugation at 20,000 × g for 1 min at 4 °C. Carefully take 0.7 mL of the upper layer and add it to the second, 1.5 mL tube containing 0.7 mL ice-cold neutralization solution. Vortex for 20 s and separate the layers by centrifugation at 20,000 × g for 1 min at 4 °C. Transfer the upper layer to a fresh tube and use for both the boronate column separation procedure and the rNTP measurement. 3.3  Separation of dNTPs from rNTPs with the Boronate Column

The following procedures should be carried out under gravity flow. 1. Wash the column with 12 mL of column washing buffer. 2. Equilibrate the column with 12 mL of ambic buffer (see Note 17). 3. Adjust 475  μL of the solution obtained in step 3 of Subheading  3.2 to pH 8–9 using 25 μl of 1 M ammonium carbonate and load onto the boronate column. Discard the flow through (see Note 18). 4. Transfer the boronate columns to 10 mL plastic tubes that have been prechilled on ice (see Note 19). 5. Elute the dNTPs with 2.5 mL of ambic buffer. 6. Remove columns from collection tubes and wash with 12 mL column washing buffer to remove rNTPs from the column (see Note 20).

3.4  HPLC Analysis of dNTPs and rNTPs

The dNTPs and rNTPs are analyzed in two separate runs. The rNTPs values are used for normalizing possible variation in cell numbers in each corresponding dNTP sample. 1. HPLC separation is carried out in the isocratic elution mode at a 1.0 mL/min flow rate. The separation is carried out at room temperature (see Note 21). 2. Transfer the dNTP standards, rNTP standards, and experimental samples prepared in Subheading 2.4 into 1.5 mL glass injection vials. Place the vials in the 4 °C auto-sampler and inject 1.0 mL onto the SAX column. Chromatograms for the analysis of dNTPs and rNTPs are shown in Figs. 1 and 2, respectively. 3. Integrate the chromatograms and calculate the amount of dNTPs in yeast cells by comparing the peak heights of the experimental samples with those of the standards. The result is then expressed as pmol/108 cells (see Note 22).

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Fig. 2 The analysis of all four rNTPs in the standards mixture solution (a) and a yeast sample (b). Peak identification: 1, CTP; 2, UTP; 3, ATP; 4, GTP

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4  Notes 1. The prepared solution should be filtered with a 0.2 μm membrane filter. The solution is chemically stable, but we suggest that it should be re-filtered or freshly made depending on evidence of microbial growth. 2. Store TCA powder and solution at 4 °C. TCA is a colorless to white crystalline solid with a sharp, pungent odor. Work with TCA in the fume hood. The prepared solution should be used within 4 weeks. 3. While measuring Freon, one has to be careful because it has low viscosity and drips very easily during transfer. The measurement should be carried out in a fume hood, and the tips and tubes used with Freon should be collected separately for disposal. Make solution fresh each time. 4. Before packing the column, block the outlet of the column and add 1.0 mL water to mark a line for the following packing. Discard the water and fill the column with the hydrated gel to the marked line. In our case, the packed column can be regenerated and reused several times before observing rNTP leakage. 5. We usually do not filter this solution. However, if filtration is desired make sure that the membrane filter is stable at high pH. The solution is chemically stable, but we suggest that it should be re-filtered or freshly made depending on the growth of microbes or any change in pH value. 6. We usually do not filter this solution. However, if filtration is desired make sure that the membrane filter is stable at high pH. The solution is chemically stable, but we suggest that it should be freshly made each time from stock components. 7. The stock solutions in water can be stored at −20 °C for 6 months. Although these solutions tolerate several freeze–thaw cycles, we divide the stock solutions into small aliquots and use the solutions for no more than three freeze–thaw cycles. 8. The pH can be easily checked by using pH test paper. After adding 6 M HCl, the samples should be vortexed thoroughly to make sure no air bubbles are sitting in the samples. The air bubbles might affect the baseline stability or suddenly increase the backpressure of the HPLC column. 9. The remaining 1.25 mL of eluted solution from the boronate column can be stored at −20 °C for 1 week in case a repeat analysis is required. After thawing on ice, the pH adjustment procedure is the same as see Note 8. 10. Because the amount of rNTPs is much higher than dNTPs, a sample volume of 23.75 μL can provide a strong signal without interference from the dNTP peaks.

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11. Make sure the adapter is fully filled with water before connecting the syringe to avoid an air-lock. Air will cause the sample to pass through very slowly. 12. A syringe barrel with a volume of 20 mL or 60 mL is commonly used in our lab. 13. The filtration should be as fast as possible [8]. If slow filtration is observed, check for air in the adapter as well as bacterial growth in the culture medium. 14. Keep the tubes on ice. 15. A short hypodermic needle works well for this. 16. Turn the tubes upside down every 5 s to mix the solutions well and do this procedure in the fume hood because of the Freon. 17. We add ambic buffer when no more washing buffer drips down. No extra pressure is needed to completely remove the washing buffer before loading the ambic buffer. 18. No dNTPs or rNTPs were found in the discarded solution when analyzed with HPLC. 19. We made holes in the lids of the tubes to support the boronate columns. 20. The manufacturer suggests low-pH buffers for eluting the bound rNTPs. However, a decreased gel volume was observed when washing with acid solution. Once the pH value of the washing buffer is adjusted to 8.9 (which is the same as ambic buffer), the rNTPs can be quickly removed without shrinking the column. 21. Because we usually analyze a large number of samples, the HPLC system is set to standby mode with a 0.05 mL/min flow rate instead of shutting down the system. The slow flow rate is used to avoid the salt-out phenomenon when using high concentration of salt in the mobile phase. 22. We have carried out method validation including accuracy, precision, and linearity. Because our HPLC system is only used for dNTP analysis, very high intraday and interday reproducibility can be obtained. As a result, method validation is not required for every batch of runs, and instead quality control samples are inserted at the beginning and the end of the sequence to ensure the accuracy of the analysis.

Acknowledgements This work was supported by the Swedish Cancer Society. SJ is a Kempe Foundation scholarship recipient.

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References 1. Reichard P (1988) Interactions between deoxyribonucleotide and DNA synthesis. Annu Rev Biochem 57:349–374 2. Kunz BA, Kohalmi SE, Kunkel TA, Mathews CK, McIntosh EM, Reidy JA (1994) Interna­ tional Commission for Protection Against Environmental Mutagens and Carcinogens. Deoxyribonucleoside triphosphate levels: a critical factor in the maintenance of genetic stability. Mutat Res 318:1–64 3. Kumar D, Viberg J, Nilsson AK, Chabes A (2010) Highly mutagenic and severely imbalanced dNTP pools can escape detection by the S-phase checkpoint. Nucleic Acids Res 38:3975–3983 4. Shewach DS (1992) Quantitation of deoxyribonucleoside 5′-triphosphates by a sequential boronate and anion-exchange high-pressure liquid chromatographic procedure. Anal Biochem 206: 178–182 5. Nick McElhinny SA, Watts BE, Kumar D, Watt DL, Lundström E-B, Burgers PMJ, Johansson

E, Chabes A, Kunkel TA (2010) Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases. Proc Natl Acad Sci U S A 107:4949–4954 6. Zhang W, Tan S, Paintsil E, Dutschman GE, Gullen EA, Chu E, Cheng Y-C (2011) Analysis of deoxyribonucleotide pools in human cancer cell lines using a liquid chromatography coupled with tandem mass spectrometry technique. Biochem Pharmacol 82:411–417 7. Cohen S, Megherbi M, Jordheim LP, Lefebvre I, Perigaud C, Dumontet C, Guitton J (2009) Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem mass spectrometry. J Chromatogr B 877:3831–3840 8. Olempska-Beer Z, Freese EB (1984) Optimal extraction conditions for high-performance liquid chromatographic determination of nucleotides in yeast. Anal Biochem 140: 236–245

Determination of deoxyribonucleoside triphosphate concentrations in yeast cells by strong anion-exchange high-performance liquid chromatography coupled with ultraviolet detection.

DNA polymerase assays are commonly used for the detection of deoxyribonucleoside triphosphates (dNTPs) in biological samples. For better specificity a...
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