Phosphorothioated DNA Is Shielded from Oxidative Damage

Bacterial strains, plasmids, and culture conditions.Bacterial strains and plasmids used in this work are listed in Table 1, and most have been described in previous publications (52). Specifically, the dndC C39A, C146A, C262A, C273A, C280A, and C283A mutants were generated using gene-specific primers (Table 2) to amplify the entire pDndBCDE_H plasmid. Primers possessed a 19-nt 5′ primer extension to facilitate cyclization of the PCR product in E. coli, and the 3-bp dndC modified sequence used in the creation of the dndC mutant was placed in the middle of the 5′ primer extension. PCR products were digested with DpnI (Thermo Fisher Scientific, Inc., MA, USA) for 4 h to remove the methylated plasmid template, and the resulting unmethylated PCR amplification product was purified using an AxyPrep DNA gel extraction kit (Axygen, Corning, NY, USA). PCR product identities were confirmed by size determination in 1% agarose gels using a DNA size marker for comparison (Invitrogen 1 Kb Plus DNA ladder; Thermo Fisher Scientific, Inc.). Once product identity had been confirmed, the desired fragment was stabilized by transformation into E. coli strain DH10B using the protocol of Inoue et al. (53). For heterologous gene expression experiments, the dndC mutant plasmid was isolated and purified using an AxyPrep plasmid extraction kit (Axygen Corning) and transformed into E. coli strain BL21(DE3)/plysS (Agilent Technologies, CA, USA).

Bacterial cells were routinely cultured in Luria broth medium (10 g/liter tryptone, 5 g/liter yeast extract, 10 g/liter sodium chloride) supplemented with 50 μg ml⁻¹ kanamycin when needed for strain selection. Specifically, E. coli strains used in protein expression experiments were grown in 1 liter of LB medium containing 50 μg ml⁻¹ kanamycin at 37°C with shaking at 250 rpm until the culture optical density at 600 nm (OD₆₀₀) reached 0.6. At this point gene expression was induced by addition of isopropyl-d-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, and the culture was allowed to incubate for a further 24 h at 16°C. After this, cells were harvested by centrifugation at 6,000 × g for 20 min and, if not used immediately in protein purification, stored flash-frozen at −80°C.

Protein expression and purification.(i) N-terminal His-tagged cysteine desulfurase, IscS. The recovery and purification of N-terminal His-tagged cysteine desulfurase followed the general protocol described by Xiong et al. (52) in which 1 g of cell pellet or frozen cell pellet was resuspended in 50 ml of lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 5% glycerol) in a sterile 50-ml polypropylene centrifuge tube (Corning, NY, USA) and sonicated on ice at 100 W using a 6.4-mm-diameter probe for a total of 10 min with 2-s bursts and 4-s intervals (Qsonica Sonicator Q500: Qsonica Sonicators, Newtown, Canada). Subsequently, insoluble cell debris was removed by centrifugation at 18,000 × g for 20 min, and the supernatant was then applied to a 1-ml Ni-nitrilotriacetic acid (NTA) column (GE Life Sciences, MA, USA), which was washed with 10 ml of Ni wash buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 40 mM imidazole, and 5% glycerol). Bound protein was then eluted with 5 ml of Ni elution buffer 2 (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 500 mM imidazole, and 5% glycerol), and 2.5 ml of the eluate was desalted using a PD-10 desalting column (GE Life Sciences, MA, USA) with 3.5 ml of storage buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5% glycerol) according to the manufacturer’s instructions. The desalted protein solution was then subjected to a Bradford assay (54).

(ii) C-terminal His-tagged Dnd protein complex DndCDE or DndCDE_Pf0. The recovery and purification of DndCDE (or DndCDE_Pf0) protein complex involved taking 5 g of cell pellet or frozen cell pellet and resuspending the cells in 25 ml of lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 5% glycerol) in a sterile 50-ml polypropylene centrifuge tube (Corning). The cell suspension was then treated in an identical manner to that described for the extraction of IscS protein except that the entire volume eluted from the Ni-NTA column was loaded onto a 1-ml heparin high-performance column (HiTrap; GE Life Sciences), which was washed with five column bed volumes (CBV) of heparin wash buffer 1 (20 mM Tris-HCl, pH 8.0, 25 mM NaCl, and 5% glycerol) followed by five CBV of heparin wash buffer 2 (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 5% glycerol). Protein was subsequently eluted with 5 ml of heparin elution buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 5% glycerol), and the eluate was subjected to size exclusion chromatography on an ÄKTA fast protein liquid chromatography system (GE Life Sciences) using a preequilibrated Superdex 200 10/30 column (GE Life Sciences) in heparin wash buffer 2. Eluate was collected in 1-ml fractions which were monitored for their absorbance at a wavelength of 280 nm (λ₂₈₀). Pooled peak fractions (3 ml) with the highest absorbance reading were collected and concentrated to 0.5 ml using a centrifugal filter at 8,000 × g and 4°C for 2 h (Amicon Ultra, Merck, Darmstadt, Germany). Concentrated, purified protein was then dialyzed in 1 liter of storage buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5% glycerol) for 6 h at 4°C, and the resulting DndCDE protein was quantified by Bradford assay (54).

(iii) Gradient native gel electrophoresis detection of both native and cross-linked DndCDE. The preparation of cross-linked DndCDE was achieved by treatment with 1 mM disuccinimidyl suberate (DSS) (Thermo Fisher Scientific, Inc., MA, USA) for 20 min at room temperature (55).

The 4% to 20% gradient native gel was prepared according to the protocol described by Wittig et al. (56). Basically, 4% acrylamide and 20% acrylamide were prepared separately on ice. Four percent acrylamide contained 1.2 ml of acrylamide-bisacrylamide mixture (total concentration of the monomers acrylamide and bisacrylamide [T], 49.5%; concentration of the cross-linker [C] relative to the total concentration 3% [Solarbio, Beijing, China]), 5 ml of 3× Tris-glycine buffer (75 mM Tris base and 57.6 mM glycine), 100 μl of 10% (wt/vol) ammonium persulfate (APS), 10 μl of tetramethylethylenediamine (TEMED), and 8.7 ml of water. Twenty percent acrylamide contained 6 ml of acrylamide-bisacrylamide mixture (49.5% T, 3% C; Solarbio, Beijing, China), 5 ml of 3× Tris-glycine buffer (75 mM Tris base and 57.6 mM glycine), 3 ml of glycerol, 75 μl of 10% (wt/vol) APS, 7.5 μl of TEMED, and 918 μl of water. Gradient was achieved by adding 20% acrylamide into 4% acrylamide while the latter solution was also pumped into the vertical electrophoresis apparatus with a peristaltic pump. Two micrograms of protein sample was used for detection. One microgram of ferritin protein (A600457-0500; Sangon Biotech, Shanghai, China) was used as the size marker.

(iv) In vitro anaerobic enzymatic formation of active Fe-S cluster Dnd protein complex, DndCDE-FeS. The formation of active DndCDE-FeS was performed under anaerobic conditions (nitrogen) in a glove box (Coy Laboratory Product, Inc., MI, USA) in a reaction catalyzed by IscS (23). Typically, 20 μM DndCDE was incubated for 30 min at room temperature under aerobic conditions with 1 mM α,α′-dipyridyl and 2 mM dithionite, after which the reaction mixture was desalted using a PD-10 desalting column (GE Life Sciences) according to the manufacturer’s instructions, and 3.5 ml of eluate was concentrated to approximately 900 μl by centrifugation in a 0.5-ml centrifugal filter at 8,000 × g and 4°C for 2 h (Amicon Ultra; Merck, Darmstadt, Germany). From this point onward, all other steps were undertaken under anaerobic conditions. The concentrated protein (20 μM) was then mixed with 20 μM purified IscS protein, 7.5 mM dithiothreitol, 0.5 mM Fe(NH₄)₂(SO₄)₂, and 0.5 mM cysteine in storage buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5% glycerol) in a total volume of 1 ml, and the sample tubes were sealed and incubated at 4°C overnight. Subsequently, the mixture was centrifuged at 15,000 × g for 10 min, and the supernatant was desalted by passage through a PD-10 desalting column (GE Life Sciences) and concentrated by centrifugation in a 0.5-ml centrifugal filter (Centricon Plus-20 [PL-10]; Millipore, MA, USA) at 4°C. Finally, the tube containing DndCDE-FeS was sealed with Parafilm before being removed from the glove box. The protein was used in the experiments immediately.

UV-Vis and EPR analysis of DndCDE and DndCDE-FeS.The UV-visible light (UV-Vis) absorption characteristics of the DndCDE protein and its Cys mutants were investigated using a microtiter plate reader (BioTek Synergy 2; BioTek Instruments, Inc., VT, USA) equipped with Gen5 software. Briefly a 200-μl sample containing 130 μM, 20 mg ml, of the protein complex in storage buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5% glycerol) in a 96-well microplate (3599; Corning, NY, USA) was scanned for its absorption between λ₂₉₀ and λ₇₀₀. This concentration of protein complex was used as it had previously been determined to be the minimum required to produce a visible spectroscopic peak for the Fe-S cluster.

Electron spin resonance (EPR) spectrum acquisitions were performed under aerobic conditions, and in these experiments 130 μM DndCDE in storage buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5% glycerol) was used. Protein was supplemented with either sodium dithionite (2 mM) or dithiothreitol (2 mM) where appropriate. EPR spectra were acquired using a Bruker EMX Plus 10/12 spectrometer (Bruker Co., Ltd., Bremen, Germany) equipped with an Oxford ESR910 liquid helium continuous-flow cryostat (Oxford Instrument Co., Ltd., Oxfordshire, UK) and Bruker WINEPR software at the National High Magnetic Field Laboratory of the Chinese Academy of Sciences (Hefei, China). Conditions used for EPR signal acquisition were the following: microwave frequency, 9.387 GHz; field modulation amplitude, 5 gauss; microwave power, 2 mW; modulation frequency, 100 kHz at either 13 K or 77 K.

EPR was also used in experiments designed to determine the possible difference in the Fe-S cluster states of DndCDE proteins during their reactions with H₂O_2. In this case 1,200 μl of DndCDE solution (130 μM in the storage buffer) was mixed with 12 μl of 5 M H₂O₂ and 60 μl of 2.5 M imidazole, pH 8.0. Samples (200 μl) of the mixture were removed into EPR tubes and snap-frozen in liquid nitrogen after 0.5 min, 1 min, 2 min, 5 min, and 10 min in readiness for EPR data acquisition, which was performed in a manner identical to that described above.

Fenton reaction and HPLC-MS detection of phosphorothioate DNA.The 100-μl reaction mixture contained 25 μM dGsA (synthesized from Sangon Biotech, Shanghai, China) and 100 μM FeCl₂. A final concentration of 1 mM H₂O₂ (from 100 mM stock) was added to initiate the Fenton reaction (31). The reaction was performed at room temperature for 30 min. Then 10 μl of the reaction solution was loaded onto a YMC-C₁₈ reverse-phase column (250 mm by 4.6 mm; 5-μm particle size; YMC Co., Ltd., Kyoto, Japan) fitted to an Agilent 1100 series LC/MSD (liquid chromatography/mass-selective detector) trap system. Sample separation was achieved by use of a two-solvent mixture (solvent A consisting of 0.1% acetic acid in water and solvent B consisting of 0.1% acetic acid in acetonitrile) at a flow rate of 0.4 ml/min and a temperature of 30°C. Gradient conditions were 1% to 13% solvent B for 35.5 min, 13% to 30% solvent B for 20 min, and finally 1% solvent B for 10 min. UV absorption was monitored at λ_254.4. Ionization was set in positive mode, gas flow was 10 liters min⁻¹, nebulizer pressure was 30 lb/in², drying gas temperature was 325°C, and capillary voltage was 3,100 V. Signals at m/z 597 (dGsA), 565 (dG_HA), and 581 (dGA) were extracted to confirm the identities of the main peaks.

EMSAs.DNA sequences used in electrophoresis mobility shift assays (EMSAs) are listed in Table 2; they were purchased from Genewiz (Jiangsu, China) and were either labeled with 5′ 6-carboxyfluorescein (FAM) and/or phosphorothioated at their GA/GT residues. The DNA fragments were prepared as 500-μl volumes which contained 50 μl of 400 μM sense and antisense oligonucleotides, 100 μl of 5× annealing buffer (150 mM Tris-HCl, pH 8.0, 50 mM MgCl₂, and 240 mM NaCl), and 300 μl of water. Prior to use, these mixtures were heated to 100°C for 10 min and then slowly cooled to room temperature. The DNA solutions were then either used immediately or stored frozen until required.

Each 20-μl EMSA mixture contained 2 μl of a specific 5′ FAM-labeled oligonucleotide (4 μM), 2 μl or 4 μl (4 μM) of complementary oligonucleotide where required, 2 μl of binding buffer (100 mM Tris-HCl, pH 8.0, 1 M KCl, 1 mM dithiothreitol, 0.1 mg ml⁻¹ bovine serum albumin [BSA], and 50% glycerol) along with 10 μl of 6.67 μM DndCDE and sterile double-distilled water to make up the volume. Mixtures were incubated at room temperature for 40 min, and then 10 μl was subjected to PAGE in a 3.96% gel (49.5% T and 3% C, acrylamide-bisacrylamide [Solarbio, Beijing, China]) in 0.5× TBE buffer (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA). The gel was electrophoresed at 100 V for 1 h after which DNA bands were detected and recorded using an FL3000 fluorescence detector (Fujifilm Corp., Tokyo, Japan).

Measurement of DndCDE and DndCDE-FeS H₂O₂ decomposition activities.(i) Colorimetric assay. H₂O₂ scavenging activity was assayed in a 100-μl reaction mixture composed of 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 125 mM imidazole, 5% glycerol, and 40 mM H₂O₂ (Sangon Biotech). Scavenging reactions were initiated by addition of 1.67 μM (0.25 mg ml⁻¹) protein and incubated at 25°C for a total of 20 min. Ten-microliter aliquots were removed from the reaction mixtures at 5-min intervals and diluted 1,000-fold with storage buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5% glycerol) to a final volume of 10 ml. Twenty microliters of the diluted reaction mixture was then added to 200 μl of ferric-xylenol orange assay mixture (Hydrogen Peroxide Quantitative Assay kit; Sangon Biotech) (18, 57), and the mixture was allowed to rest at room temperature for 30 min, after which its optical density was measured at λ₅₉₅. To investigate the impact of Fe²⁺, 10 μM or 100 μM FeCl₂ was added into the assay prior to H₂O₂. All reactions were carried out in triplicate.

(ii) Bubble test. Bubble tests (26) were performed to follow the liberation of oxygen during the decomposition of H₂O₂ in a qualitative fashion by the two proteins. In this assay the presence of Triton X-100 entraps bubbles of oxygen liberated during the reaction at its surface to form a foam layer which is photographed after 10 min. Reaction volumes were 1 ml and contained 10 μM protein, 3 M H₂O₂, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 125 mM imidazole, 5% glycerol, and 1% Triton X-100. This concentration of H₂O₂ was chosen based on the report of Iwase et al. (26) as it is the concentration required to produce bubbles and foam, which can be entrapped at the reaction’s surface by the surfactant; two negative controls were used for comparison. The first involved the use of heat-inactivated protein, and the second consisted of reaction buffer alone.

(iii) UV absorption. The H₂O₂ decomposition rates of DndCDE and DndCDE-FeS were also measured in real time in a 96-well UV-transparent microplate (Corning) using a Synergy2 microplate reader (BioTek Instruments, Inc., VT) operated at λ₂₄₀ (H₂O₂ has a characteristic absorption peak at λ₂₄₀) (58). Concentrations of H₂O₂ employed were 0, 15, 20, 24, 30, 40, 48, 60, 80, 96, 120, 160, 200, and 240 mM, and scavenging reaction mixtures (100-μl total volume) were composed of 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 125 mM imidazole, 5% glycerol, and 1.67 μM protein. All assays were performed in triplicate and initiated by addition of H₂O₂. Readings were taken at 3-s intervals over a period of 2 min, and the concentration of H₂O₂ remaining in solution was calculated by reference to a standard curve constructed previously from a known concentration series of H₂O₂. The concentration of DndCDE-FeS was half that of DndCDE, and experiments involving the use of DndCDE-FeS were terminated at 1 min, in both cases because the large volume of oxygen generated would interfere detection. Calculations relating to H₂O₂ decomposition were made using Prism 5 software (GraphPad Software, Inc., CA, USA). To investigate the impact of EDTA, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM, or 128 μM EDTA was added into the assay prior to H₂O₂.

(iv) O₂ liberation rate measurement (pO₂ oxygen electrode).Rate measurement of oxygen liberation during DndCDE H₂O₂ decomposition was also undertaken using a Clark-type oxygen electrode as described by Jacoby et al. (59). (This experiment also served to confirm that the gas generated on H₂O₂ decomposition was oxygen). Reaction mixtures (2-ml total volume) were composed of 50 mM H₂O₂ and 1.67 μM DndCDE in storage buffer supplemented with 125 mM imidazole (pH 8.0). Partial O₂ pressure (pO₂) measurement was terminated when the oxygen concentration reached saturation level, and all reactions were carried out in triplicate.