Isolation of the DNA Synthesome

HeLa cells were continuously grown in Eagle’s minimum essential medium (Joklik modification with L-glutamine without calcium chloride, Sigma-Aldrich, St. Louis, MO) containing 2 g/L sodium bicarbonate, 5% fetal bovine serum, 5% calf serum, 100 units/mL penicillin, and 0.1 mg/mL streptomycin at pH 7.4 in two 4 L spinner flasks. Cells were grown at 37°C in 5% CO₂, harvested in mid-log phase (5 x 10⁵ cells/mL), and collected by centrifugation at 250 x g for 5 min. Cell pellets were washed three times by suspending them in ice cold PBS, centrifugation, and removing the supernatant before storing the washed pellet at -80°C.

The DNA synthesome was partially purified to the level of the P4 fraction [5] from a 5 g pellet of HeLa cells with slight modifications. All purification steps were completed at 4°C. Cell pellets were homogenized using a loose-fitting Dounce homogenizer in three pellet vol of 50 mM Tris pH 7.5, 0.25 M sucrose, 5 mM EDTA, 5 mM EGTA, 1 mM PMSF, 1 mM DTT, and 1x protease inhibitor cocktail (Thermo Fisher Scientific, Grand Island, NY). The extract was initially separated by differential centrifugation starting at 570 x g for 10 min to yield a nuclear pellet (the NP fraction) and a supernatant containing the cytosolic extract (the S1 fraction). Mitochondria were pelleted by centrifugation at 10,000 x g for 15 min from the S1 fraction, and the supernatant (the S2 fraction) was further processed to remove microsomes from the S2 fraction by centrifugation at 165,000 x g for 1 h. The supernatant (the S3 fraction) was removed and placed on ice. The NP fraction was suspended in two times the pellet vol of buffer (50 mM Tris, pH 7.5, 1 mM DTT, 0.15 M KCl, 5 mM EDTA, 5 mM EGTA, 1 mM PMSF, and 1x protease inhibitor cocktail), rocked gently for 2 h, and clarified by centrifugation initially at 10,000 x g for 15 minutes to remove larger debris from the NP fraction, followed by centrifugation at 165,000 x g for 1 h (the NE fraction) to prepare a soluble protein fraction. The NE fraction was combined with the S3 fraction, and carefully layered above a sucrose cushion (2 M sucrose, 50 mM Tris, pH 7.5, 0.15 M KCl, 5 mM EDTA, 5 mM EGTA, 1 mM DTT, and 1x protease inhibitor cocktail). After ultracentrifugation using a SW40Ti rotor spun at 40,000 rpm for 17 h using maximum acceleration and no break for deceleration, the lower 20% of the soluble protein above the sucrose cushion was pooled and designated as the P4 fraction.

The P4 fraction was dialyzed into 50 mM Tris-HCl, pH 7.5, 5 mM KCl, 1 mM DTT, 1 mM EDTA, 10% glycerol, 1 mM PMSF, and 1 mM aminoacetonitrile, pH 7.5, using 12–14 kDa molecular weight cut-off (MWCO) SpectraPor 2 dialysis tubing (Spectrum Labs, Houston, TX). Aminoacetonitrile HCl (Thermo Fisher Scientific, Grand Island, NY) was added from a 0.5 M stock concentration adjusted to pH 7.5. After dialysis, the protein retentate was retrieved and stored for later use in aliquots maintained at -80°C.

PCNA-FLAG Purification

The vector pCMV6-Entry (Origene, Rockville, MD), which contains PCNA with a C-terminal Myc-FLAG (PCNA-FLAG), was stably transfected into MDA-MB-468 breast cancer cells using G418 (1 mg/ml) selection in Dulbecco’s Modified Eagle’s Medium containing 10% fetal bovine serum, 100 units/mL penicillin, and 0.1 mg/mL streptomycin. PCNA-FLAG was affinity purified from cell lysates using anti-FLAG M2 resin and following the directions provided by the manufacturer (Sigma-Aldrich, St. Louis, MO) with appropriate modifications. All steps were performed at 4°C. Cell pellets were lysed by Dounce homogenization using a loose-fitting homogenizer pestle in 50 mM Tris, pH 7.5, 0.25 M sucrose, 5 mM EDTA, 5 mM EGTA, 1 mM PMSF, and 1x protease inhibitor cocktail at 2X the pellet vol. After centrifugation at 1,000 x g for 15 min, the supernatant (the HS fraction) and pellet (the HP fraction) were saved. The HP fraction was suspended in 2 vol of 50 mM Tris, pH 7.5, 0.15 M KCl, 5 mM EDTA, 5 mM EGTA, 1 mM PMSF, and 1x protease inhibitor cocktail and rocked gently for 2 h. The HP fraction was combined with the HS fraction and centrifuged at 10,000 x g for 15 min. The resulting supernatant was clarified at 165,000 x g for 1 h to create a soluble protein fraction containing the FLAG-PCNA protein. Anti-FLAG M2 resin was added to the supernatant, and gently rotated for 1 h in batch format. The resin was loaded into a column, washed with a minimum of 50 column vol of wash buffer (50 mM Tris, pH 7.4, containing 150 mM KCl) and eluted with wash buffer supplemented with 0.1 mg/mL FLAG peptide (Sigma-Aldrich, St. Louis, MO). PCNA-FLAG was dialyzed (50 mM Tris-HCl, pH 7.5, 5 mM KCl, 1 mM DTT, 1 mM EDTA, and 10% glycerol) and frozen at -80°C in aliquots.

IZE Setup

Spacers (0.2 mm thick), electrode membrane strips, and filter paper strips (0.3 mm thick) were placed between two free flow electrophoresis plates and assembled as described by the manufacturer (FFE Service GmbH, Munich, Germany). The separation chamber temperature was maintained at 10°C. Water was pumped into inlets 1, 4, 6, and 9; while 0.01% sulfanilic acid azochromotrop (SPADNS) was pumped into inlets 2, 3, 5, 7 and 8 to provide visual assurance of an even laminar flow of the liquid phase in the separation chamber. Prior to loading the protein fractions, the separation chamber was coated with 0.2% (hydroxypropyl) methyl cellulose in a solution of 25% glycerol for 1 min at a flow-rate of 360 mL/h, followed by 30 min at a reduced flow-rate of 40 mL/h. The separation chamber was rinsed with water for 10 min at 240 mL/h and then equilibrated in separation buffers for 10 min at 240 mL/h. To establish the pH step gradient across the separation chamber, the buffers pumped through each inlet were as follows: inlet 1 (250 mM mannitol, 100 mM HCl, and 4-(2-hydroxyethyl)morpholine at pH 6.13), inlet 2 and 3 (250 mM mannitol, 20 mM α-hydroxyisobutyric acid and 4-(2-hydroxyethyl) morpholine at pH 6.05), inlet 4 and 5 (250 mM mannitol, 10 mM α-hydroxyisobutyric acid and 4-(2-hydroxyethyl)morpholine at pH 6.6), inlet 6 and 8 (250 mM mannitol, 10 mM α-hydroxyisobutyric acid and 4-(2-hydroxyethyl)morpholine at pH 7.23), inlet 7 (250 mM mannitol, 10 mM α-hydroxyisobutyric acid, 5 mM KCl and 4-(2-hydroxyethyl)morpholine at pH 7.23), and inlet 9 (250 mM mannitol, 150 mM α-hydroxyisobutyric acid, 250 mM Tris base, 50 mM ethanolamine, and 100 mM KOH at pH 9.3). The anode buffer (100 mM HCl and 4-(2-hydroxyethyl) morpholine at pH 6.13) and cathode buffer (100 mM NaOH and 200 mM glycine) were circulated at the electrodes during the entire separation. Mannitol (250 mM) was used as the counter-flow solution.

Running Samples for IZE

Immediately before IZE purification, the P4 fraction or PCNA-FLAG protein was desalted at 4°C in 5 mM Tris-acetate, pH 7.5, using either a PD SpinTrap G-25 column (GE Healthcare Life Sciences, Pittsburgh, PA) or a 7 kDa MWCO Zeba desalting column (Thermo Fisher Scientific, Grand Island, NY). The relative protein concentration was determined by Bradford assay using BSA as the standard. The P4 fraction was adjusted to a protein concentration between 2.6–12.3 mg/mL before separation.

The voltage, current, power limit, and media pump speed settings were set to 1000 V, 190 mA, 300 W, and 136 mL/h, respectively. Samples (either colored pI marker standard mix (FFE Service GmbH, Germany), the P4 fraction, or PCNA-FLAG protein) were applied to the separation chamber with a sample pump set to 2.1 mL/h with 0.5 mm inner diameter tubing. After 1.5 min, the sample pump was reversed for 1 sec and then stopped. Once the samples reach the separation region of the chamber (15 sec), the voltage was turned on for 10 s and the media pump was adjusted to 36 mL/h. After a 6 min separation, the voltage was turned off, and the samples were collected into a 96-well plate at 236 mL/h over the next 75 sec. Directly from the 96-well plate, the pH of every other well was measured using a modified Freedom EVO robot (Tecan, San Jose, CA) connected to a micro-pH electrode. The optical density of the colored pI marker standard mix was measured at 450 nm using a microplate reader to verify that the separation is routinely reproducible from day-to-day, and that the proper pH steps, timing, and voltage across the separation chamber were maintained throughout the purification.

Silver Staining

Proteins were resolved by Tris-glycine SDS-PAGE. Five and twelve percent buffered polyacrylamide was used for the 1.5 mm thick stacking and resolving gels, respectively. Selected IZE fractions (0.03 mL) were loaded into each lane. Gels were stained with the Silver Stain Plus kit from Biorad following the manufacturer's directions. Proteins were visualized following silver staining, and their location in the gel compared with the Precision Plus mol wt protein standard (Biorad, Hercules, CA).

Dot Blot

The nitrocellulose membrane used for the dot-blot analysis was briefly soaked in water followed by 25 mM Tris base, 192 mM glycine, and 20% v/v methanol, pH 8.3. All 96 fractions were passed through a 0.45 μm nitrocellulose membrane by vacuum filtration using the Convertible Filtration Manifold System (Whatman Biometra, Baltimore, MD). For each blot, either 0.1 ml or 0.2 ml of fraction was applied to the membrane. The resulting blot was incubated for 1 h in 2–5% nonfat dried milk before incubation overnight with individual primary antibodies recognizing one of the following proteins: 1:5,000 PCNA (mouse monoclonal, catalog no. SC-56, Santa Cruz Biotechnology, Dallas, TX), 1:500 Topo I (rabbit polyclonal, catalog no. TA307877, Origene, Rockville, MD), 1:500 Pol ɛ subunit 2 (rabbit polyclonal, catalog no. GTX109069, GeneTex, Irvine, CA), 1:500 RPA 32 kDa subunit (mouse monoclonal, catalog no. NA19L, Calbiochem, Darmstadt, Germany), 1:500 Pol δ catalytic subunit (mouse monoclonal, catalog no. 610972, BD Biosciences, San Jose, CA), or 1:500 RFC subunit 4 (rabbit polyclonal, catalog no. SC-20996, Santa Cruz Biotechnology, Dallas, TX). Primary antibodies were then labeled with either 1:5,000 goat anti-mouse IRDye 800CW (catalog no. 926–32210, Li-Cor, Lincoln, NE) for detection on the Odyssey infrared imaging system or 1:10,000 anti-rabbit conjugated with horseradish peroxidase (HRP) (catalog no. 7074S, Cell Signaling Technology, Beverly, MA) for imaging by enhanced chemiluminescence. The signal was quantified using Image Studio Lite (Li-Cor, Lincoln, NE) or Quantity One (Biorad, Hercules, CA) software. The data was normalized on a scale of 0 to 1 by dividing the quantified signal of the fraction with the background subtracted by the maximum quantified signal from the 96 fractions with the background subtracted.

Blue Native (BN) Gel

IZE fractions were concentrated using a 100 kDa MWCO polyethersulfone ultrafiltration device (Thermo Fisher Scientific, Grand Island, NY) at 4°C, and the final vol of each fraction were adjusted to 0.02 mL with 50 mM Tris, pH 7.5. The protein was loaded into 3–12% gradient NativePAGE Novex 10 well gels (Thermo Fisher Scientific, Grand Island, NY) using the cathode and anode buffers described by the manufacturer, except the buffers were chilled to 4°C. To reduce the potential dissociation of the large complex into component pieces while performing this electrophoresis step, the gels were run at 4°C and 35 V for 18 h using the PowerPac 3000 power supply (Biorad, Hercules, CA). For Coomassie staining, gels were fixed as described by the manufacturer, incubated in Coomassie SimplyBlue Safestain (Thermo Fisher Scientific, Grand Island, NY) for 1 h, and then destained with water.

For Western blotting, proteins were transferred to a PVDF membrane using the Pierce G2 Fast blotter (Thermo Fisher Scientific, Grand Island, NY). The membrane was fixed with 8% acetic acid for 15 min with gentle shaking, rinsed in 100% methanol until the blue color faded, and then briefly rinsed in TBS (20 mM Tris, pH 7.6, and 137 mM NaCl). Ponceau-S solution (Sigma-Aldrich, St. Louis, MO) was added to identify the mol wt marker standards (NativeMark unstained protein standard, Thermo Fisher Scientific, Grand Island, NY), and their relative position on the filter membrane was subsequently marked with a ball point pen. To identify the position of PCNA, the membranes were rinsed with TBS to remove the Ponceau-S solution, blocked for 1 h in 5% milk in TBS containing 0.05% Tween-20 detergent (TBS-T), and incubated with mouse anti-PCNA antibody at the appropriate dilution. The location of the antibody on the membrane was detected using ECL Prime, which reacts with the anti-mouse antibody conjugated with HRP (catalog no. 7076S, Cell Signaling Technology, Beverly, MA.

SV40 DNA Replication Activity Assay

IZE fractions were concentrated and then desalted into 50 mM Tris, pH 7.5, 5 mM KCl, and 1 mM DTT using 100 kDa MWCO PES concentrators (Thermo Fisher Scientific, Grand Island, NY). Concentrators were prewashed with 0.5 mL of the exchange buffer. Fractions were assayed for T-antigen dependent SV40 DNA replication activity as described previously [5] with modification. The reaction (30 mM HEPES, pH 7.5, 7 mM MgCl₂, 0.1 mM dATP, 0.1 mM dGTP, 0.1 mM dTTP, 0.1 μM dCTP, 0.2 mM GTP, 0.2 mM UTP, 0.2 mM CTP, 4 mM ATP, 0.5 mM DTT, 1 μCi [α-32P] dCTP, 40 mM phosphocreatine, 2.6 units creatine phosphokinase, 0.25 μg SV40 large T antigen and 50 ng pSVO⁺) was performed at 37°C for 1 h and spotted on either Whatman DE81 ion exchange paper (GE Healthcare Life Sciences, Pittsburgh, PA) or DEAE Filtermat (Perkin Elmer, Waltham, MA) cut to 1 cmx2 cm. After drying, the ion exchange paper was washed twice in 0.1 M sodium pyrophosphate, pH 7.4, twice in 0.3 M ammonium formate, pH 7.4, rinsed in 95% ethanol, and air dried. The activity was measured by scintillation counting. One unit was defined as 1 pmol dNMP incorporated into the plasmid pSVO⁺ DNA after 1 h. The relative protein concentration for each fraction was determined by Bradford assay on a NanoVue Plus microvolume spectrophotometer (GE Healthcare, Pittsburg, PA) at 0.5 mm path length (Biorad, Hercules, CA) using increasing amounts of BSA as the protein standard.

In-gel Digestion

After separation by IZE and concentration through a 100,000 kDa MWCO membrane, samples were resolved on two blue native (BN) gels loaded with 67% (gel 1) and 33% (gel 2) of the concentrated samples. Gel 1 was stained with Coomassie blue. Western blot analysis using anti-PCNA was performed on gel 2. The Western blot film was then superimposed onto the Coomassie stained gel by mol wt marker alignment, and the identified PCNA containing bands were excised from the native gel. Gel bands were destained three times with 200 μL destaining buffer (40% acetonitrile (ACN) and 200 mM ammonium bicarbonate) for 30 min at 37°C, reduced with 50 μL DTT (15mg/mL) at 80°C for 10 min, and then alkylated in the dark using 50 μL iodoacetamide (18mg/mL) for 45 min at rt. Gel bands were dried for 30 min under vacuum and then rehydrated on ice for 45 min with 10 ng/μL trypsin in 40 mM ammonium bicarbonate and 10% ACN, pH 8. Unabsorbed trypsin solution was discarded, and a 50 μL overlay buffer (40 mM ammonium bicarbonate in 10% ACN) was added to cover the gel to allow trypsin digestion to proceed overnight at 37°C. Peptide fragments within the gel bands were extracted three times with a solution of 50% ACN and 0.1% trifluoroacetic acid. The corresponding peptide solutions were combined, dried completely under vacuum, and stored at -80°C. Prior to LC/MS analysis, samples were dissolved in 10 μL of 1% formic acid.

Nanoflow LC/MS/MS Analysis

All LC/MS/MS experiments were performed on a Thermo Scientific Orbitrap Fusion Mass Spectrometer equipped with an Easy Spray source and an Easy-nLC1000 system. Mobile phases (MP) were composed of MP-A (0.1% formic acid in water) and MP-B (0.1% formic acid in ACN). Samples (8 μL) were loaded onto a trapping column (Acclaim PepMap100, 75μm x 2cm, C18, 3 μm, 100 Å, Thermo Scientific, Sunnyvale, CA) and separated on an integrated Easy-Spray column emitter packed with PepMap C18, 3 μm, and 100 Å particles (75 μm x 15cm). The column temperature was maintained at 45°C. Samples were desalted for 2.4 min with MP-A at a flow rate of 5 μL/min; the trypsin peptides were separated at a flow rate of 300 nL/min, using a linear gradient from 3% to 35% of MP-B over 8 min. This was followed by a fast gradient to 90% MP-B in 1 min and then 90% MP-B for another min. To improve resolution of the sample components, a longer gradient method was also used that started from 3% to 38% MP-B over 40 min, followed by a fast gradient to 85% MP-B for another min, and ended with 85% MP-B for 4 additional min.

Data was acquired on an Orbitrap Fusion Mass Spectrometer using a resolution of 120,000 (at 200 m/z) for full scans over a 400–1600 m/z range (a maximum of 0.75 s between the full scans), followed by collision induced dissociation fragmentation of the individual peptides (at the top speed setting) and detection of fragmentation ions in the ion trap. Spray voltage was set at 2300 V, S-lens RF level set to 60 and heated capillary set at 275°C. For MS, AGC target was set at 2.5 x 10⁵ and maximum injection time was 100 ms. For the MS/MS experiment, AGC was set to 1x 10⁴ and injection time to 35 ms. Isolation width was set at 2 Da, and collision energy was set to 35%. One microscan was acquired for one spectrum and dynamic exclusion was set at 15 s. XCalibur (3.0.63) software was used for data acquisition. Peptides were identified using three search engines: Proteome Discoverer, the GMP Search, and Mascot. A decoy database was used, and the false discovery rate was set to 5%.

Partial Purification using IZE

We applied IZE as a technique to increase the purity of the DNA synthesome using its net charge in a mobile liquid based system to resolve it from other proteins and protein complexes found in the soluble protein fraction obtained from mammalian cells. Prior to IZE purification, we verified proper laminar flow, pH steps, voltage, and time conditions for the purification procedure with a dye (SPADNS) and specific pI markers (S1 Fig) as described in the methods section of this manuscript. A series of different buffers were continuously pumped through the individual inlets in order to create discrete laminar flowing streams of buffer that differ in pH from one another when traveling between the anode and cathode (Fig 1A and 1B). Samples were introduced near the cathode side using a peristaltic pump until the sample reached approximately half way up the separation chamber. The sample pump was then reversed for 1 sec to curtail the sample flow before shutting-off the pump. When the samples reach between the anode and cathode with the laminar flow, the electric field was applied for 6 minutes while the flow-rate of the separation buffers was decreased. As samples continue to move through the electrical field perpendicular to the laminar flow, more negatively charged proteins migrate faster toward the anode, and more positively charged proteins move toward the cathode. The heterogeneous mixture of analytes resolve from one another and sharpen as they move towards the anode or cathode and pass through individual streams of separation buffers of differing pH. Together, the individual buffer streams form step-gradients of decreasing pH, each of which sharpens the resolution of the DNA synthesome as it moves toward the anode. After turning off the electric field, the resolved proteins and protein complexes were pumped directly into the wells of a 96-well plate, and the contents of each well were subjected to further analyses in order to determine the location of the DNA synthesome within the buffer stream and to confirm whether the components of the DNA synthesome migrated as an intact and functional protein complex. Because the separation is automated, several purification cycles were sequentially collected and processed for later analysis.

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Fig 1. Fractionation of HeLa cell proteins using IZE.

(A) Simplified diagram of the FFE separation chamber. (B) pH measured for every other fraction on the 96-well plate after protein separation. (C) Separation of fractions from the IZE by denaturing gel electrophoresis and detection by silver staining. Fraction numbers are labeled on top, and M represents the mol wt marker. P4 represents the protein before the IZE separation. The molecular masses of the markers are 250 kDa, 150 kDa, 100 kDa, 75 kDa, 50 kDa, 37 kDa, 25 kDa, and 20 kDa labeled A to H from top to bottom.

https://doi.org/10.1371/journal.pone.0169259.g001

Samples collected from the FFE instrument were visualized after resolution by SDS-PAGE using silver stain (Fig 1C). Fractions 3 and 94 represent samples nearest to the anode and cathode, respectively. Fig 1B shows the pH of every other fraction across the 96 well plate following protein sample collection.

Optimization of IZE Runs

Conditions for the IZE were optimized based on dot blot analysis. For dot blot experiments, all 96 fractions were transferred onto nitrocellulose membranes by vacuum filtration, and specific proteins were detected by Western blot analysis of these filter membranes. Two major peaks were found for PCNA (Fig 2A) from the P4 fraction following IZE separation. The first peak was found at fraction 17, while a second peak was identified at fraction 25. The PCNA-containing peak around fraction 17 from the P4 fraction corresponds to the location where purified recombinant PCNA elutes (Fig 2B). The timing, pump flow rate, and voltage conditions for IZE separation were optimized by monitoring the relative position of colored pI marker standards and the resolution of the two PCNA peaks that were detected by dot blotting. After our optimization studies, we were unable to improve the resolution of the PCNA complex by either altering the resolution time in the separation chamber of the FFE flow cell or by adjusting the voltage used to resolve PCNA and the other DNA synthesome components. In addition, the resolution did not improve by widening the pH step gradient from pH 5.4 to 7.0 (data not shown).

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Fig 2. Dot blot analysis of IZE fractions.

(A) Dot blot analysis of an IZE separation from the P4 fraction using antibodies that recognize PCNA, Topo I, Pol ɛ subunit 2, RPA subunit 2, Pol δ catalytic subunit, and RFC subunit 4. Recombinant PCNA (rPCNA) was analyzed following IZE separation of PCNA-FLAG expressed and purified protein. Fraction numbers are labeled at the top of the figure. (B) Dot blot quantification of PCNA. The Western blot signal of each fraction for PCNA from the P4 fraction (black circle) and PCNA-FLAG (blue square) was normalized from 0 to 1.

https://doi.org/10.1371/journal.pone.0169259.g002

The reproducibility of independent IZE runs performed on six different days was monitored by dot blot analysis with antibody against PCNA. The runs were performed either manually or by robotic automation. The first PCNA peak appeared in fractions 17 or 18. The second PCNA peak appeared in fraction 26 +/- 2, which represents +/- 2 out of 96 fractions or an error of approximately +/- 2.1% and a fraction range equivalent to 4 out of 96 fractions or 4.2%. Differences in the setup or minor changes in the pH or conductivity of the buffers can give rise to these minor variations in electrophoretic mobility. Analysis of the proteins resolved during the first and last purification cycle performed on the same day showed that PCNA peaks consistently in the same fraction.

In an attempt to recover higher protein yields, we loaded increasing amounts of the P4 fraction into the separation chamber and tested the resolution of proteins within the P4 fraction using dot blot analysis with anti-PCNA antibody. Our initial IZE separations injected 0.1 mg of partially purified protein from the P4 fraction (described in the methods section) for each purification cycle. Using dot blot analysis for determining the location of PCNA within the fractions eluting from the separation, we showed that PCNA consistently eluted in the same fractions, and thus the resolving power of IZE was unaffected by increasing the amount of protein loaded into the separation chamber over the six fold range we used in this study. We only checked the resolution when loading up to 0.6 mg of protein.

Known DNA Synthesome Containing Proteins Co-elute with One Another

We used dot blot analysis to show the elution position of proteins previously identified as part of the DNA synthesome. Dot blot analysis showed that known DNA synthesome proteins, including PCNA, Topo I, Pol ɛ, Pol δ, RPA, and RFC (Fig 2A), co-elute with one another after IZE separation. The results suggest that the DNA synthesome remained intact after partial purification by IZE. Other proteins, most notably PCNA and RFC subunit 4, were found to be complexed or uncomplexed with known DNA synthesome proteins. Replicate dot blot analysis performed on an independent IZE purification run showed equivalent separations of these proteins. To show that the dot blot signal is from the protein of interest and not from a nonspecific signal associated with the primary antibody, denaturing SDS PAGE Western blot analysis showed that the major band is the apparent mol wt for that protein (S2 Fig).

We performed control experiments to determine whether secondary antibodies were falsely detecting proteins being examined in the dot blot analysis. Instead of incubating the nitrocellulose membrane with primary antibody, only the secondary antibody was added. We observed that when the secondary antibody was linked with the IRDye 700, which is a near-infrared fluorescence dye, false signals were detected when scanned at 700 nm; however, when IRDye 800 or HRP was conjugated to the secondary antibody, no false signals from the nitrocellulose membrane were detected. We report here dot blot results with the secondary antibody conjugated with either IRDye 800 or HRP in Fig 2.

BN PAGE Resolves a MDa PCNA-containing Multi-Protein Complex

To visualize native protein complexes following fractionation of the partially purified cell lysates by IZE, we used Blue Native (BN) gel electrophoresis. We reasoned that BN gels at pH of 7.5 may help protein complexes remain intact, and that they are well suited for resolving MDa sized protein complexes. A feature of BN PAGE is the presence of negatively charged Coomassie G-250, which binds to proteins and migrates with them towards the anode. As proteins move from larger to smaller pores in the gradient polyacrylamide gel, they almost stop migrating when they reach a particular pore size because separation is based on size rather than charge/mass ratio [25]. Since Coomassie G-250 binds to surface hydrophobic regions and reduces protein aggregation, BN gels are often used to resolve membrane proteins in the presence of detergents [26, 27]. To further reduce dissociation of multiprotein complexes, the BN gel is run at low temperature (4°C) and low voltage (35 V) for 18 h [20]. Highly abundant proteins retained the Coomassie stain after electrophoresis, while lower abundant proteins could be detected after additional Coomassie staining (Fig 3A).

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Fig 3. Separation of IZE fractions by BN gel electrophoresis.

(A) The gels were stained with Coomassie. (B) Separate gels were transferred to PVDF membrane and probed with PCNA antibody. Fraction numbers are labeled on top of the figure.

https://doi.org/10.1371/journal.pone.0169259.g003

To estimate the size of the PCNA associated multiprotein complex, we performed Western blotting with the BN gel and probed with an antibody against PCNA. Native gel mol wt marker proteins were used to provide a relative measure of the mass at any given distance from the wells of the gel. Because of the association of PCNA within the DNA replication apparatus of the cell and the co-migration of PCNA with other proteins having a role in the DNA replication process, it became important to establish whether this PCNA containing multiprotein complex could be the functional DNA synthesome that we had previously reported. It is also important to note that PCNA serves as a processivity factor, which is an essential component of the DNA synthetic process [14] carried-out by the in vitro SV40 DNA replication model system used in the analysis of the mammalian cell DNA synthetic process [28, 29]. The native gel showed two peaks of PCNA with a relative electrophoretic mobility between 800–1,000 kDa (Fig 3B, S3 Fig), at fractions 29 and 38, and a lower mol wt PCNA species which appeared at fraction 17.

Partial IZE Purification Increases the Specific Activity of T-antigen Dependent SV40 DNA Replication

To determine whether the multiprotein PCNA associated complex eluting from the FFE chamber contained enzymatically active fractions capable of supporting the DNA replication process, we measured the ability of the complex to support in vitro SV40 DNA replication activity. To do so, we assayed every third fraction collected from the IZE separation chamber for its ability to support initiation of DNA replication, DNA unwinding, short RNA oligomer synthesis, and elongation of the DNA template. The plasmid DNA template used in the assay contains the core sequences of the SV40 replication origin [30]. Addition of the SV40 viral large T-antigen to the replication assay mixture containing the multiprotein PCNA associated protein complex results in incorporation of the radiolabeled nucleotide in a T-antigen dependent manner, showing that the multiprotein complex is fully competent to support in vitro SV40 origin and T-antigen dependent DNA replication [3].

To measure the DNA replication activity of the purified PCNA associated DNA synthetic protein complex, the PCNA containing fractions from 8 cycles were collected and concentrated through a 100,000 kDa MWCO membrane. This step served as an additional purification step because smaller proteins and protein complexes can be separated from the larger complexes. Importantly, the SV40 DNA replication activity of these IZE fractions was not lost after concentrating the protein through the 100,000 kDa MWCO membrane filter (data not shown), indicating that the proteins needed to support in vitro SV40 origin dependent DNA replication activity are associated with one another in a highly organized and functionally active large complex. Fig 4A shows one major peak for SV40 DNA replication activity at fraction 27. As shown in Fig 4B from Western blot analysis, PCNA was also found within the fractions exhibiting DNA replication activity. The specific activity of the peak for T-antigen dependent DNA replication activity was 11.26+/-0.22 units/mg. One unit is 1 pmol dNMP synthesized into DNA after 1 hour. The specific activity of the protein before IZE separation (the P4 fraction) was 2.11+/-0.06 units/mg. From our assay results, we estimated that the SV40 origin dependent in vitro DNA replication activity of IZE fraction 27 was enhanced 5.3 times over that of the activity of the P4 fraction before IZE separation.

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Fig 4. In vitro SV40 DNA replication activity relative to the position of PCNA.

(A) Circles with the solid line represent the normalized SV40 DNA replication activity labeled on the left axis. Diamonds with the dotted line represent the protein concentration in μg labeled on the right axis. (B) Squares represent dot blot quantification of PCNA levels from IZE fractions run on the same day. The data were normalized between 0–1.

https://doi.org/10.1371/journal.pone.0169259.g004

The MDa Complex Contains Known DNA Synthesome Proteins

To verify the results of the Western blot analysis from IZE fractions, we used mass spectrometry based protein sequencing to validate whether the native gel slices containing PCNA, having a relative electrophoretic mobility between 800–1000 kDa, is the replication competent DNA synthesome. We specifically identified 23 proteins or protein subunits previously reported as being required for the origin and T-antigen dependent DNA replication process; many of whom were identified as components of the DNA synthesome [2, 5–7, 9–13]. Table 1 lists the identified proteins. The roles of these proteins in mammalian DNA replication are described by Smith et al. [14].

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Table 1. Previously identified DNA synthesome proteins or protein subunits identified by mass spectrometry from BN gel with a relative electrophoretic mobility of 0.8–1 MDa.

https://doi.org/10.1371/journal.pone.0169259.t001