Experimental Procedures

1. HEK293T cells are maintained in complete DMEM/F12 (1:1) media in a humidified cell culture incubator at 37°C, 5% CO₂. Transfection efficiency is higher when cells are between 3 weeks and 3 months age, post-thaw.
2. Passage cells one day before transfection, plating approximately 1.25 x 10⁶cells per 6 cm dish, or an amount that yields 60-90% confluence the next day. The procedure may be scaled: 6 cm dishes yield 2 bead aliquots, 10cm dishes yield 5 bead aliquots, and 15 cm dishes yield 12 bead aliquots.
3. Change medium 2-3 hours before transfection.
4. Transfect each plate with half pCDNA-3FHP and half pCMV-HE (by weight) using calcium phosphate transfection (or any other suitable method). Use 10 μg total weight of DNA for 6 cm dishes, 20 μg for 10 cm dishes, and 50 μg for 15 cm dishes. Include any desired controls.
5. Calculate volumes for water, DNA, calcium solution, and 2X HBS phosphate solution. Total volume should be 500 μL for 6 cm dishes, 1 mL total for 10 cm dishes, or 2.5 mL for 15 cm dishes.
6. Add in the following order: sterile water, DNA, and calcium chloride (calcium chloride volume is 31 μL for 6 cm, 62 μL for 10 cm, or 155 μL for 15 cm dishes).
7. To the DNA-calcium tube, add 2X HBS dropwise (250 μL for 6 cm, 500 μL for 10 cm, or 1.25 μL for 15 cm dishes) while agitating the receiving tube by flicking or agitating on a vortexer set low enough that no splashing occurs.
8. After 5 to 20 minutes, apply transfection reagent dropwise onto labeled plates.
9. Incubate at 37°C, 5% CO₂for 8 hours to overnight with transfection reagent. Wash cells once with 1X PBS and apply fresh medium.
10. Allow cells to grow for two days, then lyse according to the procedure below. Alternatively, cells can be frozen at -80°C in parafilm-wrapped dishes after removing growth medium and a PBS rinse.

Preparation of FLAG antibody-bound beads

1. Resuspend immobilized protein A/G beads by inverting several times. Transfer 20 µL of the bead suspension per 6 cm plate (50 µL per 10 cm plate, 125 µL per 15 cm plate) into a single tube, which will be split into bead groups later.
2. Pellet beads by centrifugation and remove storage buffer. Bead centrifugation steps should be at 350 x g for 2 minutes. Wash beads three times with TN buffer. Wash by adding the buffer to resuspend the beads, then centrifuge and remove the buffer.
3. After washing, resuspend beads in TN buffer at half the original bead volume but at least 200 µL.
4. Bind anti-FLAG IgG antibody onto washed beads by adding 2.8 µL of anti-FLAG antibody per each 6 cm dish (7 µL per 10 cm dish, or 17.5 µL per 15 cm dish).
5. Rotate at room temperature for 3 to 4 hours. Proceed to cell lysis during incubation.
6. After the anti-FLAG antibody is bound to beads, spin and remove unbound antibody. Wash beads three times with 500 μL FLAG lysis buffer (protease inhibitors are not necessary in this buffer). When adding wash buffer the final time, resuspend beads with low retention tips, and divide equal volumes into separate tubes for each condition used in the transfection. Place tubes on ice.

HBV polymerase immunoprecipitation and purification

1. Wash cells once with 2 mL 1X PBS per 6 cm plate (4 mL per 10 cm plate, or 10 mL per 15 cm plate), being careful not to detach cells.
2. Wash cells once with 2 mL cold 1X PBX with protease inhibitors per 6 cm plate (4 mL per 10 cm plate, or 10mL per 15 cm plate). From here, keep samples RNase-free and on ice.
3. To each 6 cm plate, add 0.4 mL cold FLAG lysis buffer with inhibitors, (1 mL to 10 cm plates, or 2.5 mL to 15 cm plates). Free cells from the dish by scraping with a cell scraper or spraying with the buffer from a pipet tip.
4. Collect cells from the same treatment condition into a single chilled tube, and rotate for 20 minutes at 4°C.
5. Centrifuge lysate at 4°C for 10 minutes at maximum speed in a microcentrifuge ~18,000 x g. Supernatants represent the cytoplasmic fraction, which contains HBV polymerase.
6. Transfer supernatants to chilled tubes of prepared anti-FLAG antibody-bound beads. The pellet and some lysate (~100 μL) may be frozen together for troubleshooting.
7. Rotate antibody-bound beads and cell lysate supernatant at 4°C overnight to allow immunoprecipitation to occur.
8. The next day, spin beads at 4°C for 2 minutes at 350 x g. Remove unbound supernatant (supernatant may be saved for troubleshooting).
9. Wash beads five times with 500 μL FLAG wash buffer. When adding wash buffer the final time, resuspend beads with low retention tips, and divide equal volumes into separate chilled labeled tubes for each assay to be performed. Assays usually include a western blot (polymerase protein levels), northern blot (epsilon RNA levels), and the priming assay itself.
10. Store bead aliquots at -80°C, removing wash buffer before further experiments. Approximate bead volume is 10 μL per tube.

Downstream analysis of immunopurified HBV polymerase complexes

1. For western blotting or protein staining (Coomassie blue, silver stain):
   1. Keeping samples on ice throughout, remove the wash buffer, and add 20 μL 2X SDS lysis buffer to the ~10 μL beads.
   2. Boil for five minutes, vortex, boil another five minutes, and place samples back on ice. Spin briefly (~4,000 x g for one second) to collect condensate.
   3. Mix samples and load 20 μL on a 9% SDS-PAGE gel.
   4. If analyzing/troubleshooting each step, load lysate supernatant (30 µL lysate + 30 μL 2X SDS lysis buffer, load 30 μL) and load insoluble pellet (add 50 µL TE, vortex to re-suspend pellet, then add 200 µL 2X SDS lysis buffer, mix, boil, load 50 µL).
   5. At this point, the gel can be used for staining or transferred to a membrane for western blotting, with a 1:2000 dilution of anti-FLAG antibody and a 1:20,000 dilution of anti-mouse secondary antibody.
2. For analyzing epsilon RNA bound to immunoprecipitated HBV polymerase, please see the related protocol “Hepatitis B Virus Polymerase Epsilon RNA Binding Assays”.

Assay of in vitro protein priming and transferase activity

1. To a bead aliquot (approximately 10 μL beads and 10 μL residual buffer), add 19 μL of TMgNK (or TMnNK) priming buffer.
2. Add 1 μL of radiolabeled nucleotide triphosphates or 1 μL of a 100 μM solution of unlabeled nucleotide triphosphates. Shake at room temperature 4 hours, such as a vortexer set to 3.
3. Alternatively, 2-step incubations can be performed with nucleotide analogs (triphosphate form) or other RT inhibitors shaken for 2 hours, then dNTPs are added and samples are shaken for 2 more hours.
4. Pellet beads at 350 x g for 1 minute. Remove supernatant to radioactive waste. (Nucleotides may be removed with the Tdp2 cleavage assay at this point).
5. To visualize priming levels, wash each sample once with 500μl of TNK wash buffer. Remove the supernatant to radioactive waste. Add 20 μL 2X SDS lysis buffer. Boil 5 minutes, vortex, and boil another 5 minutes. Spin briefly to collect condensate (~4,000 x g for one second). Load 20 μL of each sample on a 9% SDS-PAGE gel.
6. Place gel onto filter paper and cover with plastic wrap. Dry for 3 hours on a vacuum gel drying apparatus at 75°C if available, otherwise proceed with imaging directly.
7. Expose the gel to film or phosphorimager screen.

Materials and Reagents

• TNK buffer: 20mM Tris pH 7.0, 15mM NaCl, 10mM KCl. For 50mL, combine 1mL 1M Tris pH 7.0, 150μL 5M NaCl, 500μL 1M KCl, and 48.35mL nuclease-free water. Store at room temperature, keep RNase-free.
• TNK wash buffer: TNK buffer plus 28μM E-64, 1mM PMSF, 5μg/ml leupeptin, and 10mM β-mercaptoethanol. To 50mL TNK buffer, add 700μL 2mM E-64, 250μL 1mg/mL leupeptin, 250μL 200mM PMSF, and 39.06μL 12.8M β‑mercaptoethanol. Prepare just before use, use on ice, do not store, keep RNase-free.
• Tyrosyl DNA phosphodiesterase 2 (Tdp2/TTRAP) (Abnova). Enzyme concentration was 0.23μg/μL but may vary by lot, suspended in 50mM Tris pH 8.0 and 10mM reduced glutathione. Store at -80°C.
• Tdp2 mock buffer: 50mM Tris pH 8.0 and 10mM reduced glutathione. Store at ‑20°C.
• 2X Tdp2 buffer: 50mM Tris pH 8, 260mM KCl, 2mM DTT, 20mM MgCl₂. For 1mL, combine 50μL 1M Tris pH 8.0, 260μL 1M KCl, 2μL 1M DTT, 20μL 1M MgCl₂, and 668μL nuclease-free water. Make fresh, do not store.
• 1X Tdp2 wash buffer: 1X Tdp2 buffer, 28μM E-64, 1mM PMSF, 5μg/mL leupeptin. For 1mL, combine 500μL 2X Tdp2 buffer, 476μL nuclease-free water, 14μL 2mM E-64, 5μL 200mM PMSF, and 5μL 1mg/mL leupeptin. Make fresh, use on ice, do not store.
• 1X Tdp2 reaction buffer: 1X Tdp2 buffer, 1X Complete EDTA-free protease cocktail inhibitor, with Tdp2 enzyme or mock buffer. For 100μL, combine 50μL 2X Tdp2 buffer, 4μL 25X Complete EDTA-free protease inhibitor cocktail, 26μL nuclease-free water and 20μL Tdp2 enzyme (or Tdp2 mock buffer). Make fresh, do not store.
• Gel loading buffer II (Invitrogen) or other formamide loading dye.

Experimental procedures

1. Perform the priming assay as described above, with sufficient aliquots that each condition can be mock treated and Tdp2 treated.
2. Wash the priming reaction three times with 500μl TNK wash buffer.
3. Wash twice with 100 μL 1X Tdp2 wash buffer.
4. Add 10 μL of Tdp2 reaction buffer (or mock buffer) to each sample.
5. Incubate at 37°C for 1 hour with shaking.
6. After incubation, spin for 1 minute at 350 x g.
7. Separate supernatants (released nucleotides) into new tubes and save bead pellets (protein). Samples may be stored at -80°C until testing.
8. For testing supernatants (released nucleotides): Add 5μl formamide gel loading buffer to a 5μl aliquot. Heat to 95°C for 5 minutes by boiling or thermal cycler. Load all 10 μL of samples while still hot onto a 20% acrylamide 8 M urea-PAGE gel in 1X TBE. Gels should be pre-run for 30 minutes prior to loading. Run until the bromophenol blue dye front reaches 2/3 the length of the gel. Expose gel to film or phosphorimager screen.
9. For testing bead pellets (polymerase protein): Add 20μl of 2X SDS lysis buffer, boil 5 minutes, vortex, and boil another 5 minutes. Spin briefly to collect condensate (~4,000 x g for one second), and load 10 μL of each sample on a 9% SDS-PAGE gel. Expose gel to film or phosphorimager screen.