Nappi

GMDCs were washed in PBS, snap-frozen in liquid nitrogen, and lysed in ice-cold buffer containing: 50 mM Hepes (pH 7.6) (Sigma-Aldrich), 50 mM NaF (Sigma-Aldrich), 50 mM KCl (MilliporeSigma), 5 mM NaPPi (Sigma-Aldrich), 1 mM EDTA (Thermo Fisher Scientific), 1 mM EGTA (Sigma-Aldrich), 1 mM DTT (Promega), 5 mM β-glycerophosphate (Sigma Aldrich), 1 mM sodium vanadate (Sigma-Aldrich), 1% NP40 (Sigma-Aldrich), and protease inhibitors cocktail (Complete, Roche). Proteins were separated by SDS-PAGE and transferred to a PVDF membrane. Membranes were blocked for 1 hour at room temperature in TTBS buffer (20 mM Tris–HCl [pH 7.6; Thermo Fisher Scientific], 137 mM NaCl [MilliporeSigma], and 0.25% [v/v] Tween 20 [MilliporeSigma]) containing 5% (w/v) fat-free milk. Membranes were incubated with primary antibodies overnight at 4°C, washed in TTBS buffer, incubated with horseradish peroxidase–conjugated secondary antibodies for 2 hours at room temperature, washed again, and developed using enhanced chemiluminescence. Primary antibodies included AMPK (Cell Signaling Technology, 2532) and HSP90 (Santa Cruz Biotechnology Inc., sc7947).

The nonnucleoside reverse transcriptase inhibitor (NNRTI) inhibition assay was similar to the polymerase assay described above. However, the DNA primer was not 5′ end labeled. The unlabeled primer was annealed to single-stranded M13mp18 DNA as described above. The annealed template/primer was suspended in a mixture of 25 mM Tris (pH 8.0), 75 mM KCl, 8.0 mM MgCl₂, 100.0 μg of BSA per ml, 10.0 mM CHAPS, and 10.0 μM (each) dATP, dTTP, dGTP, and dCTP. The radioactive triphosphate [α-³²P]dCTP was included in the reaction mixture and acted as a tracer. The reactions also contained various concentrations of the NNRTI as shown in the figures. The reactions were initiated by the addition of WT RT or an RT variant and allowed to proceed for 30 min at 37°C. The reactions were halted by the addition of 200 μl of 10.0 mg/ml of sheared/denatured salmon sperm DNA, 200 μl of 0.2 M NaPP_i (Sigma), and 3.0 ml of 10% trichloroacetic acid (TCA). The precipitated DNA was captured on Whatman GF/C glass filters by suction filtration and the filters were added to scintillation vials. After addition of scintillation fluid, the filters were counted. The number of counts in the absence of NNRTI was considered 100% activity, and the other counts obtained at the other NNRTI concentrations were normalized to this value.

For pyrophosphorolysis experiments, a 100 mM stock solution of Na-PP_i (Sigma-Aldrich) was prepared, aliquoted, and stored at −80 °C. PP_i was incubated in the absence of Mg²⁺ in the mixture containing T7 DNA polymerase for solubility reasons. Control stopped flow experiments (not shown) indicated that PP_i does not bind to the enzyme without Mg²⁺, so mixing Mg²⁺ from the other syringe starts the reaction and allows higher concentrations to be reached for short reaction times before Mg²⁺-PP_i precipitates. For the experiment with [α-³²P]-28/45 DNA in Figure 4A and the nucleotide dissociation rate experiment in Figure 2E, the formation of [α-³²P]-dATP was monitored by TLC on PEI-cellulose plates. To remove contaminants, TLC plates were first developed in ultrapure H₂O. Plates were then dried, the contaminants at the top cut off, and the plates stored at 4 °C. Samples were spotted on 1-cm lanes and developed in 0.3 M KPO₄ pH 7. Plates were then dried, visualized by phosphorimaging, and quantified in Image Quant.