PicoGreen nuclease assay.

Nuclease activity was assessed using the Quant-iT PicoGreen dsDNA reagent dye (Molecular Probes by Life Technologies catalog no. P7581) to detect dsDNA as previously reported (82, 83). Nuclease reactions were performed in 1× nuclease buffer (20 mM Tris-HCl [pH 8.2], 40 mM NaCl, 1 mM MgCl₂, 1 mM DTT) at 37°C. The DNA substrate was linearized pSAK vector (4 kbp) cut with PstI. Nuclease reaction mixtures contained 14 ng of purified UL12 protein, 1.7% DMSO, or 15 μM each α-hydroxytropolone from a stock prepared in 100% DMSO. UL12 protein was incubated with DMSO or compound for 5 min at 37°C, and the reaction was initiated with the addition of 10 ng dsDNA per 30 μl of reaction mixture. Control reaction mixtures contained protein alone without DNA and dsDNA substrate without protein. At time zero and 10 min, 30 μl was removed from each reaction tube and quenched with 3 μl of 500 mM EDTA. The quenched reaction mixtures were stored on ice until all reactions were complete.

Each sample was processed for PicoGreen fluorescence in accordance with the manufacturer's protocol. Briefly, 67 μl of TE buffer was added to each quenched reaction mixture to bring the volume up to 100 μl. Then 100 μl of the PicoGreen reagent, diluted 1:200 in TE buffer, was added to each tube and vortexed briefly to mix. Fluorescence was measured with a SpectraMax 3 plate reader in a 96-well Fluotrac 200 black plate (Greiner Bio-One catalog no. 655076/077) with excitation and emission wavelengths of 485 nm and 520 nm (530-nm cutoff), respectively.