Pol I extension.

Extension assays were performed by first annealing oJR234 and oJR145 as described above (see “RNase H assays” above) to create a 10 μM 5′-IR-labeled substrate with 1 embedded rNMP. Hydrolysis reactions were then incubated for 2 h at 37°C. The same annealing buffer was used with 2 μM the annealed substrate. The first hydrolysis reaction mixture contained only 1 mM MnCl₂ and was labeled “none.” The second contained 300 mM NaOH and was labeled “NaOH.” The third and fourth both contained 1 mM MnCl₂, with the former having 1 μM RNase HII and the latter 1 μM RNase HIII. Each reaction was subsequently stopped using 100 μl of quenching buffer (3.6 M ammonium acetate [NH₄Ac], 20 mg glycogen). A 25-fold excess of ethanol was then added and mixed, and each mixture was incubated at −80°C overnight. Precipitated DNA was pelleted at 13,000 rpm for 15 min, washed in 1 ml of 70% ethanol, and pelleted again. Liquid was aspirated, and the pellet was air dried before resuspending it to a final volume of 20 μl using double-distilled water (ddH₂O), solubilizing the hydrolyzed substrates. The substrate concentrations were normalized after visualizing each one by 20% urea-PAGE and quantifying the concentration via the Li-Cor IR Odyssey imager software. For the primer extension reaction (see Fig. S5 in the supplemental material), primer oJR247 was annealed to oJR251, NaOH treated, and purified as described above.

Extension reactions with each substrate were performed with 100 nM each hydrolyzed substrate in extension buffer (40 mM Tris-acetate, pH 7.8, 12 mM magnesium acetate, 300 mM potassium glutamate, 3 μM ZnSO₄, 2% polyethylene glycol [PEG], 1 mM dithiothreitol [DTT]) with or without 1 μM Pol I at 25°C. Samples were removed and quenched with an equal volume of formamide loading dye (95% formamide, 1% SDS, 5 mM EDTA, 0.01% bromophenol blue) at 5, 25, and 75 min. Next, 300 mM NaOH was added to the remaining sample and incubated at 45°C for 60 min. One-fifth the volume of 2 M Tris-HCl, pH 7, was added to neutralize the pH, and the reactions were quenched with an equal volume of formamide loading dye. Samples were boiled for 2 min at 100°C and snap-cooled in an ice bath. The reaction products were separated by 20% urea-PAGE, followed by imaging with a Li-Cor Odyssey imager.