Structural Analysis of DnaT-ssDNA Complexes

Electron microscopy was used to examine the DnaT-phiX174 ssDNA complex with a published method that was applied for the analysis of the (39 (link)) PriB-phiX174 ssDNA complex (21 (link)). For preparing the DnaT-phiX174 ssDNA nucleoprotein filaments, the purified DnaT protein (8.0 μL; 10 mg/ml) was incubated with phiX174 ssDNA (1.6 μL; 50 μg/ml) and added buffer A to a final volume of 50 μl for 30 min at 20°C. The reaction products were diluted 4-fold with buffer A for Electron microscopy (EM) study. The final concentration of DnaT applied in the EM study was around 20 μM (400 μg/ml) and the substrate, circular phiX-174 ssDNA (5386 nt), was 0.24 nM (0.4 μg/ml) which was quite low. Other samples were prepared similar to this. For each sample, ∼3 μl protein-ssDNA complex was applied to a glow-discharged carbon-coated 400-mesh Cu EM specimen grid. Then, the sample was stained by 0.7% (w/w) uranyl formate. We imaged the protein-ssDNA particles under a low-dose condition using an FEI Tecnai F20 microscope (200 kV accelerating voltage, ∼0.6–0.8 μm underfocus) with an FEI Eagle CCD camera at 62 000× or 150 000× magnification. The final pixel sizes were 3.54 or 1.46 Å after 2-fold pixel averaging.