FIGURE 6.

D403E Pol III* can exchange into Pol III HE during elongation on a long single-stranded template in the absence of helicase. Protein components required for a holoenzyme reconstitution experiment were added to an M13Gori template. Initiation complexes were allowed to form for 3 min at 30 °C in the presence of 1 mm ATP. The reaction was started by addition of 18 μm dNTPs, [³H]dTTP, and a competitor Pol III* (0, 24, 48, 94, 188, 375, and 750 nm). DNA synthesis was quenched after 30 s by the addition of EDTA to 140 mm. Reactions were run in triplicate. Shown are competitor mutant D403E Pol III* (blue), WT Pol III* (red), and buffer control (green). The buffer control samples were prepared identically to the Pol III* samples except that no Pol III* was added.