Long-Range PCR Amplification of DNA by DNA Polymerase III Holoenzyme from Thermus thermophilus

Figure 3

Requirements for individual components of the Tth pol III holoenzyme. DNA synthesis was used to monitor the activity of the subunits [7]. The pol III holoenzyme was reconstituted using native forms of each subunit. The components that were not being tested were held constant at saturating concentrations (0.05 μM , 0.01 μM , 1.4 μM β (monomer), 0.05 μM DnaX (monomer), and 0.5 μM α) while the test subunit was titrated into the reactions as indicated. Reactions were at 55°C. The subunits titrated into the reactions were (a) α, (b) β, (c) DnaX, (d) , and (e) . The concentrations of proteins were calculated based on the molecular mass of single subunits. “Activity” is defined as the total pmol of nucleotides incorporated.

(a)

(b)

(c)

(d)

(e)