Figure 4. Primer extension assays for optimizing enzymatic activity of Sau-PolC-ΔNΔExo.

(A) Duplex DNA sequence used for all primer extension assays performed in this study. “*/” at 5′ end of primer indicates 6-FAM label. (B) Effect of pH (C) Effect of NaCl concentration (D) Effect of Mg²⁺ concentration and (E) Effect of temperature on Sau-PolC-ΔNΔExo activity. Primer extension assays were carried out under steady-state conditions by adding 1 mM dTTP (the correct incoming dNTP) to a pre-incubated solution of 400 nM p/t DNA and 1 nM Sau-PolC-ΔNΔExo. Reactions were quenched after 2 minutes by addition of an equal volume of 250 mM EDTA. Unextended and extended primers were separated by gel electrophoresis on a 17% denaturing TBE-acrylamide gel. Fraction of primer DNA extended was determined by measuring the relative intensity of the extended primer band with respect to the total labeled DNA (extended and unextended primer).