Figure EV3. Polymerase and exonuclease activity assays in the presence of α‐Thio dNTPs.

1. Phosphorothioate linkage is resistant to 3′–5′ exonuclease activity of T7 DNAP. (i) and (ii) Design of the DNA substrates used for the experiments. A mismatched 35‐mer/65‐mer primer/template with the primer having a fluorescein moiety at 5′ end and a phosphorothioate linkage at 3′ end was used. Control primer/template did not have a phosphorothioate linkage. Excision reactions were conducted on a rapid quenched‐flow apparatus and analyzed on 24% denaturing polyacrylamide gels. (iii) Relative intensities of 35‐mer primer left at different time points.
2. Real‐time helicase unwinding traces (i) and determined unwinding rates with dTTP and dTTPαS (ii). Moles of nucleotides incorporated (iii) and excised (iv) per mole of substrate DNA during leading strand synthesis reactions performed in the presence of α‐thio‐dNTP (dNTPαS).

Data information: Bars show the data from two individual experiments. Circles in two shades of the same color show data points from two individual experiments. Mean data are fitted to linear trends.