Figure 1.

DNA polymerase activity of PrimPol is most strongly promoted by manganese. (A) Recombinant full-length human PrimPol (a.a. 1–560) was purified from Escherichia coli. (B) The polymerase activity of purified recombinant PrimPol was confirmed with an active-site titration. PrimPol (0.25 μM, ●; 0.5 μM, ■; 1 μM, ▲) was incubated with p/t-DNA (2.5 μM) and dCTP insertion was measured. Linear regression analysis resulted in a y-intercept of 0.32 ± 0.04, 0.53 ± 0.01, and 0.99 ± 0.13 μM for PrimPol concentrations of 0.25, 0.5 and 1 μM, respectively. (C) PrimPol (250 nM) was incubated with 13/18-mer p/t-DNA (2.5 μM), a mixture of all four dNTPs (250 μM each) and seven different metal ions at the indicated concentrations. The reactions were allowed to proceed for 30 min, and the products were separated by 16% (w/v) polyacrylamide/7 M urea gel electrophoresis. DNA synthesis is most robust with Mn²⁺ as the cofactor.