Zn2+ ions improve the fidelity of metal-mediated primer extension while suppressing intrinsic and Mn2+-induced mutagenic effects by DNA polymerases.

While Mn²⁺ ions are well-established for reducing the fidelity of DNA polymerases, leading to the misincorporation of nucleotides, our investigation of the effects of metal ions revealed a contrasting role of Zn²⁺. Here, we demonstrate that Zn²⁺ ions enhance the fidelity of DNA polymerases (the 3' → 5' exonuclease-deficient Klenow fragment and Taq DNA polymerase) by suppressing misincorporation during primer extension reactions. Remarkably, Zn²⁺ ions inhibit both intrinsic misincorporation and Mn²⁺-induced misincorporation of nucleotides. Furthermore, Zn²⁺ ions also effectively suppressed misincorporation during metal-mediated primer extension reactions, which involved forming Ag⁺ and Hg²⁺ ion-mediated base pairs. These findings suggest that Zn²⁺ ions inhibit both intrinsic and Mn²⁺-induced mismatched base pair formation. Consequently, the combined use of Mn²⁺ and Zn²⁺ ions may offer a strategy for precisely regulating the fidelity of DNA polymerases. Remarkably, Zn²⁺ ions even suppress misincorporation in primer extension reactions that rely on metal-mediated base pairs, and conversely, this suggests that DNA polymerases recognize metal-mediated base pairs such as T-Hg²⁺-T, C-Ag⁺-A, and C-Ag⁺-T as relatively stable base pairs. These results imply that Zn²⁺ ions may also enhance the fidelity of DNA polymerases when incorporating non-canonical nucleobases, potentially paving the way for the expansion of the genetic alphabet.