The effect of different divalent cations on the kinetics and fidelity of Bacillus stearothermophilus DNA polymerase.

Abstract

Although Mg²⁺ is the metal ion that functions as the cofactor for DNA polymerases (DNA pols) in vivo, Mn²⁺ can also serve in this capacity but it reduces base discrimination. Metal ions aside from Mg²⁺ or Mn²⁺ can act as cofactors for some DNA pols but not for others. Here we report on the ability of several divalent metal ions to substitute for Mg²⁺ or Mn²⁺ with BST DNA polymerase (BST pol), an A family DNA pol. We selected the metal ions based on whether they had previously been shown to be effective with other DNA pols. We found that Co²⁺ and Cd²⁺ were the only cations tested that could replace Mg²⁺ or Mn²⁺. When Co²⁺ was substituted for Mg²⁺, the incorporation efficiency for correct dNTPs increased 6-fold but for incorrect dNTPs there was a decrease which depended on the incoming dNTP. With Mn²⁺, base selectivity was impaired compared to Co²⁺ and Cd²⁺. In addition, Co²⁺ and Mn²⁺ helped BST pol to catalyze primer-extension past a mismatch. Finally both Co²⁺ and Mn²⁺ enhanced ground-state binding of both correct and incorrect dNTPs to BST pol: Dideoxy terminated primer-template complexes.