The effect of different divalent cations on the kinetics and fidelity of <em>Bacillus stearothermophilus</em> DNA polymerase

Although Mg 2+ is the metal ion that functions as the cofactor for DNA polymerases (DNA pols) in vivo , Mn 2+ can also serve in this capacity but it reduces base discrimination. Metal ions aside from Mg 2+ or Mn 2+ 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 Mg 2+ or Mn 2+ 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 Co 2+ and Cd 2+ were the only cations tested that could replace Mg 2+ or Mn 2+ . When Co 2+ was substituted for Mg 2+ , the incorporation efficiency for correct dNTPs increased 6-fold but for incorrect dNTPs there was a decrease which depended on the incoming dNTP. With Mn 2+ , base selectivity was impaired compared to Co 2+ and Cd 2+ . In addition, Co 2+ and Mn 2+ helped BST pol to catalyze primer-extension past a mismatch. Finally both Co 2+ and Mn 2+ enhanced ground-state binding of both correct and incorrect dNTPs to BST pol: Dideoxy terminated primer-template complexes.