Effect of different divalent cations on the kinetics and fidelity of DNA polymerases

DNA polymerases (DNA pols) are essential for accurately copying genomes of all organisms. The polymerase and exonuclease activities associated with DNA pols require the presence of two divalent cations which occupy the A and B metal ion sites. Even though the two-metal ion mechanism is generally applicable for all DNA pols, a third metal ion was proposed to be essential for phosphoryl transfer reaction. The metal ion in the A site is coordinated by six ligands including the 3' hydroxyl group of the primer, the carboxylates of two aspartic acid residues, as well as water molecules and the metal ion exhibits a distorted octahedral geometry. This metal ion plays a crucial role in lowering the pK(a) of the 3' hydroxyl group of the primer increasing its nucleophilicity for attack on a phosphorous atom of the incoming dNTP. The metal ion occupying the B site stabilizes the transition state and is coordinated to the non-bridging oxygen atoms of the incoming dNTP and carboxylates of aspartic acid residues along with carboxyl oxygen of an adjacent peptide bond. In a similar fashion, two divalent cations are required for the 3'-5' exonuclease activity of DNA pols. Analogous to their role in the polymerase active site, one divalent cation lowers the pK(a) of the water molecule making it a more potent nucleophile and the other cation helps to stabilize the transition state, assisting in excision of the 3' terminal nucleotide. These divalent cations affect the various fidelity checkpoints along minimal kinetic scheme for DNA pols. The effect of different divalent cations on various steps in the kinetic scheme, including their influence on ground-state binding affinity, base selectivity, efficiency of extension past a mismatch and the exonuclease activity are discussed. We have also attempted to explain why only certain divalent cations act as cofactors for various DNA pols based on their properties including ionic radii, coordination geometry, and their ability to lower the pK(a) of the 3' hydroxyl group of primer strand.