Different Divalent Cations Alter the Kinetics and Fidelity of DNA Polymerases

Abstract

Divalent metal ions are essential components of DNA polymerases both for catalysis of the nucleotidyl transfer reaction and for base excision. They occupy two sites, A and B, for DNA synthesis. Recently, a third metal ion was shown to be essential for phosphoryl transfer reaction. The metal ion in the A site is coordinated by the carboxylate of two highly conserved acidic residues, water molecules, and the 3'-hydroxyl group of the primer so that the A metal is in an octahedral complex. Its catalytic function is to lower the pK_a of the hydroxyl group, making it a highly effective nucleophile that can attack the α phosphorous atom of the incoming dNTP. The metal ion in the B site is coordinated by the same two carboxylates that are affixed to the A metal ion as well as the non-bridging oxygen atoms of the incoming dNTP. The carboxyl oxygen of an adjacent peptide bond serves as the sixth ligand that completes the octahedral coordination geometry of the B metal ion. Similarly, two metal ions are required for proofreading; one helps to lower the pK_a of the attacking water molecule, and the other helps to stabilize the transition state for nucleotide excision. The role of different divalent cations are discussed in relation to these two activities as well as their influence on base selectivity and misincorporation by DNA polymerases. Some, but not all, of the effects of these different metal ions can be rationalized based on their intrinsic properties, which are tabulated in this review.

Keywords: calcium; magnesium; manganese; nickel; protein complex.

SCHEME 1.

Minimal kinetic scheme for DNA polymerases depicting various fidelity checkpoints along the reaction pathway. EDN represents the open conformation of the ternary collision complex, whereas FDN represents the closed conformation. Additional details are provided in the text.

FIGURE 1.

The structure of the tm RB69 pol ternary complex (Protein Data Bank (PDB) accession number 3SJJ). a, ternary complex showing dUpNpp bound in the active site with metal ions A and B. b, close-up of the tm RB69 pol active site showing the B metal ion in perfect octahedral geometry and the A metal ion in a distorted octahedral geometry. c, predicted position of 3′-OH group in an idealized octahedron during the transition state. d, a close-up of the coordination complex showing the two metal ions, the 3′-OH group, and the α phosphate of the incoming dNTP.

FIGURE 2.

Comparison of metal ion bound structures of the tm RB69 pol with other DNA polymerases. a, superposition of the tm RB69 pol with T7 DNA pol (PDB accession number: 1T7P, green), HIV reverse transcriptase (HIV RT) (PDB accession number: 1RTD, cyan), and Dpo4 (PDB accession number: 2AGQ, magenta). b, superposition of the tm RB69 pol with pol β (PDB accession number: 2FMS, golden).

SCHEME 2.

Minimal Kinetic scheme for DNA polymerases depicting the ground-state binding affinity (K_d,_g) and apparent binding affinity (K_d,app) for an incoming dNTP. EDN represents the open conformation of the ternary collision complex, whereas FDN represents the closed conformation.