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.
Highlights
DNA polymerases replicate genomic DNA with extremely high fidelity [1]
Attempts have been made in the past to find out why these metal ions foster the mutagenic behaviors of DNA pols but most of these efforts have focused on Mn2+ [11,12,22]
For example: 1) metal ions can affect the ground-state binding affinity of the correct and incorrect dNTPs to pol/P/T binary complexes; 2) they can promote misincorporation during primer extension; 3) intrinsic exonuclease activity can be diminished resulting in the failure to remove incorrectly incorporated dNMPs; 4) the efficiency of extension beyond the mismatch could be affected upon encountering a mismatch at the P/T terminus, resulting in mutations being embedded in the DNA; 5) the metal ion could affect enzymes in the DNA repair pathways [16]
Summary
DNA polymerases replicate genomic DNA with extremely high fidelity [1]. A two metal ion catalytic mechanism has been widely accepted for DNA pols [2]. All DNA polymerases known to date require two and sometimes three divalent cations (usually Mg2+) for the nucleotidyl transfer reaction but only two are needed to catalyze the 3′→5′ exonuclease activity associated with replicative DNA pols. Even though DNA pols utilize the physiologically relevant Mg2+, Mg2+ can substitute for Mg2+ in the nucleotidyl transfer reaction but, when this occurs, it tends to lower the base selectivity dramatically [3,4,5,6]. The metal ion in the “B” site coordinates the α-, β-, and γ-phosphate oxygens of the incoming dNTPs, assists in neutralizing the developing negative charge in the transition state, and assists the departure of the PPi product. The third metal ion likely helps to neutralize the negative charge built up in the transition state and may help in protonating the leaving PPi [8]. A number of metal ions have been shown to be mutagens and carcinogens and some may act by reducing the accuracy of DNA replication [15], there are many other possibilities that would produce the same results such as interference by metal ions in DNA repair pathways [16]
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