Date Published: May 23, 2018
Author(s): Ashwani Kumar Vashishtha, William H. Konigsberg.
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.
DNA polymerases replicate genomic DNA with extremely high fidelity . A two metal ion catalytic mechanism has been widely accepted for DNA pols . 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 [3–6]. Two metal ions play an important role in assembling the catalytic groups, the metal ion present in the “A” site helps to lower the pKa of the terminal 3′ OH group on the primer-terminus and coordinates both the 3′-OH of the primer strand and the α-phosphate of incoming dNTP facilitating the nucleophilic attack of the 3′-OH on the α-phosphorous of incoming dNTPs . 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 also help in protonating the leaving PPi . The effect of metal ion cofactors on the fidelity of DNA replication has been studied for various pols, including E. coli DNA pol I , AMV DNA polymerase , Klenow fragment of E. coli DNA pol I , T4 polymerase , T7 DNA polymerase , human DNA polymerase α, human DNA polymerase β , Sulfolobus solfataricus DNA pol IV (Dpo4)  and RB69pol . A number of metal ions have been shown to be mutagens and carcinogens and some may act by reducing the accuracy of DNA replication , although there are many other possibilities that would produce the same results such as interference by metal ions in DNA repair pathways .
The effect of divalent metal ions on the replication fidelity of various pols has been studied previously but the rates of incorporation of correct and incorrect nucleotides have not been determined under single-turnover conditions in the presence of different metal ion cofactors except for Mn2+ [2,22] and Co2+ . Co2+, Mn2+, and Ni2+ have been characterized as “mutagens”, since they reduce the fidelity of DNA synthesis [3,4,9–13]. 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]. Metal ions can affect the behavior of high fidelity polymerases by altering the various checkpoints along the reaction pathway. 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 also affect enzymes in the DNA repair pathways . Here, we used BST pol, a high fidelity A family DNA replicative polymerase, to systematically study the effects of various divalent metal ions on the fidelity checkpoints during DNA replication.
Several divalent cations including Co2+, Mn2+, and Ni2+ have been characterized as “mutagenic”, since they reduce the fidelity of DNA synthesis [3,9–13,15]. Previous attempts to address the reasons for the mutagenic behavior of these metal ions have mainly focused on Mn2+ [11,12]. For example, translesion DNA synthesis in herpes simplex virus Type I was promoted by Mn2+ . Also, the rate of misincorporation opposite an abasic site by T4 DNA pol was enhanced by 11–34 fold when Mn2+ was substituted for Mg2+ . In addition, studies on pol β using blunt-ended DNA showed primer-extension in the presence of Mn2+ rather than Mg2+ . Moreover, the rates of incorporation of correct and incorrect nucleotides have not been determined under single-turnover conditions in the presence of different metal ions except for Mn2+ , and Co2+ .