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Effect of Different Divalent Cations on the Kinetics and Fidelity of RB69 DNA Polymerase

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journal contribution
posted on 20.04.2016, 00:00 by Ashwani Kumar Vashishtha, William H. Konigsberg
Although Mg2+ is the cation that functions as the cofactor for the nucleotidyl transfer reaction for almost all DNA polymerases, Mn2+ can also serve, but when it does, the degree of base discrimination exhibited by most DNA polymerases (pols) is diminished. Metal ions other than Mg2+ or Mn2+ can also act as cofactors depending on the specific DNA polymerase. Here, we tested the ability of several divalent metal ions to substitute for Mg2+ or Mn2+ with RB69 DNA polymerase (RB69pol), a model B-family pol. Our choice of metal ions was based on previous studies with other DNA pols. Co2+, and to a lesser extent Ni2+, were the only cations among those tested besides Mg2+ and Mn2+ that could serve as cofactors with RB69pol. The incorporation efficiency of correct dNMPs increased by 5-fold with Co2+, relative to that of Mg2+. The incorporation efficiencies of incorrect dNMPs increased by 2–17-fold with Co2+, relative to that with Mg2+ depending on the incoming dNTP. Base selectivity was reduced even further with Mn2+ compared to that observed with Co2+. Substitution of Mn2+, Co2+, or Ni2+ for Mg2+ reduced the exonuclease activity of RB69pol by 2-, 6-, and 33-fold, respectively, contributing to the frequency of misincorporation. In addition, Co2+ and Mn2+ were better able to extend a primer past a mismatch than Mg2+. Finally, Co2+ and Mn2+ enhanced ground-state binding of both correct and incorrect dNTPs to RB69pol:dideoxy-terminated primer–template complexes.

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