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

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• 作者：Ashwani Kumar Vashishtha ; William H. Konigsberg
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：May 10, 2016
• 年：2016
• 卷：55
• 期：18
• 页码：2661-2670
• 全文大小：444K
• 年卷期：0
• ISSN：1520-4995

文摘

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.