No abstract

Divalent metal ions, usually Mg2+, are required for both DNA synthesis and proofreading functions by DNA polymerases (DNA Pol). Although used as a non-reactive cofactor substitute for binding and crystallographic studies, Ca2+ supports DNA polymerization by only one DNA Pol, Dpo4. Here, we explore whether Ca2+-driven catalysis might apply to high-fidelity (HiFi) family B DNA Pols. The consequences of replacing Mg2+ by Ca2+ on base pairing at the polymerase active site as well as the editing of terminal nucleotides at the exonuclease active site of the archaeal Pyrococcus abyssi DNA Pol (PabPolB) are characterized and compared to other (families B, A, Y, X, D) DNA Pols. Based on primer extension assays, steady-state kinetics and ion-chased experiments, we demonstrate that Ca2+ (and other metal ions) activates DNA synthesis by PabPolB. While showing a slower rate of phosphodiester bond formation, nucleotide selectivity is improved over that of Mg2+. Further mechanistic studies show that the affinities for primer/template are higher in the presence of Ca2+ and reinforced by a correct incoming nucleotide. Conversely, no exonuclease degradation of the terminal nucleotides occurs with Ca2+. Evolutionary and mechanistic insights among DNA Pols are thus discussed.