Calcium-driven DNA synthesis by a high-fidelity DNA polymerase

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.

Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase

“…2). Ca 2+ -mediated catalysis of DNA polymerase activity, which we incidentally observed here, has been previously reported (18,19), but the precise role of Ca 2+ in the phosphodiesterforming mechanism remains to be elucidated. By cocrystallization, we observe Ca 2+ bound to the substrate triphosphate moiety in a manner similar to that seen for Mg 2+ crystallized under similar conditions, but there remains the possibility that our structures do not capture transiently bound metal ions that are nevertheless critical to the reaction mechanism, as has recently been proposed for certain eukaryotic polymerase family members (34,35).…”

“…2 B and C), maximally ∼4.5 nt/h with Ca 2+ . Phosphodiester-forming activity with Ca 2+ has been reported (18,19). Here, two additions of dCTP were observed on a GG-containing template in the presence of either Ca 2+ or Mg 2+ , where the first is linked via a phosphoramidate ester and the second via a phosphodiester ( Fig.…”

Role of RadA and DNA Polymerases in Recombination-Associated DNA Synthesis in Hyperthermophilic Archaea

“…The p41/p46 complex is involved in RNA primer synthesis but is also a potential DNA repair enzyme [37][38][39][40]. Several biochemical characterization studies contributed to show that both PolB and PolD are processive enzymes with high nucleotide selectivity and 3 -5 proofreading activities [32][33][34]39,[41][42][43][44][45][46].…”

Liquid drop of DNA libraries reveals total genome information

“…quasi single-molecule amplification | uniform distribution of amplicons | diversity degeneration | emulsion PCR modeling | template mispairing U nlike the tightly controlled replication of DNA in living cells, PCR amplification, a "workhorse" of molecular biology, balances between simplicity and accuracy. While sequence accuracy can be achieved by using high-fidelity polymerases (1)(2)(3) and specific amplification protocols (4)(5)(6), the uniform amplification of complex DNA templates remains challenging. The difficulties are associated with variable amplification rates of different templates and uncontrolled mispairings that are caused by DNA repeats or homologous sequences, commonly encountered, respectively, during genomic or antibody library preparation.…”