Modulation of DNA Polymerase Noncovalent Kinetic Transitions by Divalent Cations

Replicative DNA polymerases (DNAPs) require divalent metal cations for phosphodiester bond formation in the polymerase site and for hydrolytic editing in the exonuclease site. Me2+ ions are intimate architectural components of each active site, where they are coordinated by a conserved set of amino acids and functional groups of the reaction substrates. Therefore Me2+ ions can influence the noncovalent transitions that occur during each nucleotide addition cycle. Using a nanopore, transitions in individual phi 29 DNAP complexes are resolved with single-nucleotide spatial precision and sub-millisecond temporal resolution. We studied Mg2+ and Mn2+, which support catalysis, and Ca2+, which supports deoxynucleoside triphosphate (dNTP) binding but not catalysis. We examined their effects on translocation, dNTP binding, and primer strand transfer between the polymerase and exonuclease sites. All three metals cause a concentration-dependent shift in the translocation equilibrium, predominantly by decreasing the forward translocation rate. Me2+ also promotes an increase in the backward translocation rate that is dependent upon the primer terminal 3-OH group. Me2+ modulates the translocation rates but not their response to force, suggesting that Me2+ does not affect the distance to the transition state of translocation. Absent Me2+, the primer strand transfer pathway between the polymerase and exonuclease sites displays additional kinetic states not observed at >1 mm Me2+. Complementary dNTP binding is affected by Me2+ identity, with Ca2+ affording the highest affinity, followed by Mn2+, and then Mg2+. Both Ca2+ and Mn2+ substantially decrease the dNTP dissociation rate relative to Mg2+, while Ca2+ also increases the dNTP association rate.