Modulation of DNA Polymerase Noncovalent Kinetic Transitions by Divalent Cations.
Abstract
Replicative DNA polymerases (DNAPs) require divalent metal cations for phosphodiester bond formation in the polymerase site and for hydrolytic editing in the exonuclease site. Me(2+) 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 Me(2+) ions can influence the noncovalent transitions that occur during each nucleotide addition cycle. Using a nanopore, transitions in individual Φ29 DNAP complexes are resolved with single-nucleotide spatial precision and sub-millisecond temporal resolution. We studied Mg(2+) and Mn(2+), which support catalysis, and Ca(2+), 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. Me(2+) also promotes an increase in the backward translocation rate that is dependent upon the primer terminal 3'-OH group. Me(2+) modulates the translocation rates but not their response to f...Continue Reading
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