Capture of a third Mg2+ is essential for catalyzing DNA synthesis

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Science  10 Jun 2016:
Vol. 352, Issue 6291, pp. 1334-1337
DOI: 10.1126/science.aad9633

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A hit-and-run metal ion

DNA polymerase is an enzyme that uses existing DNA as a template to build new DNA by adding new nucleotides to the end of the newly forming daughter strand. The enzyme mechanism that catalyzes formation of a phosphodiester bond is known to require two Mg2+ ions, and recent crystal structures have shown that a third metal ion is present after bond formation. Gao et al. used time-resolved crystallography to visualize bond formation. The enzyme-substrate complex captures a third cation before bond formation occurs, and DNA synthesis cannot occur without the third metal ion. Binding of this metal ion requires thermal motion of the enzyme-substrate complex, so that catalysis is achieved by acquiring a transient cofactor.

Science, this issue p. 1334


It is generally assumed that an enzyme-substrate (ES) complex contains all components necessary for catalysis and that conversion to products occurs by rearrangement of atoms, protons, and electrons. However, we find that DNA synthesis does not occur in a fully assembled DNA polymerase–DNA–deoxynucleoside triphosphate complex with two canonical metal ions bound. Using time-resolved x-ray crystallography, we show that the phosphoryltransfer reaction takes place only after the ES complex captures a third divalent cation that is not coordinated by the enzyme. Binding of the third cation is incompatible with the basal ES complex and requires thermal activation of the ES for entry. It is likely that the third cation provides the ultimate boost over the energy barrier to catalysis of DNA synthesis.

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