Watching DNA polymerase η make a phosphodiester bond

DNA synthesis has been extensively studied, but the chemical reaction itself has not been visualized. Here we follow the course of phosphodiester bond formation using time-resolved X-ray crystallography. Native human DNA polymerase η, DNA and dATP were co-crystallized at pH 6.0 without Mg2+. The polymerization reaction was initiated by exposing crystals to 1 mM Mg2+ at pH 7.0, and stopped by freezing at desired time points for structural analysis. The substrates and two Mg2+ ions are aligned within 40 s, but the bond formation is not evident until 80 s. From 80 to 300 s structures show a mixture of decreasing substrate and increasing product of the nucleotidyl-transfer reaction. Transient electron densities indicate that deprotonation and an accompanying C2′-endo to C3′-endo conversion of the nucleophile 3′-OH are rate limiting. A third Mg2+ ion, which arrives with the new bond and stabilizes the intermediate state, may be an unappreciated feature of the two-metal-ion mechanism.