Watching DNA polymerase η make a phosphodiester bond

Abstract

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. Atomic-resolution time courses of phosphodiester bond formation catalysed by DNA polymerase η reveal transient intermediate states and an unexpected third metal ion in the reaction mechanism. Chemists would like to be able to determine the structures of true transition states in chemical reactions, but the high energy and unstable nature of transition states had made this goal unattainable. Using a repair reaction catalysed by DNA polymerase η (Pol η) as their model, Wei Yang and colleagues have extended the use of flash–freeze technology to observe DNA synthesis in real time and at atomic resolution using X-ray crystallography to analyse the trapped covalent intermediates. Pol η is particularly well suited to this approach because it has a slow rate of reaction and a relatively rigid catalytic centre. The observed reaction intermediates reveal several unanticipated transient states, and implicate an unexpected third magnesium ion in the reaction mechanism.