National Center for
4ECW: Human Dna Polymerase Eta - Dna Ternary Complex: Reaction In The At Crystal At Ph 7.0 For 250 Sec
Nature (2012) 487 p.196-201
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 eta, DNA and dATP were co-crystallized at pH 6.0 without Mg(2+). The polymerization reaction was initiated by exposing crystals to 1 mM Mg(2+) at pH 7.0, and stopped by freezing at desired time points for structural analysis. The substrates and two Mg(2+) 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 Mg(2+) ion, which arrives with the new bond and stabilizes the intermediate state, may be an unappreciated feature of the two-metal-ion mechanism.