Download MendeleyWatching DNA polymerase eta make a phosphodiester bond
Nakamura, T., Zhao, Y., Yamagata, Y., Hua, Y.J., Yang, W.(2012) Nature 487: 196-201
- PubMed: 22785315
- DOI: https://doi.org/10.1038/nature11181
- Primary Citation of Related Structures:
4ECQ, 4ECR, 4ECS, 4ECT, 4ECU, 4ECV, 4ECW, 4ECX, 4ECY, 4ECZ, 4ED0, 4ED1, 4ED2, 4ED3, 4ED6, 4ED7, 4ED8 - PubMed 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 ¦Ç, 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.
Organizational Affiliation:
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
