Watching DNA polymerase eta make a phosphodiester bond

Article

关键词: CRYSTAL-STRUCTURES;    2-METAL-ION MECHANISM;    TERNARY COMPLEXES;    TRANSITION-STATE;    STRUCTURAL BASIS;    ACTIVE-SITE;    SUBSTRATE;    FIDELITY;    METAL;    BINDING;   

DOI  :  10.1038/nature11181

来源: SCIE

【 摘 要 】

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 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.

【 授权许可】

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