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Watching DNA polymerase ? 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 ?, 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.

SUBMITTER: Nakamura T

PROVIDER: S-EPMC3397672 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Watching DNA polymerase η make a phosphodiester bond.

Nakamura Teruya T Zhao Ye Y Yamagata Yuriko Y Hua Yue-jin YJ Yang Wei W

Nature 20120711 7406

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 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 align ...[more]

PMID: 22785315

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