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- Title
Watching DNA polymerase ? make a phosphodiester bond.
- Authors
Nakamura, Teruya; Zhao, Ye; Yamagata, Yuriko; Hua, Yue-jin; Yang, Wei
- 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 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.
- Subjects
DNA polymerases; X-ray crystallography; PHOSPHODIESTERASE inhibitors; POLYMERASE chain reaction; NUCLEOTIDYLTRANSFERASES; PROTON transfer reactions
- Publication
Nature, 2012, Vol 487, Issue 7406, p196
- ISSN
0028-0836
- Publication type
Article
- DOI
10.1038/nature11181