Watching DNA polymerase η make a phosphodiester bond

Nature. 2012 Jul 11;487(7406):196-201. doi: 10.1038/nature11181.


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 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.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalysis
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / metabolism*
  • Humans
  • Hydrogen Bonding
  • Hydrogen-Ion Concentration
  • Magnesium / chemistry
  • Metals / chemistry
  • Models, Molecular*
  • Nucleic Acid Conformation
  • Water / chemistry


  • Metals
  • Water
  • DNA-Directed DNA Polymerase
  • POLN protein, human
  • Magnesium

Associated data

  • PDB/4ECQ
  • PDB/4ECR
  • PDB/4ECS
  • PDB/4ECT
  • PDB/4ECU
  • PDB/4ECV
  • PDB/4ECW
  • PDB/4ECX
  • PDB/4ECY
  • PDB/4ECZ
  • PDB/4ED0
  • PDB/4ED1
  • PDB/4ED2
  • PDB/4ED3
  • PDB/4ED6
  • PDB/4ED7