Watching DNA polymerase η make a phosphodiester bond.
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.
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Teruya
T
Nakamura
Ye
Y
Zhao
Yuriko
Y
Yamagata
Yue-jin
YJ
Hua
Wei
W
Yang
D002384:Catalysis; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D014867:Water / Q000737:chemistry
D008670:Metals; D014867:Water; D004259:DNA-Directed DNA Polymerase; C546205:POLN protein, human; D008274:Magnesium