Watching DNA polymerase η make a phosphodiester bond

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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 η, 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.

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Figure 1: pH and metal-ion dependence of Pol η.
Figure 2: Reaction time course.
Figure 3: Deprotonation of the 3′-OH.
Figure 4: The third Mg 2+ ion in Pol η catalysis.
Figure 5: A proposed mechanism for metal-ion-dependent polymerase reaction.

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Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data Bank with accession codes from 4ECQ to 4ECZ, 4ED0 to 4ED3, and 4ED6 to 4ED8.

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Acknowledgements

We thank D. Leahy, M. Gellert and R. Craigie for editing the manuscript. The research was supported by the intramural research program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (W.Y., T.N. and Y.Z.); the Japan Society for the Promotion of Science Institutional Program for Young Researcher Overseas visits, Kumamoto University, and the Kumayaku Alumni Research Fund (T.N.); Chinese Ministry of Education scholarship (Y.Z.); National Natural Science Foundation of China (Y.-J.H.); and Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Y.Y.).

Author information

T.N. and Y.Z. carried out crystallography. Y.Z. did the mutagenesis and kinetic analyses. T.N. handled crystals, refined structures and analysed reaction time courses. W.Y. designed the project. T.N., Y.Z. and W.Y. prepared the manuscript. Y.Y. supervised T.N. and Y.-J.H. advised Y.Z. All authors discussed the results and commented on the manuscript.

Correspondence to Wei Yang.

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This file contains an animation of the chemical bond formation. (MOV 15373 kb)

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Nakamura, T., Zhao, Y., Yamagata, Y. et al. Watching DNA polymerase η make a phosphodiester bond. Nature 487, 196–201 (2012) doi:10.1038/nature11181

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