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Watching DNA polymerase η make a phosphodiester bond

Nature volume 487, pages 196–201 (2012)Cite this article

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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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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.

References

  1. Echols, H. & Goodman, M. F. Fidelity mechanisms in DNA replication. Annu. Rev. Biochem. 60, 477–511 (1991)

    Article  CAS  Google Scholar 

  2. Rothwell, P. J. & Waksman, G. Structure and mechanism of DNA polymerases. Adv. Protein Chem. 71, 401–440 (2005)

    Article  CAS  Google Scholar 

  3. Brody, R. S. & Frey, P. A. Unambiguous determination of the stereochemistry of nucleotidyl transfer catalyzed by DNA polymerase I from Escherichia coli . Biochemistry 20, 1245–1252 (1981)

    Article  CAS  Google Scholar 

  4. Steitz, T. A. & Steitz, J. A. A general two-metal-ion mechanism for catalytic RNA. Proc. Natl Acad. Sci. USA 90, 6498–6502 (1993)

    Article  ADS  CAS  PubMed Central  Google Scholar 

  5. Yang, W., Lee, J. Y. & Nowotny, M. Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity. Mol. Cell 22, 5–13 (2006)

    Article  CAS  Google Scholar 

  6. Johnson, K. A. Role of induced fit in enzyme specificity: a molecular forward/reverse switch. J. Biol. Chem. 283, 26297–26301 (2008)

    Article  CAS  PubMed Central  Google Scholar 

  7. Warshel, A. et al. Electrostatic basis for enzyme catalysis. Chem. Rev. 106, 3210–3235 (2006)

    Article  CAS  Google Scholar 

  8. Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H. & Kraut, J. Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP. Science 264, 1891–1903 (1994)

    Article  ADS  CAS  Google Scholar 

  9. Doublie, S., Tabor, S., Long, A. M., Richardson, C. C. & Ellenberger, T. Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution. Nature 391, 251–258 (1998)

    Article  ADS  CAS  Google Scholar 

  10. Huang, H., Chopra, R., Verdine, G. L. & Harrison, S. C. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science 282, 1669–1675 (1998)

    Article  ADS  CAS  Google Scholar 

  11. Li, Y., Korolev, S. & Waksman, G. Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation. EMBO J. 17, 7514–7525 (1998)

    Article  CAS  PubMed Central  Google Scholar 

  12. Franklin, M. C., Wang, J. & Steitz, T. A. Structure of the replicating complex of a pol α family DNA polymerase. Cell 105, 657–667 (2001)

    Article  CAS  Google Scholar 

  13. Ling, H., Boudsocq, F., Woodgate, R. & Yang, W. Crystal structure of a Y-family DNA polymerase in action: a mechanism for error-prone and lesion-bypass replication. Cell 107, 91–102 (2001)

    Article  CAS  Google Scholar 

  14. Berman, A. J. et al. Structures of phi29 DNA polymerase complexed with substrate: the mechanism of translocation in B-family polymerases. EMBO J. 26, 3494–3505 (2007)

    Article  CAS  PubMed Central  Google Scholar 

  15. Johnson, S. J., Taylor, J. S. & Beese, L. S. Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations. Proc. Natl Acad. Sci. USA 100, 3895–3900 (2003)

    Article  ADS  CAS  Google Scholar 

  16. Evans, R. J. et al. Structure of PolC reveals unique DNA binding and fidelity determinants. Proc. Natl Acad. Sci. USA 105, 20695–20700 (2008)

    Article  ADS  CAS  Google Scholar 

  17. Wang, F. & Yang, W. Structural insight into translesion synthesis by DNA Pol II. Cell 139, 1279–1289 (2009)

    Article  PubMed Central  Google Scholar 

  18. Batra, V. K. et al. Magnesium-induced assembly of a complete DNA polymerase catalytic complex. Structure 14, 757–766 (2006)

    Article  CAS  PubMed Central  Google Scholar 

  19. Patel, S. S., Wong, I. & Johnson, K. A. Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. Biochemistry 30, 511–525 (1991)

    Article  CAS  Google Scholar 

  20. Joyce, C. M. & Benkovic, S. J. DNA polymerase fidelity: kinetics, structure, and checkpoints. Biochemistry 43, 14317–14324 (2004)

    Article  CAS  Google Scholar 

  21. Showalter, A. K. & Tsai, M. D. A reexamination of the nucleotide incorporation fidelity of DNA polymerases. Biochemistry 41, 10571–10576 (2002)

    Article  CAS  Google Scholar 

  22. Shah, A. M., Li, S. X., Anderson, K. S. & Sweasy, J. B. Y265H mutator mutant of DNA polymerase β. Proper teometric alignment is critical for fidelity. J. Biol. Chem. 276, 10824–10831 (2001)

    Article  CAS  Google Scholar 

  23. Fiala, K. A. & Suo, Z. Mechanism of DNA polymerization catalyzed by Sulfolobus solfataricus P2 DNA polymerase IV. Biochemistry 43, 2116–2125 (2004)

    Article  CAS  Google Scholar 

  24. Cramer, J. & Restle, T. Pre-steady-state kinetic characterization of the DinB homologue DNA polymerase of Sulfolobus solfataricus . J. Biol. Chem. 280, 40552–40558 (2005)

    Article  CAS  Google Scholar 

  25. Choi, J. Y. & Guengerich, F. P. Adduct size limits efficient and error-free bypass across bulky N2-guanine DNA lesions by human DNA polymerase η. J. Mol. Biol. 352, 72–90 (2005)

    Article  CAS  Google Scholar 

  26. Rothwell, P. J., Mitaksov, V. & Waksman, G. Motions of the fingers subdomain of klentaq1 are fast and not rate limiting: implications for the molecular basis of fidelity in DNA polymerases. Mol. Cell 19, 345–355 (2005)

    Article  CAS  Google Scholar 

  27. Zhang, H., Cao, W., Zakharova, E., Konigsberg, W. & De La Cruz, E. M. Fluorescence of 2-aminopurine reveals rapid conformational changes in the RB69 DNA polymerase-primer/template complexes upon binding and incorporation of matched deoxynucleoside triphosphates. Nucleic Acids Res. 35, 6052–6062 (2007)

    Article  CAS  PubMed Central  Google Scholar 

  28. Yang, W. & Woodgate, R. What a difference a decade makes: insights into translesion DNA synthesis. Proc. Natl Acad. Sci. USA 104, 15591–15598 (2007)

    Article  ADS  CAS  Google Scholar 

  29. Biertümpfel, C. et al. Structure and mechanism of human DNA polymerase η. Nature 465, 1044–1048 (2010)

    Article  ADS  PubMed Central  Google Scholar 

  30. Zhao, Y., Biertümpfel, C., Gregory, M. T., Hua, Y. J., Hanaoka, F. & Wang, W. Structural basis of human DNA polymerase η-mediated chemoresistance to cisplatin. Proc. Natl Acad. Sci. USA 109, 7269–7274 (2012)

    Article  ADS  CAS  Google Scholar 

  31. Castro, C. et al. Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases. Proc. Natl Acad. Sci. USA 104, 4267–4272 (2007)

    Article  ADS  CAS  Google Scholar 

  32. Garcia-Diaz, M., Bebenek, K., Krahn, J. M., Pedersen, L. C. & Kunkel, T. A. Role of the catalytic metal during polymerization by DNA polymerase lambda. DNA Repair 6, 1333–1340 (2007)

    Article  CAS  PubMed Central  Google Scholar 

  33. Le Du, M. H. et al. Artificial evolution of an enzyme active site: structural studies of three highly active mutants of Escherichia coli alkaline phosphatase. J. Mol. Biol. 316, 941–953 (2002)

    Article  CAS  Google Scholar 

  34. Graham, D. L. et al. MgF3 − as a transition state analog of phosphoryl transfer. Chem. Biol. 9, 375–381 (2002)

    Article  CAS  Google Scholar 

  35. Lee, J. Y. & Yang, W. UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke. Cell 127, 1349–1360 (2006)

    Article  CAS  PubMed Central  Google Scholar 

  36. Cavanaugh, N. A., Beard, W. A. & Wilson, S. H. DNA polymerase β ribonucleotide discrimination: insertion, misinsertion, extension, and coding. J. Biol. Chem. 285, 24457–24465 (2010)

    Article  CAS  PubMed Central  Google Scholar 

  37. Wang, L., Broyde, S. & Zhang, Y. Polymerase-tailored variations in the water-mediated and substrate-assisted mechanism for nucleotidyl transfer: insights from a study of T7 DNA polymerase. J. Mol. Biol. 389, 787–796 (2009)

    Article  CAS  PubMed Central  Google Scholar 

  38. Wang, L., Yu, X., Hu, P., Broyde, S. & Zhang, Y. A water-mediated and substrate-assisted catalytic mechanism for Sulfolobus solfataricus DNA polymerase IV. J. Am. Chem. Soc. 129, 4731–4737 (2007)

    Article  CAS  PubMed Central  Google Scholar 

  39. Polesky, A. H., Dahlberg, M. E., Benkovic, S. J., Grindley, N. D. & Joyce, C. M. Side chains involved in catalysis of the polymerase reaction of DNA polymerase I from Escherichia coli . J. Biol. Chem. 267, 8417–8428 (1992)

    CAS  PubMed  Google Scholar 

  40. Copeland, W. C. & Wang, T. S. Mutational analysis of the human DNA polymerase α. The most conserved region in α-like DNA polymerases is involved in metal-specific catalysis. J. Biol. Chem. 268, 11028–11040 (1993)

    CAS  PubMed  Google Scholar 

  41. Zakharova, E., Wang, J. & Konigsberg, W. The activity of selected RB69 DNA polymerase mutants can be restored by manganese ions: the existence of alternative metal ion ligands used during the polymerization cycle. Biochemistry 43, 6587–6595 (2004)

    Article  CAS  Google Scholar 

  42. Kim, E. E. & Wyckoff, H. W. Reaction mechanism of alkaline phosphatase based on crystal structures. Two-metal ion catalysis. J. Mol. Biol. 218, 449–464 (1991)

    Article  CAS  Google Scholar 

  43. Romier, C., Dominguez, R., Lahm, A., Dahl, O. & Suck, D. Recognition of single-stranded DNA by nuclease P1: high resolution crystal structures of complexes with substrate analogs. Proteins 32, 414–424 (1998)

    Article  CAS  Google Scholar 

  44. Garcin, E. D. et al. DNA apurinic–apyrimidinic site binding and excision by endonuclease IV. Nature Struct. Mol. Biol. 15, 515–522 (2008)

    Article  CAS  Google Scholar 

  45. Shan, S., Yoshida, A., Sun, S., Piccirilli, J. A. & Herschlag, D. Three metal ions at the active site of the Tetrahymena group I ribozyme. Proc. Natl Acad. Sci. USA 96, 12299–12304 (1999)

    Article  ADS  CAS  Google Scholar 

  46. Stahley, M. R. & Strobel, S. A. Structural evidence for a two-metal-ion mechanism of group I intron splicing. Science 309, 1587–1590 (2005)

    Article  ADS  CAS  Google Scholar 

  47. Ding, X., Rasmussen, B. F., Petsko, G. A. & Ringe, D. Direct structural observation of an acyl-enzyme intermediate in the hydrolysis of an ester substrate by elastase. Biochemistry 33, 9285–9293 (1994)

    Article  CAS  Google Scholar 

  48. Scott, W. G., Murray, J. B., Arnold, J. R., Stoddard, B. L. & Klug, A. Capturing the structure of a catalytic RNA intermediate: the hammerhead ribozyme. Science 274, 2065–2069 (1996)

    Article  ADS  CAS  Google Scholar 

  49. Murray, J. B., Szoke, H., Szoke, A. & Scott, W. G. Capture and visualization of a catalytic RNA enzyme–product complex using crystal lattice trapping and X-ray holographic reconstruction. Mol. Cell 5, 279–287 (2000)

    Article  CAS  Google Scholar 

  50. Yang, W. Nucleases: diversity of structure, function and mechanism. Q. Rev. Biophys. 44, 1–93 (2011)

    Article  PubMed Central  Google Scholar 

  51. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  PubMed Central  Google Scholar 

  52. Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010)

    Article  CAS  PubMed Central  Google Scholar 

  53. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  54. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    Article  CAS  Google Scholar 

  55. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot . Acta Crystallogr. D 66, 486–501 (2010)

    Article  CAS  PubMed Central  Google Scholar 

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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.).

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Author notes

  1. Teruya Nakamura and Ye Zhao: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Teruya Nakamura, Ye Zhao & Wei Yang

  2. Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, 862-0973, Japan

    Teruya Nakamura & Yuriko Yamagata

  3. Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, 310029, China

    Ye Zhao & Yue-jin Hua

Authors
  1. Teruya Nakamura
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Contributions

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.

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Correspondence to Wei Yang.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-3 and Supplementary Figures 1-5. (PDF 2963 kb)

Supplementary Movie 1

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). https://doi.org/10.1038/nature11181

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