Article | Published:

Self-correcting mismatches during high-fidelity DNA replication

Nature Structural & Molecular Biology volume 24, pages 140143 (2017) | Download Citation

Abstract

Faithful DNA replication is essential to all forms of life and depends on the action of 3′–5′ exonucleases that remove misincorporated nucleotides from the newly synthesized strand. However, how the DNA is transferred from the polymerase to the exonuclease active site is not known. Here we present the cryo-EM structure of the editing mode of the catalytic core of the Escherichia coli replisome, revealing a dramatic distortion of the DNA whereby the polymerase thumb domain acts as a wedge that separates the two DNA strands. Importantly, NMR analysis of the DNA substrate shows that the presence of a mismatch increases the fraying of the DNA, thus enabling it to reach the exonuclease active site. Therefore the mismatch corrects itself, whereas the exonuclease subunit plays a passive role. Hence, our work provides unique insights into high-fidelity replication and establishes a new paradigm for the correction of misincorporated nucleotides.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

References

  1. 1.

    The kinetic and chemical mechanism of high-fidelity DNA polymerases. Biochim. Biophys. Acta 1804, 1041–1048 (2010).

  2. 2.

    & Structures of mismatch replication errors observed in a DNA polymerase. Cell 116, 803–816 (2004).

  3. 3.

    , , , & Identification of the epsilon-subunit of Escherichia coli DNA polymerase III holoenzyme as the dnaQ gene product: a fidelity subunit for DNA replication. Proc. Natl. Acad. Sci. USA 80, 7085–7089 (1983).

  4. 4.

    & DNA polymerase III accessory proteins. V. Theta encoded by holE. J. Biol. Chem. 268, 11785–11791 (1993).

  5. 5.

    & DNA polymerase III of Escherichia coli: purification and identification of subunits. J. Biol. Chem. 254, 1748–1753 (1979).

  6. 6.

    , & Mechanism of the sliding beta-clamp of DNA polymerase III holoenzyme. J. Biol. Chem. 266, 11328–11334 (1991).

  7. 7.

    , & Mechanism of the E. coli tau processivity switch during lagging-strand synthesis. Mol. Cell 11, 315–327 (2003).

  8. 8.

    et al. Mechanism of polymerase collision release from sliding clamps on the lagging strand. EMBO J. 28, 2981–2991 (2009).

  9. 9.

    & The Escherichia coli preprimosome and DNA B helicase can form replication forks that move at the same rate. J. Biol. Chem. 262, 16644–16654 (1987).

  10. 10.

    , , & Characterization of a triple DNA polymerase replisome. Mol. Cell 27, 527–538 (2007).

  11. 11.

    et al. Fidelity of Escherichia coli DNA polymerase III holoenzyme: the effects of beta, gamma complex processivity proteins and epsilon proofreading exonuclease on nucleotide misincorporation efficiencies. J. Biol. Chem. 272, 27919–27930 (1997).

  12. 12.

    , , & Cryo-EM structures of the E. coli replicative DNA polymerase reveal its dynamic interactions with the DNA sliding clamp, exonuclease and τ. eLife 4, e11134 (2015).

  13. 13.

    & Protein-protein HADDocking using exclusively pseudocontact shifts. J. Biomol. NMR 50, 263–266 (2011).

  14. 14.

    , & DNA-protein π-interactions in nature: abundance, structure, composition and strength of contacts between aromatic amino acids and DNA nucleobases or deoxyribose sugar. Nucleic Acids Res. 42, 6726–6741 (2014).

  15. 15.

    , , & DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis. Cell 113, 183–193 (2003).

  16. 16.

    , & Structure of DNA polymerase I Klenow fragment bound to duplex DNA. Science 260, 352–355 (1993).

  17. 17.

    et al. Structure of a sliding clamp on DNA. Cell 132, 43–54 (2008).

  18. 18.

    & Phosphoesterase domains associated with DNA polymerases of diverse origins. Nucleic Acids Res. 26, 3746–3752 (1998).

  19. 19.

    et al. A structural role for the PHP domain in E. coli DNA polymerase III. BMC Struct. Biol. 13, 8 (2013).

  20. 20.

    et al. DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader. Nat. Genet. 47, 677–681 (2015).

  21. 21.

    , , & Molecular recognition of canonical and deaminated bases by P. abyssi family B DNA polymerase. J. Mol. Biol. 423, 315–336 (2012).

  22. 22.

    , , & Measurement of hydrogen exchange rates using 2D NMR spectroscopy. J. Magn. Reson. 69, 201–209 (1986).

  23. 23.

    & Effects of sequence and length on imino proton exchange and base pair opening kinetics in DNA oligonucleotide duplexes. Nucleic Acids Res. 20, 5339–5343 (1992).

  24. 24.

    & Kinetic mechanism of the 3′→5′ proofreading exonuclease of DNA polymerase III: analysis by steady state and pre-steady state methods. Biochemistry 35, 12919–12925 (1996).

  25. 25.

    , , & Architecture of the Pol III-clamp-exonuclease complex reveals key roles of the exonuclease subunit in processive DNA synthesis and repair. EMBO J. 32, 1334–1343 (2013).

  26. 26.

    et al. Structure of the theta subunit of Escherichia coli DNA polymerase III in complex with the epsilon subunit. J. Bacteriol. 188, 4464–4473 (2006).

  27. 27.

    et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).

  28. 28.

    Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

  29. 29.

    RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

  30. 30.

    Beam-induced motion correction for sub-megadalton cryo-EM particles. eLife 3, e03665 (2014).

  31. 31.

    et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013).

  32. 32.

    & Prevention of overfitting in cryo-EM structure determination. Nat. Methods 9, 853–854 (2012).

  33. 33.

    & Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

  34. 34.

    , , , & Sampling the conformational space of the catalytic subunit of human γ-secretase. eLife 4, e11182 (2015).

  35. 35.

    , & Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

  36. 36.

    , , , & Structural basis for proofreading during replication of the Escherichia coli chromosome. Structure 10, 535–546 (2002).

  37. 37.

    , , & Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

  38. 38.

    et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D Biol. Crystallogr. 71, 136–153 (2015).

  39. 39.

    , & Structure-based design of Taq DNA polymerases with improved properties of dideoxynucleotide incorporation. Proc. Natl. Acad. Sci. USA 96, 9491–9496 (1999).

  40. 40.

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

  41. 41.

    , & Spin-lattice relaxation measurements in slowly relaxing complex spectra. J. Chem. Phys. 55, 3604–3605 (1971).

  42. 42.

    , , & The MPI bioinformatics Toolkit as an integrative platform for advanced protein sequence and structure analysis. Nucleic Acids Res. 44, W410–W415 (2016).

  43. 43.

    & MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).

  44. 44.

    , , & WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).

Download references

Acknowledgements

We thank D. Neuhaus for suggestions and D. Neuhaus and R. Williams for reading of the manuscript. This work was supported by the UK Medical Research Council through grants U105197143 to M.H.L. and MC_UP_A025_1013 to S.H.W.S.

Author information

Author notes

    • Julian Conrad

    Present address: Science for Life Laboratory, Solna, Sweden.

Affiliations

  1. MRC laboratory of Molecular Biology, Cambridge, UK.

    • Rafael Fernandez-Leiro
    • , Julian Conrad
    • , Ji-Chun Yang
    • , Stefan M V Freund
    • , Sjors H W Scheres
    •  & Meindert H Lamers

Authors

  1. Search for Rafael Fernandez-Leiro in:

  2. Search for Julian Conrad in:

  3. Search for Ji-Chun Yang in:

  4. Search for Stefan M V Freund in:

  5. Search for Sjors H W Scheres in:

  6. Search for Meindert H Lamers in:

Contributions

R.F.-L. and M.H.L. designed and directed experiments. R.F.-.L. and J.C. collected and processed cryo-EM data. S.H.W.S. assisted in data processing. R.F.-L. purified proteins and performed biochemical assays. S.M.V.F. and J.-C.Y. collected and processed NMR data. M.H.L. and R.F.-L. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Meindert H Lamers.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–4 and Supplementary Tables 1 and 2

  2. 2.

    Supplementary Data Set 1

    Uncropped gel images for Figures 2 and 3

Videos

  1. 1.

    Structure of the PolIIα–clamp–exonuclease–θ–DNA complex.

    Fitting of the high-resolution structures into the cryo-EM map.

  2. 2.

    Morphing of the complex in between the polymerization and editing modes.

    The θ subunit was omitted for clarity. Template strand nucleotide 18 is colored in magenta to better visualize the screw motion of the DNA.

  3. 3.

    Interdomain movement within the complex.

    Three cryo-EM maps are shown, highlighting the inter-domain movement within the complex. Maps were generated after particle alignment by focused alignment on the clamp region using signal subtraction. Following this, particles were sorted by 3D classification without re-alignment. (See Methods for details).

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb.3348

Further reading