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Self-correcting mismatches during high-fidelity DNA replication

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.

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Figure 1: Cryo-EM structure of the E. coli PolIIIα–clamp–exonuclease–θ–DNA complex in editing mode.
Figure 2: Protein-DNA contacts in editing mode.
Figure 3: A terminal mismatch increases fraying of the DNA.

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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.

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Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Meindert H Lamers.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Microscopy data analysis and validation.

(a) Representative micrograph of the PolIIIα-clamp-exonuclease-θ-τ500-DNA complex. (b) 2D class averages derived from the initial 148,532-particle dataset. (c) Schematic representation of the cryo-EM data processing strategy. (d) Fourier shell correlation for the PolIIIα-clamp-exonuclease-θ-DNA complex with and without focussed classification. The correlation between two independently refined halves of the data is indicated in solid lines (gold-standard FSC) and the correlation between the rigid-body docked model and the map in dashed line. (e) Local resolution map for the whole reconstruction and the map after focussed classification and alignment of clamp-signal subtracted particles. See also Supplementary Video 3. (f) Example of the PolIIIα subunit fitted into the cryo-EM map. (g) Examples of the DNA fitted into the cryo-EM map.

Source data

Supplementary Figure 2 Sequence analysis of PolIIIα homologs.

(a) Sequence logo showing conservation profile of tyrosine 453 in the replicative C-family polymerases (top panel) and non-replicative C-family polymerases (bottom panel). (b) Sequence logo for exonuclease residues lysine 141 and arginine 142 from bacteria that use a separate exonuclease like E. coli (top panel) and from bacteria that use an intrinsic PHP-like exonuclease (Rock, J. M. et al., Nat. Genet. 47, 677–681, 2015).

Supplementary Figure 3 DNA polymerization and editing modes of A-, B-, and C-family DNA polymerases.

(a) Polymerization and (b) editing modes of A, B, and C-family DNA polymerases are shown in cartoon representation. Polymerase domains are colored in orange and exonuclease domains in yellow. Structures for polymerization mode are: T. aquaticus Pol I (1QTM (Li, Y. et al., Proc. Natl. Acad. Sci. USA 96, 9491–9496, 1999)), E. coli Pol II (3K57 (Wang, F. et al., Cell 139, 1279–1289, 2009)), E. coli PolIIIα (5KFW (Fernandez-Leiro R. et al., Elife 4, 459, 2015)). Structure for the editing mode are: E. coli Pol I (1KLN)(Beese, L. S. et al., Science 260, 352–355, 1993)), P. abyssi Pol B (4FLW (Gouge, J., et al., J. Mol. Biol. 423, 315–336, 2012)), E. coli PolIIIα (this work) (c) Comparison of the DNA molecules in both modes. Light gray: polymerase mode, black: editing mode. (d) Close-up of exonuclease active sites of Pol I, Pol B, and Pol III. Catalytic residues are shown in stick representation and colored green. D357 in Pol I (shown in white sticks) was mutated to alanine in the structure and was mutated back to the original aspartate for clarity.

Supplementary Figure 4 2D NOESY spectrum at 278 K of the matched DNA substrate.

(a) Diagonal peaks are observed for all imino protons except for the terminal base pairs. Off-diagonal cross-peaks indicate through-space connectivities between neighboring imino protons. See Methods for further experimental details

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Tables 1 and 2 (PDF 1342 kb)

Supplementary Data Set 1

Uncropped gel images for Figures 2 and 3 (PDF 568 kb)

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

Fitting of the high-resolution structures into the cryo-EM map. (MP4 15156 kb)

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. (MP4 2712 kb)

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). (MP4 74 kb)

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Fernandez-Leiro, R., Conrad, J., Yang, JC. et al. Self-correcting mismatches during high-fidelity DNA replication. Nat Struct Mol Biol 24, 140–143 (2017). https://doi.org/10.1038/nsmb.3348

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