A conformational switch in response to Chi converts RecBCD from phage destruction to DNA repair

Abstract

The RecBCD complex plays key roles in phage DNA degradation, CRISPR array acquisition (adaptation) and host DNA repair. The switch between these roles is regulated by a DNA sequence called Chi. We report cryo-EM structures of the Escherichia coli RecBCD complex bound to several different DNA forks containing a Chi sequence, including one in which Chi is recognized and others in which it is not. The Chi-recognized structure shows conformational changes in regions of the protein that contact Chi and reveals a tortuous path taken by the DNA. Sequence specificity arises from interactions with both the RecC subunit and the sequence itself. These structures provide molecular details for how Chi is recognized and insights into the changes that occur in response to Chi binding that switch RecBCD from bacteriophage destruction and CRISPR spacer acquisition to constructive host DNA repair.

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Fig. 1: Structural changes associated with Chi binding.
Fig. 2: Details of the Chi-binding site interactions.
Fig. 3: Details of the full Chi-binding site.
Fig. 4: Comparison of RecBCD (top) and AddAB (bottom) Chi-bound complexes.
Fig. 5: The nuclease active site in the Chi-bound RecBCD and AddAB complexes.

Data availability

The cryo-electron density maps and coordinates of the final refined models have been deposited at the EMDB and wwPDB, respectively, with the following accession codes: EMD-10214 and PDB 6SJB (Chi-recognized); EMD-10215, PDB 6SJE (Chi-intermediate); EMD-10216, PDB 6SJF (Chi-unrecognized); EMD-10369, PDB 6T2U (Chi-minus 2); EMD-10370, PDB 6T2V (Chi-plus 2).

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Acknowledgements

We thank Diamond for access to and the support of the cryo-EM facilities at the UK National Electron Bio-Imaging Centre (eBIC), funded by the Wellcome Trust, MRC and BBSRC. The work was funded by the Medical Research Council (MR/N009258/1). Cryo-EM facilities at the Imperial College Centre for Structural Biology are supported by the Wellcome Trust.

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Authors

Contributions

K.C. conducted biochemical experiments and prepared samples. K.C., M.W. and Y.C. collected cryo-EM data. K.C. and M.W. processed data. K.C., M.W. and D.B.W. analysed data. D.B.W. wrote the manuscript with input from K.C. and M.W.

Corresponding author

Correspondence to Dale B. Wigley.

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The authors declare no competing interests.

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Peer review information Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 CryoEM processing flow chart (Chi-containing substrate).

a, Scheme overview of the cryoEM processing steps for RecBCD with the Chi-containing substrate. Masks used during 3D classifications are shown in green. To display the separation of particles with ordered and disordered Chi sequences, 2D central slices are displayed of each respective masked 3D model. Final maps are coloured by local resolution as estimated by ResMap40. The scale bar shows the colour scale with resolution in Å. b, Upper, an example of a typical micrograph showing particle distribution. Lower, a selection of 2D class averages representing different particle views. c, ‘Gold standard’ FSC curve showing estimated global resolution of the three structures.

Extended Data Fig. 2 CryoEM processing flow chart (Chi-minus substrate).

a, Scheme overview of the cryoEM processing steps for the RecBCD with the Chi-minus (20 base T residue 3’-tail) control substrate. Masks used during 3D classifications are shown in green. Classification with a mask around Chi showed one class with no Chi density as indicated in the displayed 2D central slice of the masked 3D model. The final map is coloured by local resolution as estimated by ResMap45. The scale bar shows the colour scale with resolution in Å. b, Upper, an example of a typical micrograph showing particle distribution. Lower, a selection of 2D class averages representing different particle views. c, ‘Gold standard’ FSC curve showing estimated global resolution of the structure.

Extended Data Fig. 3 DNA density of the six structures.

The DNA density of the Chi-recognised, Chi-intermediate, Chi-unrecognised, Chi-minus, Chi-minus 2 and Chi-plus 2 complexes are shown as transparent surfaces with the built models in cartoon and sticks. The Chi sequence, where present, is coloured in red. All the maps are contoured to same level in Chimera (0.008).

Extended Data Fig. 4 CryoEM processing flow chart (minus 2 substrate).

a, Scheme overview of the cryoEM processing steps for the RecBCD with the Chi-minus 2 substrate (a 3’-tail contained Chi with 6 base T before Chi and 4 base T after Chi, the rest are the same as Chi-containing or Chi-minus substrates). Masks used during 3D classifications are shown in green. Classification with a mask around Chi showed one class with no Chi density as indicated in the displayed 2D central slice of the masked 3D model. The final map is coloured by local resolution as estimated by ResMap45. The scale bar shows the colour scale with resolution in Å. b, Upper, an example of a typical micrograph showing particle distribution. Lower, a selection of 2D class averages representing different particle views. c, ‘Gold standard’ FSC curve showing estimated global resolution of the structure.

Extended Data Fig. 5 CryoEM processing flow chart (plus 2 substrate).

a, Scheme overview of the cryoEM processing steps for the RecBCD with the Chi-plus 2 substrate (a 3’-tail contained Chi with 10 base T before Chi and 4 base T after Chi, the rest are the same as Chi-containing or Chi-minus substrates). Masks used during 3D classifications are shown in green. Classification with a mask around Chi showed one class with no Chi density as indicated in the displayed 2D central slice of the masked 3D model. The final map is coloured by local resolution as estimated by ResMap45. The scale bar shows the colour scale with resolution in Å. b, Upper, an example of a typical micrograph showing particle distribution. Lower, a selection of 2D class averages representing different particle views. c, ‘Gold standard’ FSC curve showing estimated global resolution of the structure.

Extended Data Fig. 6 Euler angle distribution of the cryo-EM reconstructions.

Euler angle distribution of (a) the Chi-recognised complex, (b) Chi-intermediate complex, (c) Chi-unrecognised complex, (d) Chi-minus complex, (e) Chi-minus 2 complex, (f) Chi-plus 2 complex (f).

Supplementary information

41594_2019_355_MOESM2_ESM.mov

Rocking model of the Chi sequence (red) within the bound ssDNA (yellow). Side chains (cyan) from RecC (blue ribbon) that contact the Chi bases are shown with hydrogen bonds as black dotted lines. DNA density is in blue mesh and the protein in black mesh.

41594_2019_355_MOESM3_ESM.mov

Morph movie from the Chi unrecognized model, first to the intermediate model, showing conformational changes in the bound ssDNA, followed by morphing to the Chi-recognized model, showing the protein conformational changes. RecB is in orange, RecC in blue, RecD in green, the Chi bases in red, those after Chi in yellow and the remaining DNA bases in gray.

41594_2019_355_MOESM4_ESM.mov

Rocking model of the Chi bases interacting with side chains of RecC colored according to mutant properties using the same color scheme as Fig. 3.

Reporting Summary

Supplementary Video 1

Rocking model of the Chi sequence (red) within the bound ssDNA (yellow). Side chains (cyan) from RecC (blue ribbon) that contact the Chi bases are shown with hydrogen bonds as black dotted lines. DNA density is in blue mesh and the protein in black mesh.

Supplementary Video 2

Morph movie from the Chi unrecognized model, first to the intermediate model, showing conformational changes in the bound ssDNA, followed by morphing to the Chi-recognized model, showing the protein conformational changes. RecB is in orange, RecC in blue, RecD in green, the Chi bases in red, those after Chi in yellow and the remaining DNA bases in gray.

Supplementary Video 3

Rocking model of the Chi bases interacting with side chains of RecC colored according to mutant properties using the same color scheme as Fig. 3.

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Cheng, K., Wilkinson, M., Chaban, Y. et al. A conformational switch in response to Chi converts RecBCD from phage destruction to DNA repair. Nat Struct Mol Biol 27, 71–77 (2020). https://doi.org/10.1038/s41594-019-0355-2

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