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Mechanism of RecF–RecO–RecR cooperation in bacterial homologous recombination

Nature Structural & Molecular Biology volume 30, pages 650–660 (2023)Cite this article

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Abstract

In bacteria, one type of homologous-recombination-based DNA-repair pathway involves RecFOR proteins that bind at the junction between single-stranded (ss) and double-stranded (ds) DNA. They facilitate the replacement of SSB protein, which initially covers ssDNA, with RecA, which mediates the search for homologous sequences. However, the molecular mechanism of RecFOR cooperation remains largely unknown. We used Thermus thermophilus proteins to study this system. Here, we present a cryo-electron microscopy structure of the RecF–dsDNA complex, and another reconstruction that shows how RecF interacts with two different regions of the tetrameric RecR ring. Lower-resolution reconstructions of the RecR–RecO subcomplex and the RecFOR–DNA assembly explain how RecO is positioned to interact with ssDNA and SSB, which is proposed to lock the complex on a ssDNA–dsDNA junction. Our results integrate the biochemical data available for the RecFOR system and provide a framework for its complete understanding.

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Fig. 1: Overall structures of complexes of RecFOR proteins with DNA.
Fig. 2: RecF–DNA interactions in RecF–DNA and RecFR–DNA complexes.
Fig. 3: RecF–RecR interfaces.
Fig. 4: Biochemical validation of the contacts within RecFOR–DNA complexes.
Fig. 5: Structure of the RecFOR–DNA complex.

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Data availability

Atomic models are available in the Protein Data Bank (PDB) under the accession codes 8A8J (RecF–DNA), 8A93 (RecFR–DNA), 8AB0 (RecOR–DNA) and 8BPR (RecFOR–DNA). The corresponding cryo-EM reconstructions are available in the EM Data Bank under the accession codes EMD-15231, EMD-15267, EMD-15308 and EMD-16164. This study also used publicly available models of Rec proteins with the following PDB accession codes: 5Z68, 5ZVQ, 4JCV and 5Z2V. The raw data for fluorescence anisotropy, pull-down assays, glycerol gradient sedimentation and fourier-transform infrared spectroscopy have been uploaded in public repository on the Zenodo website (accession number 7515083). Source data are provided with this paper.

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Acknowledgements

We thank W. Yang for critically reading the manuscript and M. Arends for proofreading the manuscript. This work was financed by the MAESTRO grant from the National Science Center, Poland (2017/26/A/NZ1/01098). This publication was developed under the provision of the Polish Ministry of Education and Science project, ‘Support for research and development with the use of research infrastructure of the National Synchrotron Radiation Centre SOLARIS,’ under contract no. 1/SOL/2021/2. We acknowledge the SOLARIS Centre for access to the cryo-EM Beamline, where the measurements were performed.

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Authors and Affiliations

  1. Laboratory of Protein Structure, International Institute of Molecular and Cell Biology, Warsaw, Poland

    Shivlee Nirwal, Mariusz Czarnocki-Cieciura, Anuradha Chaudhary, Weronika Zajko, Sebastian Chamera, Małgorzata Figiel & Marcin Nowotny

  2. Biophysics and Bioanalytics Facility, International Institute of Molecular and Cell Biology, Warsaw, Poland

    Krzysztof Skowronek

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Contributions

S.N. prepared the cryo-EM sample and solved the structure of the RecFOR–DNA complexes. S.N. and M.C.-C. analyzed cryo-EM data. A.C. and W.Z. purified proteins. S.N. performed biochemical studies. K.S. performed biophysical protein characterization. S.C. and M.F. performed initial protein production. S.N., M.C.-C. and M.N. wrote the manuscript.

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Correspondence to Marcin Nowotny.

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Nature Structural & Molecular Biology thanks Michael Cox and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Florian Ullrich and Dimitris Typas, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Cryo-EM data processing.

(a) Three-dimensional reconstruction pipeline, showing initial processing steps (pre-processing, particle picking, and initial 3D classification). (b) Representative cryo-EM micrograph (out of 7,217 collected). (c) Representative 2D classes for RecF-DNA particles. (d) Representative 2D classes for RecFOR-DNA particles.

Extended Data Fig. 2 Three-dimensional reconstruction pipeline and quality of cryo-EM maps.

(a) RecF-DNA, (b) RecF-RecR-DNA, (c) RecO-RecR-DNA, and (d) RecF-RecO-RecR-DNA. (e) Top: gold-standard Fourier Shell Correlation (FSC) curves between two half maps, model-to-map FSC curves, and histograms of directional FSC (calculated by the 3DFSC web-server64). Horizontal lines represent a value of 0.143. Bottom: viewing direction distribution graphs. (f–i) Local resolution calculated from half maps in cryoSPARC for RecF-DNA (f), RecF-RecR-DNA (g), RecO-RecR-DNA (h) and RecF-RecO-RecR-DNA (i) reconstructions.

Extended Data Fig. 3 Overall structure of RecF-DNA complex and comparison with the crystal structure.

(a) High-resolution cryo-EM potential map of RecF-DNA complex. The two RecF protomers are displayed as orange and dark pink, and DNA is in white. (b) Two views of the RecF-DNA complex shown in cartoon representation. The color scheme is the same as in (a). The DNA is in black. (c) Side view of superposition of RecF structures. RecF-RecR-DNA reconstruction (present study) is shown in yellow/sand for RecF and purple/cyan/pink for RecR. The DNA is in black. RecF structure from RecF-DNA reconstruction (present study) is shown in same color scheme as in (a). The apo RecF structure (PDB ID: 5Z68) is shown in green. The proteins were superimposed using the ATPase domains and are shown in wire representation. The helical clamp is shown in cartoon representation. (d) Bottom view of superposition of RecF structures to show the difference in clamp placement. The color scheme is the same as in (c).

Extended Data Fig. 4 Quality of cryo-EM maps and model-to-map fits.

(a) RecF-DNA with close-up views of the selected secondary structures. (b) RecFR-DNA with close-up views of the DNA (left) and the interface between RecF and RecR proteins. (c) RecOR-DNA with close-up views of the selected parts of the model. (d) RecFOR-DNA with close-up views of the selected parts of the model. High resolution models (RecF, DNA, and part of RecR) are shown in wire and stick and lower-resolution models (RecO and RecR) are in cartoon representation.

Extended Data Fig. 5 Multiple sequence alignment of RecF protein.

Sequences aligned with Promals3D66. Residues in cyan are involved in DNA binding. Residues in gray are involved in RecR binding.

Extended Data Fig. 6 Multiple sequence alignment of RecR protein.

Sequences aligned with Promals3D66. Residues in gray are involved in RecF binding.

Extended Data Fig. 7 Comparison of RecR structures.

(a) Crystal structure of Tt-RecR (PDB ID: 5ZVQ) in surface representation, with a cartoon of tetramer formation. (b) Structures of RecR rings shown in the same orientation after they were superimposed using cyan, yellow, and green chains (marked with asterisk) from each structure.

Extended Data Fig. 8 Modeling of the complete RecR ring.

The flexible RecR protomer that was only partially visible in the cryo-EM reconstruction was modeled by superimposing the crystal structure of the Tt-RecR monomer (PDB ID: 5ZVQ) on the incomplete RecR subunit of the RecFOR-DNA model using N-terminal HhH motifs. The modeled RecR chain is shown in lightblue. (a) RecF in surface representation and RecR as cartoon. (b) Two views with RecR in surface representation. RecO has been omitted for clarity.

Extended Data Fig. 9 Secondary structure content and structural integrity of RecF and RecR tryptophan variants.

Fourier-transform infrared spectra of RecF wildtype (WT), RecF A170W, RecR WT, and RecR A147W are shown.

Extended Data Fig. 10 Control experiments for the glycerol density gradient sedimentation analysis of RecFOR proteins in the presence of 3′ overhang dsDNA.

Silver-stained SDS-PAGE analysis of the fractions from the control experiments are shown. Fractions from low to high molecular weight were analyzed and their numbers are given on the top of the gels. The proteins used in each experiment are indicated on the left of each gel. Each experiment was repeated three times. ‘M’ lane shows the loading control. The gel at the top shows standard protein ruler and loading control. RecF, RecO and RecR proteins were applied in 2:1:4 molar ratio in loading controls.

Source data

Supplementary information

Reporting Summary

Supplementary Table

List of the primers used for cloning and mutagenesis.

Source data

Source Data Fig. 4

Uncropped gels used for Figs. 4a,b (quantification),g,h.

Source Data Fig. 4

Densitometry results for the SDS–PAGE analysis of RecF–RecR pull-down fractions.

Source Data Extended Data Fig. 10

Uncropped gels.

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Nirwal, S., Czarnocki-Cieciura, M., Chaudhary, A. et al. Mechanism of RecF–RecO–RecR cooperation in bacterial homologous recombination. Nat Struct Mol Biol 30, 650–660 (2023). https://doi.org/10.1038/s41594-023-00967-z

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