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Cryogenic electron microscopy structures reveal how ATP and DNA binding in MutS coordinates sequential steps of DNA mismatch repair

Nature Structural & Molecular Biology volume 29, pages 59–66 (2022)Cite this article

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Abstract

DNA mismatch repair detects and corrects mismatches introduced during DNA replication. The protein MutS scans for mismatches and coordinates the repair cascade. During this process, MutS undergoes multiple conformational changes in response to ATP binding, hydrolysis and release, but how ATP induces the various MutS conformations is incompletely understood. Here we present four cryogenic electron microscopy structures of Escherichia coli MutS at sequential stages of the ATP hydrolysis cycle that reveal how ATP binding and hydrolysis induce closing and opening of the MutS dimer, respectively. Biophysical analysis demonstrates how DNA binding modulates the ATPase cycle by prevention of hydrolysis during scanning and mismatch binding, while preventing ADP release in the sliding clamp state. Nucleotide release is achieved when MutS encounters single-stranded DNA that is produced during removal of the daughter strand. The combination of ATP binding and hydrolysis and its modulation by DNA enables MutS to adopt the different conformations needed to coordinate the sequential steps of the mismatch repair cascade.

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Fig. 1: Structures of MutS at sequential steps of the ATP hydrolysis cycle.
Fig. 2: Closing of the MutS dimer completes the ATPase active site.
Fig. 3: Two ATPs are needed for MutS clamp formation.
Fig. 4: Release of MutS from DNA at a single-stranded gap.
Fig. 5: ATP and DNA combine to create sequential steps of the repair cascade.

Data availability

Cryo-EM maps and atomic models have been deposited in the Electron Microscopy Database and Protein Data Bank, respectively, under accession code nos. EMD-13073, PDB 7OU2, EMD-13074, PDB 7OU4, EMD-13063, PDB 7OTO, EMD-13071 and PDB 7OU0. Source data for the graphs in Figs. 3 and 4 are available with the paper online. Other requests should be addressed to Meindert Lamers (m.h.lamers@lumc.nl). Source data are provided with this paper.

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Acknowledgements

We thank the staff of the LUMC EM facility and NeCEN for help with data collection and data processing. We thank R. Fernandez-Leiro for advice on data processing. This work was supported by a LUMC Research Fellowship to M.H.L., and a European Community’s Horizon2020 Innovative Training Network Grant (no. 722433) to M.H.L. and P.F. Access to NeCEN was supported by Netherlands Electron Microscopy Infrastructure (project no. 184.034.014) of the National Roadmap for Large-Scale Research Infrastructure of the Dutch Research Council.

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Authors and Affiliations

  1. Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands

    Alessandro Borsellini & Meindert H. Lamers

  2. Institute for Biochemistry, Justus-Liebig University, Giessen, Germany

    Vladislav Kunetsky & Peter Friedhoff

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  1. Alessandro Borsellini
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Contributions

M.H.L. and A.B. conceived the overall experimental design. A.B. prepared samples and collected and processed cryo-EM data. A.B. purified proteins and performed BLI experiments. P.F. and V.K. designed and analyzed FRET experiments. V.K. performed FRET experiments, which were supervised by P.F. A.B. and M.H.L. wrote the manuscript with contributions from all authors.

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Correspondence to Meindert H. Lamers.

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Nature Structural and Molecular Biology thanks Jean-Baptiste Charbonnier and Keith Weninger for their contribution to the peer review of this work. Carolina Perdigoto and Beth Moorefield were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 CryoEM data analysis of ADP bound MutS.

a, Representative micrograph. b, 2D class averages from full dataset. c, Schematic representation of main data processing procedures. See methods section for more details. d, Fourier Shell Correlation between half-maps from subsequent refinements in the processing procedures. e, Detail of model fit to map. f, Final map obtained applying SuperEM code to Relion post-processed map. g, final map colored by local resolution. h, Orientation distribution in final set of refined particles.

Extended Data Fig. 2 CryoEM data analysis of ADP-ATP bound MutS.

a, Representative micrograph. b, 2D class averages from full dataset. c, Schematic representation of main data processing procedures. See methods section for more details. d, Fourier Shell Correlation between half-maps from subsequent refinements in the processing procedures. e, Detail of model fit to map. f, Final map obtained applying SuperEM code to Relion post-processed map. g, final map colored by local resolution. h, Orientation distribution in final set of refined particles.

Extended Data Fig. 3 CryoEM data analysis of ANPPNP bound MutS.

a, Representative micrograph. b, 2D class averages from full dataset. c, Schematic representation of main data processing procedures. See methods section for more details. d, Fourier Shell Correlation between half-maps from subsequent refinements in the processing procedures. e, Detail of model fit to map. f, Final map obtained applying SuperEM code to Relion post-processed map. g, final map colored by local resolution. h, Orientation distribution in final set of refined particles.

Extended Data Fig. 4 CryoEM data analysis of ADP-Vi bound MutS.

a, Representative micrograph. b, 2D class averages from full dataset. c, Schematic representation of main data processing procedures. See methods section for more details. d, Fourier Shell Correlation between half-maps from subsequent refinements in the processing procedures. e, Detail of model fit to map. f, Superimposition of nucleotide binding domains of MutS in ADP-Vi conformation and MutS in sliding clamp MutL bound conformation (Fernandez-Leiro 2021). g, Final map obtained applying SuperEM code to Relion post-processed map. h, final map colored by local resolution. i, Orientation distribution in final set of refined particles.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2 and references.

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Supplementary Video 1

Side view of ATPase domain closing upon binding of ATP and AMPPNP.

Supplementary Video 2

Top view of ATPase domain closing upon binding of ATP and AMPPNP.

Supplementary Video 3

Overview of conformational changes in MutS upon ATP and AMPPNP binding.

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Borsellini, A., Kunetsky, V., Friedhoff, P. et al. Cryogenic electron microscopy structures reveal how ATP and DNA binding in MutS coordinates sequential steps of DNA mismatch repair. Nat Struct Mol Biol 29, 59–66 (2022). https://doi.org/10.1038/s41594-021-00707-1

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