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ma=86400 The selection process of licensing a DNA mismatch for repair | Nature Structural & Molecular Biology
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The selection process of licensing a DNA mismatch for repair

Nature Structural & Molecular Biology volume 28, pages 373–381 (2021)Cite this article

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Abstract

DNA mismatch repair detects and removes mismatches from DNA by a conserved mechanism, reducing the error rate of DNA replication by 100- to 1,000-fold. In this process, MutS homologs scan DNA, recognize mismatches and initiate repair. How the MutS homologs selectively license repair of a mismatch among millions of matched base pairs is not understood. Here we present four cryo-EM structures of Escherichia coli MutS that provide snapshots, from scanning homoduplex DNA to mismatch binding and MutL activation via an intermediate state. During scanning, the homoduplex DNA forms a steric block that prevents MutS from transitioning into the MutL-bound clamp state, which can only be overcome through kinking of the DNA at a mismatch. Structural asymmetry in all four structures indicates a division of labor between the two MutS monomers. Together, these structures reveal how a small conformational change from the homoduplex- to heteroduplex-bound MutS acts as a licensing step that triggers a dramatic conformational change that enables MutL binding and initiation of the repair cascade.

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Fig. 1: Structures of MutS at consecutive steps of the repair cascade.
Fig. 2: Licensing of DNA mismatch repair.
Fig. 3: An intermediate state between mismatch and MutL binding.
Fig. 4: MutS−DNA−MutLLN40 complex.
Fig. 5: Kinking of the lever domain promotes clamp formation.
Fig. 6: MutS heterodimers are mismatch-repair competent.

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Data availability

Cryo-EM maps and atomic models have been deposited in the Electron Microscopy Database and Protein Data Bank, respectively, under accession codes EMD-11791, PDB 7AI5, EMD-11792, PDB 7AI6, EMD-11793, PDB 7AI7, EMD-11794, PDB 7AIB, EMD-11795 and PDB 7AIC. Other requests should be addressed to Meindert Lamers (m.h.lamers@lumc.nl), Titia K. Sixma (t.sixma@nki.nl) or Rafael Fernández-Leiro (rfleiro@cnio.es). Source data are provided with this paper.

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Acknowledgements

All cryo-EM data were collected at the LMB cryo-EM facility. We thank C. Savva for cryo-EM support; and J. Grimmett, T. Darling, J. L. Fernández and P. Reviriego for IT support. This work has been supported by a UK Medical Research Council grant U105197143 and LUMC Research Fellowship to M.H.L.; the Oncode Institute, NWO-TOP 714.016.002 and NWO-Gravity CGC.nl to T.K.S.; a Spanish Ministry of Economy and Competitiveness Grant BFU2017-87316 to R.F.L.; NWO-Gravity CGC.nl and the Oncode Institute to J.H.G.L.; and European Community’s Horizon2020 Marie Skłodowska Curie grant [722433] to P.F.

Author information

Authors and Affiliations

  1. MRC Laboratory of Medical Research, Cambridge, UK

    Rafael Fernandez-Leiro & Meindert H. Lamers

  2. Spanish National Cancer Research Centre, Madrid, Spain

    Rafael Fernandez-Leiro

  3. Division of Biochemistry and Oncode Institute, Netherlands Cancer Institute, Amsterdam, the Netherlands

    Doreth Bhairosing-Kok, Herrie H. Winterwerp, Flora Groothuizen, Alexander Fish & Titia K. Sixma

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

    Vladislav Kunetsky & Peter Friedhoff

  5. Department of Molecular Genetics and Oncode Institute, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, the Netherlands

    Charlie Laffeber & Joyce H. G. Lebbink

  6. Department of Radiation Oncology, Erasmus University Medical Center, Rotterdam, the Netherlands

    Joyce H. G. Lebbink

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

    Meindert H. Lamers

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Contributions

R.F.-L., T.K.S. and M.H.L. conceived the overall experimental design; R.F.L. prepared samples and collected and processed cryo-EM data; D.B.-K. purified proteins and performed SPR experiments. A.F. helped with SPR data analysis. V.K. performed crosslinking experiments and MutH nicking assays on crosslinked MutS; P.F. designed crosslinking experiments; J.H.G.L and C.L. performed MutH nicking assays on MutS heterodimers; F.G. and H.H.W. prepared the MutS−MutLLN40 crosslinked complex; R.F.-L., T.K.S and M.H.L. wrote the manuscript, with contributions from all authors.

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Correspondence to Rafael Fernandez-Leiro, Titia K. Sixma or Meindert H. Lamers.

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The authors declare no competing interests.

Additional information

Peer review information Nature Structural & Molecular Biology thanks Jean-Baptiste Charbonnier, Elizabeth Kellogg and Cynthia McMurray for their contribution to the peer review of this work. Peer reviewer reports are available. Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 CryoEM data analysis of scanning state MutS.

a, Representative micrograph (0° tilt). Plasmid DNA can be observed on the micrograph with MutS dimers bound like beads-on-string. b, 2D class averages from full dataset. c, Schematic representation of main data processing procedures. See methods section for more details. d, Principle Component Analysis of multibody refinement data. e, Detail of model fit to map. f, Final refinement map colored by local resolution. g, Fourier Shell Correlation between half-maps from final refinement. Green line: unmasked. Blue line: masked. Red line: phase randomised. Grey line: corrected h, Model-vs-map Fourier Shell Correlation. Grey line: model vs full-map. Blue line: model refined against half-map 1, FSC against half-map 1. Red-line: shaked model refined against half-map 1, FSC against half-map 2.

Extended Data Fig. 2 CryoEM data analysis of mismatch-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, Detail of model fit to map. e, Final refinement map colored by local resolution. f, Fourier Shell Correlation between half-maps from final refinement. Green line: unmasked. Blue line: masked. Red line: phase randomized. Grey line: corrected g, Model-vs-map Fourier Shell Correlation. Grey line: model vs full-map. Blue line: model refined against half-map 1, FSC against half-map 1. Red line: shaken model refined against half-map 1, FSC against half-map 2.

Extended Data Fig. 3 CryoEM data analysis of intermediate state 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, Detail of model fit to map. e, Final refinement map colored by local resolution. f, Fourier Shell Correlation between half-maps from final refinement. Green line: unmasked. Blue line: masked. Red line: phase randomized. Grey line: corrected g, Model-vs-map Fourier Shell Correlation. Grey line: model vs full-map. Blue line: model refined against half-map 1, FSC against half-map 1. Red line: shaken model refined against half-map 1, FSC against half-map 2.

Extended Data Fig. 4 CryoEM data analysis of MutLLN40-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, Detail of model fit to map. e, Final refinement map colored by local resolution. f, Fourier Shell Correlation between half-maps from final refinement. Green line: unmasked. Blue line: masked. Red line: phase randomized. Grey line: corrected g, Model-vs-map Fourier Shell Correlation. Grey line: model vs full-map. Blue line: model refined against half-map 1, FSC against half-map 1. Red-line: shaken model refined against half-map 1, FSC against half-map 2.

Extended Data Fig. 5 Comparison of crystal and cryo-EM structure of mismatch-bound MutS.

a, Front view of the crystal structure of mismatch-bound MutS (1E3M) fitted into in the cryo-EM map of mismatch-bound MutS. b, Side view, with additional stretches of DNA shown in red. c, Close up of the mismatch binding domain of monomer B with former position of the mismatch binding domain shown in yellow and the ~22o domain rotation indicated by a black arrow. Additional visible loops, modeled after the mismatch binding of monomer A, are highlighted in red. d, Same view as in panel c, showing the cryo-EM map for the DNA, mismatch binding domain and connector domain.

Extended Data Fig. 6 Mismtach and MutL binding surfaces in MutS.

a, Mismatch-bound MutS showing the mismatch and MutL binding surfaces. MutS monomer A in green, monomer B in light-blue, mismatch binding in dark blue, MutL Interface 1 in dark red, and MutL Interface 2 in light red. b, Binding of ATP transforms MutS into a sliding clamp that brings MutL-Interface 1 and 2 together, creating the binding site for MutL c, MutS bound to MutLLN40.

Extended Data Fig. 7 Engineered MutS homo- and heterodimers.

Front view (top row) and reverse view (bottom row) of the MutS dimer a, WT MutS showing mismatch and MutL binding surface on both sides of the dimer b, Interface 1 mutant lacks one half of MutL-binding site on either side of the MutS dimer. c, Similarly, Interface 2 mutant also lacks one half of the MutL-binding site on either side of the MutS dimer. d, Mismatch mutant lacks the mismatch binding site in both monomers. e, Mixing of Interface 1 and Interface 2 mutant creates a heterodimer with an intact MutL binding site on one side, but lacks the MutL-binding interface on the reverse side. f, A double Mismatch-Interface 1 mutant mixed with an Interface 2 mutant generates a MutS dimer with an intact MutL binding site on one side and the mismatch binding site on the reverse side. This configuration is similar to the eukaryotic MSH2-MSH6 and MSH2-MSH3 hetero dimers. g, A double Mismatch-Interface 2 mutant mixed with an Interface 1 mutant generates a MutS dimer with both MutL and mismatch binding sites on the same face of the MutS dimer.

Supplementary information

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Supplementary Table 1.

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Supplementary Video 1

Molecular mechanism of DNA mismatch repair initiation, related to Fig. 1. Front and side views of MutS passing through the first four stages of the repair cascade: DNA scanning, mismatch recognition, intermediate state, and MutL recruitment. The movements show a computational morphing between the four cryo-EM structures described in this work and are labeled in the movie. MutS monomer A is shown in a pale-green color, monomer B in pale blue, DNA in dark gray and MutLLN40 in yellow.

Supplementary Video 2

Mismatch repair licensing at a mismatch, related to Fig. 2. Top and side views of MutS as it transforms from the DNA-scanning state to the mismatch-bound state. The initial part of the movie represents the movement of monomer B relative to monomer A during the scanning state, as derived from the principal component analysis of the multibody refinement. Note that the MutS dimer explores multiple conformations, attempting to distort the DNA, without crossing over the opposite monomer. When a mismatch is present on the DNA, it allows MutS to deform and kink the DNA and the two MutS monomers to cross over in a clockwise manner. Movements show a computational morphing between the different states. MutS monomer A is shown in a pale-green color, monomer B in pale blue, and DNA in gray. The DNA mismatch is highlighted in pink.

Supplementary Video 3

Multiple conformational changes of mismatch and connector domains tracking DNA, related to Fig. 3. Front and side views of MutS as it goes from the mismatch-bound state to the MutLLN40-bound clamp state via the intermediate state. MutS monomer A is shown in a pale green color, monomer B in pale blue, and DNA in dark gray. DNA mismatch is highlighted in pink. The mismatch domain is shown in dark green and the connector domain in light green. The ends of a central helix in the connector domain are colored in red and blue for clarity. Movements show a computational morphing between the different states.

Source data

Source Data Fig. 3

Uncropped gels.

Source Data Fig. 4

Uncropped gels.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data for SPR.

Source Data Fig. 6

Uncropped gels for nicking assays.

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Fernandez-Leiro, R., Bhairosing-Kok, D., Kunetsky, V. et al. The selection process of licensing a DNA mismatch for repair. Nat Struct Mol Biol 28, 373–381 (2021). https://doi.org/10.1038/s41594-021-00577-7

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