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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.

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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 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.

References

  1. Li, Z., Pearlman, A. H. & Hsieh, P. DNA mismatch repair and the DNA damage response. DNA Repair (Amst.) 38, 94–101 (2016).

    Article  Google Scholar 

  2. Jiricny, J. Postreplicative mismatch repair. Cold Spring Harb. Perspect. Biol. 5, a012633 (2013).

  3. Welsh, K. M., Lu, A. L., Clark, S. & Modrich, P. Isolation and characterization of the Escherichia coli mutH gene product. J. Biol. Chem. 262, 15624–15629 (1987).

    Article  CAS  PubMed  Google Scholar 

  4. Junop, M. S., Yang, W., Funchain, P., Clendenin, W. & Miller, J. H. In vitro and in vivo studies of MutS, MutL and MutH mutants: correlation of mismatch repair and DNA recombination. DNA Repair (Amst.) 2, 387–405 (2003).

    Article  CAS  Google Scholar 

  5. Hall, M. C. & Matson, S. W. The Escherichia coli MutL protein physically interacts with MutH and stimulates the MutH-associated endonuclease activity. J. Biol. Chem. 274, 1306–1312 (1999).

    Article  CAS  PubMed  Google Scholar 

  6. Kadyrov, F. A., Dzantiev, L., Constantin, N. & Modrich, P. Endonucleolytic function of MutLα in human mismatch repair. Cell 126, 297–308 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Fukui, K., Nishida, M., Nakagawa, N., Masui, R. & Kuramitsu, S. Bound nucleotide controls the endonuclease activity of mismatch repair enzyme MutL. J. Biol. Chem. 283, 12136–12145 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Pluciennik, A. et al. PCNA function in the activation and strand direction of MutL endonuclease in mismatch repair. Proc. Natl Acad. Sci. USA 107, 16066–16071 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Pillon, M. C., Miller, J. H. & Guarné, A. The endonuclease domain of MutL interacts with the β sliding clamp. DNA Repair (Amst.) 10, 87–93 (2011).

    Article  CAS  Google Scholar 

  10. Yang, H., Yung, M., Sikavi, C. & Miller, J. H. The role of Bacillus anthracis RecD2 helicase in DNA mismatch repair. DNA Repair (Amst.) 10, 1121–1130 (2011).

    Article  CAS  Google Scholar 

  11. Walsh, B. W. et al. RecD2 helicase limits replication fork stress in Bacillus subtilis. J. Bacteriol. 196, 1359–1368 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Burdett, V., Baitinger, C., Viswanathan, M., Lovett, S. T. & Modrich, P. In vivo requirement for RecJ, ExoVII, ExoI, and ExoX in methyl-directed mismatch repair. Proc. Natl Acad. Sci. USA 98, 6765–6770 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Yamaguchi, M., Dao, V. & Modrich, P. MutS and MutL activate DNA helicase II in a mismatch-dependent manner. J. Biol. Chem. 273, 9197–9201 (1998).

    Article  CAS  PubMed  Google Scholar 

  14. Lahue, R. S., Au, K. G. & Modrich, P. DNA mismatch correction in a defined system. Science 245, 160–164 (1989).

    Article  CAS  PubMed  Google Scholar 

  15. Goellner, E. M., Putnam, C. D. & Kolodner, R. D. Exonuclease 1-dependent and independent mismatch repair. DNA Repair (Amst.) 32, 24–32 (2015).

    Article  CAS  Google Scholar 

  16. Schofield, M. J., Nayak, S., Scott, T. H., Du, C. & Hsieh, P. Interaction of Escherichia coli MutS and MutL at a DNA Mismatch. J. Biol. Chem. 276, 28291–28299 (2001).

    Article  CAS  PubMed  Google Scholar 

  17. Acharya, S., Foster, P. L., Brooks, P. & Fishel, R. The coordinated functions of the E. coli MutS and MutL proteins in mismatch repair. Mol. Cell 12, 233–246 (2003).

  18. Gradia, S. et al. hMSH2-hMSH6 forms a hydrolysis-independent sliding clamp on mismatched DNA. Mol. Cell 3, 255–261 (1999).

    Article  CAS  PubMed  Google Scholar 

  19. Blackwell, L. J., Bjornson, K. P., Allen, D. J. & Modrich, P. Distinct MutS DNA-binding modes that are differentially modulated by ATP binding and hydrolysis. J. Biol. Chem. 276, 34339–34347 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. Heo, S. D., Cho, M., Ku, J. K. & Ban, C. Steady-state ATPase activity of E. coli MutS modulated by its dissociation from heteroduplex DNA. Biochem. Biophys. Res. Commun. 364, 264–269 (2007).

    Article  CAS  PubMed  Google Scholar 

  21. Jeong, C. et al. MutS switches between two fundamentally distinct clamps during mismatch repair. Nat. Struct. Mol. Biol. 18, 379–385 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Fernandez-Leiro, R. et al. The selection process of licensing a DNA mismatch for repair. Nat. Struct. Mol. Biol. 28, 373–381 (2021).

    Article  CAS  PubMed  Google Scholar 

  23. Hingorani, M. M. Mismatch binding, ADP-ATP exchange and intramolecular signaling during mismatch repair. DNA Repair (Amst.) 38, 24–31 (2016).

    Article  CAS  Google Scholar 

  24. Davies, D. R. & Hol, W. G. J. The power of vanadate in crystallographic investigations of phosphoryl transfer enzymes. FEBS Lett. 577, 315–321 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Lamers, M. H. et al. The crystal structure of DNA mismatch repair protein MutS binding to a G x T mismatch. Nature 407, 711–717 (2000).

    Article  CAS  PubMed  Google Scholar 

  26. Obmolova, G., Ban, C., Hsieh, P. & Yang, W. Crystal structures of mismatch repair protein MutS and its complex with a substrate DNA. Nature 407, 703–710 (2000).

    Article  CAS  PubMed  Google Scholar 

  27. Kato, R., Kataoka, M., Kamikubo, H. & Kuramitsu, S. Direct observation of three conformations of MutS protein regulated by adenine nucleotides. J. Mol. Biol. 309, 227–238 (2001).

    Article  CAS  PubMed  Google Scholar 

  28. Hura, G. L. et al. Comprehensive macromolecular conformations mapped by quantitative SAXS analyses. Nat. Methods 10, 453–454 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Qiu, R. et al. Large conformational changes in MutS during DNA scanning, mismatch recognition and repair signalling. EMBO J. https://doi.org/10.1038/emboj.2012.95 (2012).

  30. Sharma, A., Doucette, C., Biro, F. N. & Hingorani, M. M. Slow conformational changes in MutS and DNA direct ordered transitions between mismatch search, recognition and signaling of DNA repair. J. Mol. Biol. 425, 4192–4205 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Lamers, M. H. et al. ATP increases the affinity between MutS ATPase domains: Implications for ATP hydrolysis and conformational changes. J. Biol. Chem. 279, 43879–43885 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. Nirwal, S., Kulkarni, D. S., Sharma, A., Rao, D. N. & Nair, D. T. Mechanism of formation of a toroid around DNA by the mismatch sensor protein. Nucleic Acids Res. 46, 256–266 (2018).

    Article  CAS  PubMed  Google Scholar 

  33. Groothuizen, F. S. et al. MutS/MutL crystal structure reveals that the MutS sliding clamp loads MutL onto DNA. eLife 4, e06744 (2015).

  34. Hopfner, K. P. Invited review: architectures and mechanisms of ATP binding cassette proteins. Biopolymers 105, 492–504 (2016).

    Article  CAS  PubMed  Google Scholar 

  35. Hol, W. G. J. The role of the α-helix dipole in protein function and structure. Prog. Biophys. Mol. Biol. 45, 149–195 (1985).

    Article  CAS  PubMed  Google Scholar 

  36. Hopfner, K. P. & Tainer, J. A. Rad50/SMC proteins and ABC transporters: unifying concepts from high-resolution structures. Curr. Opin. Struct. Biol. 13, 249–255 (2003).

    Article  CAS  PubMed  Google Scholar 

  37. Acharya, S. Mutations in the signature motif in MutS affect ATP-induced clamp formation and mismatch repair. Mol. Microbiol. 69, 1544–1559 (2008).

    Article  CAS  PubMed  Google Scholar 

  38. Bjornson, K. P., Allen, D. J. & Modrich, P. Modulation of MutS ATP hydrolysis by DNA cofactors. Biochemistry 39, 3176–3183 (2000).

    Article  CAS  PubMed  Google Scholar 

  39. Antony, E. & Hingorani, M. M. Asymmetric ATP binding and hydrolysis activity of the Thermus aquaticus MutS dimer is key to modulation of its interactions with mismatched DNA. Biochemistry 43, 13115–13128 (2004).

    Article  CAS  PubMed  Google Scholar 

  40. Lamers, M., Winterwerp, H. & Sixma, T. The alternating ATPase domains of the DNA mismatch repair enzyme MutS control DNA mismatch repair. EMBO J. 22, 746–756 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Hopfner, K. P. et al. Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily. Cell 101, 789–800 (2000).

    Article  CAS  PubMed  Google Scholar 

  42. Zhou, Y., Ojeda-May, P. & Pu, J. H-loop histidine catalyzes ATP hydrolysis in the E. coli ABC-transporter HlyB. Phys. Chem. Chem. Phys. 15, 15811–15815 (2013).

    Article  CAS  PubMed  Google Scholar 

  43. Groothuizen, F. S. et al. Using stable MutS dimers and tetramers to quantitatively analyze DNA mismatch recognition and sliding clamp formation. Nucleic Acids Res. 41, 8166–8181 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Mazur, D. J., Mendillo, M. L. & Kolodner, R. D. Inhibition of Msh6 ATPase activity by mispaired DNA induces a Msh2(ATP)-Msh6(ATP) state capable of hydrolysis-independent movement along DNA. Mol. Cell 22, 39–49 (2006).

    Article  CAS  PubMed  Google Scholar 

  45. Galio, L., Bouquet, C. & Brooks, P. ATP hydrolysis-dependent formation of a dynamic ternary nucleoprotein complex with MutS and MutL. Nucleic Acids Res. 27, 2325–2331 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Biswas, I. & Vijayvargia, R. Heteroduplex DNA and ATP induced conformational changes of a MutS mismatch repair protein from Thermus aquaticus. Biochem. J. 347, 881–886 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Cristóvão, M. et al. Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA. Nucleic Acids Res. 40, 5448–5464 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Iaccarino, I., Marra, G., Palombo, F. & Jiricny, J. hMSH2 and hMSH6 play distinct roles in mismatch binding and contribute differently to the ATPase activity of hMutSα. EMBO J. 17, 2677–2686 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Studamire, B., Quach, T. & Alani, E. Saccharomyces cerevisiae Msh2p and Msh6p ATPase activities are both required during mismatch repair. Mol. Cell. Biol. 18, 7590–7601 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Monti, M. C. et al. Native mass spectrometry provides direct evidence for DNA mismatch-induced regulation of asymmetric nucleotide binding in mismatch repair protein MutS. Nucleic Acids Res. 39, 8052–8064 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Gradia, S., Acharya, S. & Fishel, R. The human mismatch recognition complex hMSH2-hMSH6 functions as a novel molecular switch. Cell 91, 995–1005 (1997).

    Article  CAS  PubMed  Google Scholar 

  52. Hao, P. et al. Recurrent mismatch binding by MutS mobile clamps on DNA localizes repair complexes nearby. Proc. Natl Acad. Sci. USA 117, 17775–17784 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. LeBlanc, S. J. et al. Coordinated protein and DNA conformational changes govern mismatch repair initiation by MutS. Nucleic Acids Res. 46, 10782–10795 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Au, K. G., Welsh, K. & Modrich, P. Initiation of methyl-directed mismatch repair. J. Biol. Chem. 267, 12142–12148 (1992).

    Article  CAS  PubMed  Google Scholar 

  55. Luna-Vargas, M. P. A. et al. Enabling high-throughput ligation-independent cloning and protein expression for the family of ubiquitin specific proteases. J. Struct. Biol. 175, 113–119 (2011).

    Article  CAS  PubMed  Google Scholar 

  56. Feng, G. & Winkler, M. E. Single-step purifications of His6-MutH, His6-MutL and His6-MutS repair proteins of escherichia coli K-12. Biotechniques 19, 956–965 (1995).

    CAS  PubMed  Google Scholar 

  57. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

  58. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Zhang, K. gCTF: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Nicholls, R. A., Tykac, M., Kovalevskiy, O. & Murshudov, G. N. Current approaches for the fitting and refinement of atomic models into cryo-em maps using CCP-EM. Acta Crystallogr. D Struct. Biol. 74, 492–505 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D Struct. Biol. 75, 861–877 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Pettersen, E. F. et al. UCSF Chimera – a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  65. Venkata Subramaniya, S. R. M., Terashi, G. & Kihara, D. Super resolution Cryo-EM maps with 3D deep generative networks. Preprint at bioRxiv https://doi.org/10.1101/2021.01.12.426430 (2021).

  66. Nicholls, R. A., Fischer, M., Mcnicholas, S. & Murshudov, G. N. Conformation-independent structural comparison of macromolecules with ProSMART. Acta Crystallogr. D Biol. Crystallogr. 70, 2487–2499 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).

    Article  CAS  PubMed  Google Scholar 

  68. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

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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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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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