Letter | Published:

Cascading MutS and MutL sliding clamps control DNA diffusion to activate mismatch repair

Nature volume 539, pages 583587 (24 November 2016) | Download Citation

Abstract

Mismatched nucleotides arise from polymerase misincorporation errors, recombination between heteroallelic parents and chemical or physical DNA damage1. Highly conserved MutS (MSH) and MutL (MLH/PMS) homologues initiate mismatch repair and, in higher eukaryotes, act as DNA damage sensors that can trigger apoptosis2. Defects in human mismatch repair genes cause Lynch syndrome or hereditary non-polyposis colorectal cancer and 10–40% of related sporadic tumours3. However, the collaborative mechanics of MSH and MLH/PMS proteins have not been resolved in any organism. We visualized Escherichia coli (Ec) ensemble mismatch repair and confirmed that EcMutS mismatch recognition results in the formation of stable ATP-bound sliding clamps that randomly diffuse along the DNA with intermittent backbone contact. The EcMutS sliding clamps act as a platform to recruit EcMutL onto the mismatched DNA, forming an EcMutS–EcMutL search complex that then closely follows the DNA backbone. ATP binding by EcMutL establishes a second long-lived DNA clamp that oscillates between the principal EcMutS–EcMutL search complex and unrestricted EcMutS and EcMutL sliding clamps. The EcMutH endonuclease that targets mismatch repair excision only binds clamped EcMutL, increasing its DNA association kinetics by more than 1,000-fold. The assembly of an EcMutS–EcMutL–EcMutH search complex illustrates how sequential stable sliding clamps can modulate one-dimensional diffusion mechanics along the DNA to direct mismatch repair.

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Acknowledgements

We would like to thank C. Bell and our laboratory colleagues for many helpful insights and discussions. This work was supported by NRF of Korea Grant No. 2011-001309 (J.-B.L) and NIH grant CA67007 (R.F.).

Author information

Author notes

    • Jiaquan Liu
    •  & Jeungphill Hanne

    These authors contributed equally to this work.

Affiliations

  1. Department of Cancer Biology and Genetics, The Ohio State University Wexner Medical Center, Columbus, Ohio 43210, USA

    • Jiaquan Liu
    • , Jeungphill Hanne
    • , Brooke M. Britton
    • , Jared Bennett
    •  & Richard Fishel
  2. Department of Physics, Pohang University of Science and Technology (POSTECH), Pohang, Kyungbuk 790-784, Korea

    • Daehyung Kim
    •  & Jong-Bong Lee
  3. School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang, Kyungbuk, 790-784, Korea

    • Jong-Bong Lee
  4. Department of Physics, The Ohio State University, Columbus, Ohio 43210, USA

    • Richard Fishel

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Contributions

J.L., J.H., J.-B.L. and R.F. designed the experiments; B.M.B., and J.B., performed genetic analysis; J.L. purified and labelled the proteins; J.L. and J.H. performed the single-molecule studies; J.L., J.H., J.-B.L. and R.F. analysed the data; D.K. developed the Matlab script for diffusion analysis and particle co-localization. J.L., J.H., J.-B.L. and R.F. wrote the paper and all authors participated in critical discussions.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Jong-Bong Lee or Richard Fishel.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains the Source Data for Extended Data Figures 2a and 5a. This file was corrected on 17 November 2016 to include the Supplementary Note 1.

Videos

  1. 1.

    Representative video (10 frames per sec) of EcMutL binding to an EcMutS sliding clamp on the mismatched DNA (Extended Data Fig 4a, right).

    Two channels were merged. EcMutS is shown as red and EcMutL is shown as green. Approximate position of DNA is shown as a white line. Co-localization between EcMutS and EcMutL is observed as yellow.

  2. 2.

    Representative video (10 frames per sec) of an oscillating EcMutS-EcMutL complex (Extended Data Fig 4c, bottom).

    Two channels were merged. EcMutS is shown as red and EcMutL is shown as green. Approximate position of DNA is shown as a white line. Co-localization between EcMutS and EcMutL is observed as yellow.

  3. 3.

    Representative slow-motion (5 frames per sec) video of an oscillating EcMutS-EcMutL complex (Fig. 2b).

    Two channels were merged. EcMutS is shown as red and EcMutL is shown as green. Approximate position of DNA is shown in a white line. Co-localization between EcMutS and EcMutL is observed as yellow.

  4. 4.

    Representative video (10 frames per sec) of fast diffusing EcMutL dissociated from EcMutS-EcMutL complex (Fig. 2e).

    Two channels were merged. EcMutS is shown as red and EcMutL is shown as green. Approximate position of DNA is shown as a white line. Co-localization between EcMutS and EcMutL is observed as yellow.

  5. 5.

    Representative video (10 frames per sec) of oscillating EcMutS-EcMutL-EcMutH complex (Fig. 4d).

    Two channels were merged. EcMutH is shown as red and EcMutS is shown as green. Approximate position of DNA is shown as a white line. Co-localization between EcMutS and EcMutH is observed as yellow.

  6. 6.

    Representative video (10 frames per sec) of FRET between EcMutS and EcMutL (Extended Data Fig 5k).

    Two channels were merged. EcMutS is shown as green and EcMutL is shown as red. Approximate position of DNA is shown as a white line. FRET between EcMutS and EcMutL is observed as yellow or red depending on the FRET efficiency.

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DOI

https://doi.org/10.1038/nature20562

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