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

Nature volume 539, pages 583–587 (2016)Cite this article

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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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Figure 1: The formation of an EcMutS–EcMutL complex alters the diffusion properties of EcMutS.
Figure 2: The formation of an oscillating EcMutS–EcMutL complex and fast-diffusing EcMutL.
Figure 3: ATP binding by EcMutL results in formation of a ring-like sliding clamp.
Figure 4: EcMutH binds EcMutL sliding clamps.

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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

  1. Jiaquan Liu and Jeungphill Hanne: These authors contributed equally to this work.

Authors and Affiliations

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

    Jiaquan Liu, Jeungphill Hanne, Brooke M. Britton, Jared Bennett & Richard Fishel

  2. Department of Physics, Pohang University of Science and Technology (POSTECH), Pohang, 790-784, Kyungbuk, Korea

    Daehyung Kim & Jong-Bong Lee

  3. School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang, 790-784, Kyungbuk, Korea

    Jong-Bong Lee

  4. Department of Physics, The Ohio State University, Columbus, 43210, Ohio, USA

    Richard Fishel

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  1. Jiaquan Liu
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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.

Corresponding authors

Correspondence to Jong-Bong Lee or Richard Fishel.

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Extended data figures and tables

Extended Data Figure 1 The construction of mismatched DNA used in single-molecule total internal reflection fluorescence (smTIRF) microscopy.

a, A schematic illustration for the construction of a 17.3-kb mismatched DNA. L or R (blue) indicates the orientation of the DNA relative to the L and R cos end of λ-phage DNA. P (red) indicates the 5′-phosphate of the DNA. b, A schematic illustration of 17.3-kb mismatched DNA observation by prism-based smTIRF microscopy. c, Representative mismatched DNA visualized by smTIRF microscopy in the absence of flow. The DNA was stained with Sytox Orange and a 40 × 85 μm field of view is shown. d, A schematic illustration of the DNA length determination. e, The length distribution of the mismatched DNA observed by smTIRF microscopy. Gaussian fit of the data are shown along with the mean ± s.d.

Extended Data Figure 2 The fluorophore-labelled E. coli MMR proteins used in these studies and the formation of an EcMutS sliding clamp on DNA.

a, Coomassie stained (top) and fluorescent (bottom) SDS–PAGE gels of labelled MMR proteins. For gel source data, see Supplementary Fig. 1. b, A crystal structure of the N-terminal domain of EcMutL bound to AMP-PNP (top) and magnification of the binding domain (bottom; PDB ID: 1B63). AMP-PNP is shown in green and Arg-95 (R95) is shown in magenta24. c, An illustration of the kymograph construction of three separate EcMutS sliding clamps on a single mismatched DNA. d, The distribution of diffusion coefficients for the EcMutS sliding clamp. The data were fit to a Gaussian with the mean ± s.d. e, The distribution of dwell times (mean ± s.e.m.) for the EcMutS sliding clamp.

Extended Data Figure 3 EcMutL does not bind DNA in physiological ionic conditions.

a, b, Representative kymographs and dwell-times (mean ± s.e.m.) for EcMutL binding to a mismatched DNA at various conditions. c–h, The distributions of dwell times (mean ± s.e.m.) for EcMutL on mismatched DNA at different biochemical conditions as indicated.

Extended Data Figure 4 Representative kymographs of EcMutS–EcMutL complex and EcMutL particles.

a, Representative kymographs showing the loading of EcMutL (green) on DNA by EcMutS sliding clamp (red). b, Representative kymographs of a diffusing EcMutS–EcMutL complex (merged channels) and a non-diffusing EcMutS (red) in the same field of view. The static kymograph of a non-diffusing EcMutS indicates that the change in protein position caused by microscope stage drifting is negligible. c, Representative kymographs of oscillating EcMutS–EcMutL complexes. Two channels (red, EcMutS; green, EcMutL) were merged. d, The distribution of the association times (mean ± s.e.m.) for EcMutS–EcMutL complexes during the oscillating phase. e, Representative kymographs of fast-diffusing EcMutL dissociation from EcMutS–EcMutL complexes. Two channels (red, EcMutS; green, EcMutL) were merged.

Extended Data Figure 5 EcMutH lifetime on DNA and diffusion coefficient of EcMutS–EcMutL–EcMutH and/or EcMutL–EcMutH complex.

a, A schematic illustration of EcMutH endonuclease assay (left) and a comparison of labelled or unlabelled EcMutH endonuclease activities (right). For gel source data, see Supplementary Fig. 1. b, c, Representative kymographs and dwell times (mean ± s.e.m.) showing EcMutH on a single mismatched DNA under various ionic and magnesium conditions. d–g, The distributions of dwell times (mean ± s.e.m.) for EcMutH on a single mismatched DNA at different biochemical conditions as indicated. h, Box plots showing D for oscillating (EcMutS)–EcMutLCy3–EcMutHAF647 ((S)–L–H) complex; the established EcMutSAF555–EcMutL–EcMutHAF647 complex (S–L–H); and free EcMutLCy3–EcMutHAF647 complex (L–H) at 100 mM NaCl. i, Box plots showing D for established EcMutS–EcMutL–EcMutH complex at different NaCl concentrations. Two-sample t-test showed no significant difference between diffusion coefficients (P > 0.1). j, Box plots showing D for free EcMutL–EcMutH complex at different NaCl concentrations. Two-sample t-test showed significant differences between diffusion coefficients (P < 0.05). k, Top left, representative kymographs showing FRET between C-terminal AF555-labelled EcMutS and N-terminal AF647-labelled EcMutL (N-ter). Bottom, fluorescent intensities of EcMutS–AF555 (donor, green), EcMutL–AF647 (acceptor, red) and FRET (blue) between them when only the green laser was used for illumination. Right, a schematic illustration of kymographs. Experimental FRET measure (EEcMutS–EcMutL = 0.48 ± 0.05; mean ± s.d.) and theoretical FRET (EEcMutS–EcMutL = 0.56) based on crosslink structure22 appeared comparable. n = number of molecules throughout.

Extended Data Figure 6 The interactions and kinetic properties of the molecular switch/sliding clamp mechanism for E. coli MMR.

a, Illustration of the kinetics and diffusion properties of EcMutS. b, Illustration of the kinetics and diffusion of EcMutS with EcMutL. c, Illustration of the kinetics and diffusion of EcMutS, EcMutL and EcMutH. d, Oscillation dynamics of the EcMutS–EcMutL–EcMutH complex (see main text). Coil, 1D-diffusion search along the backbone; dashed straight arrow, rotation-independent 1D-diffusion; black curved arrow, binding; red curved arrow, oscillating complex; dashed curved arrow, binding-dissociation; binding times and ATP (•) are indicated.

Extended Data Table 1 The oligonucleotides and fluorophore-labelled MMR proteins used in these studies
Full size table
Extended Data Table 2 Cellular complementation and mutation rates of MMR proteins
Full size table
Extended Data Table 3 The frequency of particle varieties observed by single-molecule analysis
Full size table
Extended Data Table 4 Diffusion coefficients
Full size table

Supplementary information

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. (PDF 1135 kb)

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. (MOV 2715 kb)

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. (MOV 2505 kb)

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. (MOV 7731 kb)

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. (MOV 1563 kb)

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. (MOV 3211 kb)

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. (MOV 5982 kb)

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Liu, J., Hanne, J., Britton, B. et al. Cascading MutS and MutL sliding clamps control DNA diffusion to activate mismatch repair. Nature 539, 583–587 (2016). https://doi.org/10.1038/nature20562

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