Article | Published:

Visualizing one-dimensional diffusion of eukaryotic DNA repair factors along a chromatin lattice

Nature Structural & Molecular Biology volume 17, pages 932938 (2010) | Download Citation

Abstract

DNA-binding proteins survey genomes for targets using facilitated diffusion, which typically includes a one-dimensional (1D) scanning component for sampling local regions. Eukaryotic proteins must accomplish this task while navigating through chromatin. Yet it is unknown whether nucleosomes disrupt 1D scanning or eukaryotic DNA-binding factors can circumnavigate nucleosomes without falling off DNA. Here we use single-molecule microscopy in conjunction with nanofabricated curtains of DNA to show that the postreplicative mismatch repair protein complex Mlh1–Pms1 diffuses in 1D along DNA via a hopping/stepping mechanism and readily bypasses nucleosomes. This is the first experimental demonstration that a passively diffusing protein can traverse stationary obstacles. In contrast, Msh2–Msh6, a mismatch repair protein complex that slides while maintaining continuous contact with DNA, experiences a boundary upon encountering nucleosomes. These differences reveal important mechanistic constraints affecting intranuclear trafficking of DNA-binding proteins.

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Acknowledgements

We thank members of our laboratories for carefully reading the manuscript and providing suggestions throughout this study. This work was supported by the Howard Hughes Medical Institute, by a US National Science Foundation Presidential Early Career Award for Scientists and Engineers, by US National Institutes of Health (NIH) grant GM082848 to E.C.G. and by NIH grant GM53085 to E.A. J.G. is supported by an NIH training grant for Cellular and Molecular Foundations of Biomedical Sciences (T32GM00879807). A.J.P. was supported by a State University of New York fellowship. This work was partially supported by the Initiatives in Science and Engineering program through Columbia University, the Nanoscale Science and Engineering Initiative of the National Science Foundation under US National Science Foundation Award Number CHE-0641523 and by the New York State Office of Science, Technology, and Academic Research.

Author information

Author notes

    • Jason Gorman
    •  & Aaron J Plys

    These authors contributed equally to this work.

Affiliations

  1. Department of Biological Sciences, Columbia University, New York, New York, USA.

    • Jason Gorman
  2. Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.

    • Aaron J Plys
    •  & Eric Alani
  3. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA.

    • Mari-Liis Visnapuu
    •  & Eric C Greene
  4. Howard Hughes Medical Institute, Columbia University, New York, New York, USA.

    • Eric C Greene

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Contributions

J.G. designed the TIRFM experiments and collected and analyzed the single-molecule data; A.J.P. designed and engineered all constructs, expressed and purified the proteins, did all ensemble-level characterization and conducted the immunoprecipitation experiments; M.-L.V. made and characterized all chromatin substrates; J.G., A.J.P., M.-L.V., E.A. and E.C.G. discussed the data and cowrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Eric C Greene.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–4, Supplementary Table 1, Supplementary Discussion and Supplementary Methods

Videos

  1. 1.

    Supplementary Video 1

    Double-tethered DNA curtains.

  2. 2.

    Supplementary Video 2

    Double-tethered DNA curtains with diffusing Mlh1-Pms1.

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DOI

https://doi.org/10.1038/nsmb.1858

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