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

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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Figure 1: Nanofabricated racks of DNA for visualizing 1D diffusion of Mlh1–Pms1.
Figure 2: DNA-binding activity of fluorescently tagged Mlh1–Pms1.
Figure 3: Quantitative analysis of Mlh1–Pms1 diffusion.
Figure 4: End-dependent dissociation of Mlh1–Pms1 from DNA.
Figure 5: Diffusion of Mlh1–Pms1 and Msh2–Msh6 along nucleosome-bound DNA.

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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.

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Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Eric C Greene.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Table 1, Supplementary Discussion and Supplementary Methods (PDF 3298 kb)

Supplementary Video 1

Double-tethered DNA curtains. (MOV 811 kb)

Supplementary Video 2

Double-tethered DNA curtains with diffusing Mlh1-Pms1. (MOV 16938 kb)

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Gorman, J., Plys, A., Visnapuu, ML. et al. Visualizing one-dimensional diffusion of eukaryotic DNA repair factors along a chromatin lattice. Nat Struct Mol Biol 17, 932–938 (2010). https://doi.org/10.1038/nsmb.1858

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