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Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers

Nature Structural Biology volume 8, pages 606–610 (2001)Cite this article

Abstract

Single chromatin fibers were assembled directly in the flow cell of an optical tweezers setup. A single λ phage DNA molecule, suspended between two polystyrene beads, was exposed to a Xenopus laevis egg extract, leading to chromatin assembly with concomitant apparent shortening of the DNA molecule. Assembly was force-dependent and could not take place at forces exceeding 10 pN. The assembled single chromatin fiber was subjected to stretching by controlled movement of one of the beads with the force generated in the molecule continuously monitored with the second bead trapped in the optical trap. The force displayed discrete, sudden drops upon fiber stretching, reflecting discrete opening events in fiber structure. These opening events were quantized at increments in fiber length of ∼65 nm and are attributed to unwrapping of the DNA from around individual histone octamers. Repeated stretching and relaxing of the fiber in the absence of egg extract showed that the loss of histone octamers was irreversible. The forces measured for individual nucleosome disruptions are in the range of 20–40 pN, comparable to forces reported for RNA- and DNA-polymerases.

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Figure 1: Experimental setup and the specially designed flow cell.
Figure 2: Schematic representing chromatin assembly on a single λ DNA molecule (a–c) and stretching of the assembled chromatin fiber (d–f).
Figure 3: Stretching chromatin fibers reveals discrete opening events not present during stretching of naked DNA.
Figure 4: Disruption events are quantized.
Figure 5: The unraveling of nucleosomes during stretching is irreversible, as evidenced by performing successive stretch-relax cycles on the same chromatin fiber.

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Acknowledgements

We thank Z.H. Lu for preparation of the extract, M. Tomschik for biochemical characterization of the assembled chromatin fibers and M. Karymov for help with the mathematical modeling. Presented research is supported by the Dutch Foundation for Fundamental Research on Matter (M.L.B.) and the National Science Foundation (G.H.L.). S.H.L. is a National Cancer Institute Scholar. A collection of movies and animations demonstrating real-time attaching of the single λ DNA to the beads and chromatin assembly are at the following web sites: http://tnweb.tn.utwente.nl/top/ and http://rex.nci.nih.gov/RESEARCH/basic/lrbge/leuba.html.

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Author notes

  1. Gregory H. Leno

    Present address: Infigen, DeForest, Wisconsin, 53532, USA

  2. Bart G. de Grooth: This work is dedicated to the memory of Bart G. de Grooth.

Authors and Affiliations

  1. Department of Applied Physics, iBME Research Institute, University of Twente, PO Box 217, AE, 7500, Enschede, The Netherlands

    Martin L. Bennink, Bart G. de Grooth & Jan Greve

  2. Physical Molecular Biology, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, NIH, Building 41, Room B507, Bethesda, 20892, Maryland, USA

    Sanford H. Leuba

  3. Department of Biochemistry, University of Mississippi Medical Center, Jackson, 39216, Mississippi, USA

    Gregory H. Leno

  4. Department of Chemical Engineering, Chemistry, and Materials Science, Polytechnic University, Six MetroTech Center, Brooklyn, 11201, New York, USA

    Jordanka Zlatanova

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  1. Martin L. Bennink
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Correspondence to Martin L. Bennink or Jan Greve.

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Bennink, M., Leuba, S., Leno, G. et al. Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers. Nat Struct Mol Biol 8, 606–610 (2001). https://doi.org/10.1038/89646

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