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Rapid spontaneous accessibility of nucleosomal DNA

Nature Structural & Molecular Biology volume 12, pages 46–53 (2005)Cite this article

Abstract

DNA wrapped in nucleosomes is sterically occluded, creating obstacles for proteins that must bind it. How proteins gain access to DNA buried inside nucleosomes is not known. Here we report measurements of the rates of spontaneous nucleosome conformational changes in which a stretch of DNA transiently unwraps off the histone surface, starting from one end of the nucleosome, and then rewraps. The rates are rapid. Nucleosomal DNA remains fully wrapped for only ∼250 ms before spontaneously unwrapping; unwrapped DNA rewraps within ∼10–50 ms. Spontaneous unwrapping of nucleosomal DNA allows any protein rapid access even to buried stretches of the DNA. Our results explain how remodeling factors can be recruited to particular nucleosomes on a biologically relevant timescale, and they imply that the major impediment to entry of RNA polymerase into a nucleosome is rewrapping of nucleosomal DNA, not unwrapping.

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Figure 1: Mechanism of site exposure in nucleosomes deduced from earlier studies11,14.
Figure 2: Equilibrium titration of nucleosomes with LexA, detected by FRET.
Figure 3: Kinetics of LexA binding to DNA monitored by stopped-flow fluorescence anisotropy.
Figure 4: Equilibrium binding of LexA to DNA.
Figure 5: Stopped-flow FRET analysis of LexA binding to nucleosomes.
Figure 6: Spontaneous nucleosome opening and rewrapping fluctuations detected by fluorescence correlation spectroscopy.

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Acknowledgements

We are grateful to S. Huang for valuable discussions and comments on the manuscript. We thank J. Little for the LexA expression plasmid, and the Keck Biophysics Facility at Northwestern University for the use of instruments. This work was supported by US National Institutes of Health (NIH) grants GM54692 and GM58617 to J.W., and by NIH grant GM32543, and US Department of Energy grants DE-AC03-76DF00098, GTL2BN Microscopies of Molecular Machines, and SNANOB Design of Autonomous Nanobots to C.B.

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Authors and Affiliations

  1. Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, 60208-3500, Illinois, USA

    Gu Li & Jonathan Widom

  2. Department of Physics, University of California, Berkeley, 94720, California, USA

    Marcia Levitus & Carlos Bustamante

  3. Department of Molecular and Cell Biology, University of California, Berkeley, 94720, California, USA

    Carlos Bustamante

  4. Biophysics Graduate Group, University of California, Berkeley, 94720, California, USA

    Carlos Bustamante

  5. Howard Hughes Medical Institute, University of California, Berkeley, 94720, California, USA

    Marcia Levitus & Carlos Bustamante

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  1. Gu Li
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Correspondence to Jonathan Widom.

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Supplementary information

Supplementary Fig. 1

Stopped-flow FRET as a function of [LexA]. (PDF 257 kb)

Supplementary Fig. 2

Cooperative nonspecific binding by LexA. (PDF 243 kb)

Supplementary Fig. 3

Stopped-flow FRET at elevated [Na+]. (PDF 138 kb)

Supplementary Table 1

Summary of kinetic analyses. (PDF 48 kb)

Supplementary Data (PDF 77 kb)

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Li, G., Levitus, M., Bustamante, C. et al. Rapid spontaneous accessibility of nucleosomal DNA. Nat Struct Mol Biol 12, 46–53 (2005). https://doi.org/10.1038/nsmb869

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