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Conformation of Reconstituted Mononucleosomes and Effect of Linker Histone H1 Binding Studied by Scanning Force Microscopy

Kepert, Jochen Felix and Fejes Tóth, Katalin and Caudron, Maïwen and Mücke, Norbert and Langowski, Jörg and Rippe, Karsten (2003) Conformation of Reconstituted Mononucleosomes and Effect of Linker Histone H1 Binding Studied by Scanning Force Microscopy. Biophysical Journal, 85 (6). pp. 4012-4022. ISSN 0006-3495. PMCID PMC1303702. doi:10.1016/s0006-3495(03)74815-2. https://resolver.caltech.edu/CaltechAUTHORS:20200421-111931607

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Abstract

The conformation of mononucleosome complexes reconstituted with recombinant core histones on a 614-basepair-long DNA fragment containing the Xenopus borealis 5S rRNA nucleosome positioning sequence was studied by scanning/atomic force microscopy in the absence or presence of linker histone H1. Imaging without prior fixation was conducted with air-dried samples and with mononucleosomes that were injected directly into the scanning force microscopy fluid cell and visualized in buffer. From a quantitative analysis of ∼1,700 complexes, the following results were obtained: i), In the absence of H1, a preferred location of the nucleosome at the X. borealis 5S rRNA sequence in the center of the DNA was detected. From the distribution of nucleosome positions, an energy difference of binding to the 5S rRNA sequence of ΔΔG ≈ 3 kcal mol⁻¹ as compared to a random sequence was estimated. Upon addition of H1, a significantly reduced preference of nucleosome binding to this sequence was observed. ii), The measured entry-exit angles of the DNA at the nucleosome in the absence of H1 showed two maxima at 81 ± 29° and 136 ± 18° (air-dried samples), and 78 ± 25° and 137 ± 25° (samples imaged in buffer solution). In the presence of H1, the species with the smaller entry-exit angle was stabilized, yielding average values of 88 ± 34° for complexes in air and 85 ± 10° in buffer solution. iii), The apparent contour length of the nucleosome complexes was shortened by 34 ± 13 nm as compared to the free DNA due to wrapping of the DNA around the histone octamer complex. Considering an 11 nm diameter of the nucleosome core complex, this corresponds to a total of 145 ± 34 basepairs that are wound around the nucleosome.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/s0006-3495(03)74815-2DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1303702PubMed CentralArticle
ORCID:
AuthorORCID
Fejes Tóth, Katalin0000-0001-6558-2636
Additional Information:© 2003 The Biophysical Society. Published by Elsevier Inc. Received 24 June 2003, Accepted 11 August 2003, Available online 6 January 2009. We gratefully acknowledge the support of Tom Jovin and Peter Lichter, and thank Gudrun Heim and Nathalie Brun for their help, Karolin Luger for providing the histone expression vectors, and Jacek Mazurkiewicz for critical reading of the manuscript. Part of the work by J.F.K., K.F.T., and K.R., and the work of M.C., was done at the Division Biophysics of Macromolecules of the Deutsches Krebsforschungszentrum. J.F.K. did some of the SFM imaging at the Department of Molecular Biology at the Max-Planck-Institut für biophysikalische Chemie in Göttingen. The project was supported by the Volkswagen Foundation in the program “Junior Research Groups at German Universities”.
Funders:
Funding AgencyGrant Number
Volkswagen FoundationUNSPECIFIED
Issue or Number:6
PubMed Central ID:PMC1303702
DOI:10.1016/s0006-3495(03)74815-2
Record Number:CaltechAUTHORS:20200421-111931607
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200421-111931607
Official Citation:Jochen Felix Kepert, Katalin Fejes Tóth, Maïwen Caudron, Norbert Mücke, Jörg Langowski, Karsten Rippe, Conformation of Reconstituted Mononucleosomes and Effect of Linker Histone H1 Binding Studied by Scanning Force Microscopy, Biophysical Journal, Volume 85, Issue 6, 2003, Pages 4012-4022, ISSN 0006-3495, https://doi.org/10.1016/S0006-3495(03)74815-2. (http://www.sciencedirect.com/science/article/pii/S0006349503748152)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:102697
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:21 Apr 2020 18:29
Last Modified:16 Nov 2021 18:14

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