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标题：Assignment of structural transitions during mechanical unwrapping of nucleosomes and their disassembly products
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作者：César Díaz-Celis ; Cristhian Cañari-Chumpitaz ; Robert P. Sosa 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：33
DOI：10.1073/pnas.2206513119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance Nucleosomes, the fundamental structural unit of chromatin, consists of ∼147 DNA base pairs wrapped around a histone protein octamer. To characterize the strength of the nucleosomal barrier and its contribution as a mechanism of control of gene expression, it is essential to determine the forces required to unwrap the DNA from the core particle and the stepwise transitions involved. In this study, we performed combined optical tweezers and single-molecule fluorescence measurements to identify the specific DNA segments unwrapped during the force transitions observed in mechanical stretching of nucleosomes. Furthermore, we characterize the mechanical signatures of subnucleosomal hexasomes and tetrasomes. The characterization performed in this work is essential for the interpretation of ongoing studies of chromatin remodelers, polymerases, and histone chaperones. Nucleosome DNA unwrapping and its disassembly into hexasomes and tetrasomes is necessary for genomic access and plays an important role in transcription regulation. Previous single-molecule mechanical nucleosome unwrapping revealed a low- and a high-force transitions, and force-FRET pulling experiments showed that DNA unwrapping is asymmetric, occurring always first from one side before the other. However, the assignment of DNA segments involved in these transitions remains controversial. Here, using high-resolution optical tweezers with simultaneous single-molecule FRET detection, we show that the low-force transition corresponds to the undoing of the outer wrap of one side of the nucleosome (∼27 bp), a process that can occur either cooperatively or noncooperatively, whereas the high-force transition corresponds to the simultaneous unwrapping of ∼76 bp from both sides. This process may give rise stochastically to the disassembly of nucleosomes into hexasomes and tetrasomes whose unwrapping/rewrapping trajectories we establish. In contrast, nucleosome rewrapping does not exhibit asymmetry. To rationalize all previous nucleosome unwrapping experiments, it is necessary to invoke that mechanical unwrapping involves two nucleosome reorientations: one that contributes to the change in extension at the low-force transition and another that coincides but does not contribute to the high-force transition.
关键词：ennucleosomenucleosome unwrappingnucleosome disassemblyoptical trappingFRET