Internal Motion of Chromatin Fibers Is Governed by Dynamics of Uncompressed Linker Strands

Basak, Rajib; Rosencrans, William M.; Yadav, Indresh; Yan, Peiyan; Berezhnoy, Nikolay V.; Chen, Qinming; Kan, J.A. van; Nordenskiöld, Lars; Zinchenko, Anatoly; Maarel, Johan R. C. van der

doi:10.1016/j.bpj.2020.10.018

Cited by 6 publications

(9 citation statements)

References 49 publications

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“…The qualitative behavior of the fibers was similar in the 60-nm and 125-nm channels, though the molecules were stretched more in narrower channels. Previously, similar results were reported for chromatin reconstituted with bacteriophage T4-DNA 27 . The primary reorganization process is the association and dissociation of distant clutches of nucleosomes, mediated by the looping of the connecting DNA and chromatin.…”

Section: Resultssupporting

confidence: 87%

“…The observed difference is similar to the behavior observed for compaction of the longer T4 DNA chains ( ca. 166 kbp) and corresponding reconstituted chromatin fibers reported in our previous studies 25 , 27 . It was earlier demonstrated that a significant HO loading (HO:DNA > 0.7) is necessary for the condensation of short nucleosome arrays 53 .…”

Section: Resultssupporting

confidence: 62%

“…Nanofluidic devices were fabricated in PDMS elastomer as previously described 27 . The device consisted of an array of 90 μm long, rectangular channels with a cross-section of 50 × 70 nm 2 (60-nm channel system) or 120 × 130 nm 2 (125-nm system) and uncertainty in width and depth of ± 5 nm.…”

Section: Methodsmentioning

confidence: 99%

“…We have previously demonstrated 24 – 27 that T4 DNA (~ 166 kbp) with natural nucleotide sequence can be reconstituted to chromatin fibers with controlled nucleosome occupancy and used as in vitro model of long TAD-sized chromatin fibers, suitable for single-molecule studies. λ-phage DNA (~ 48.5 kbp, λ-DNA) chromatin displayed similar properties as T4 DNA chromatin 26 .…”

Section: Introductionmentioning

confidence: 99%

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Reconstituted TAD-size chromatin fibers feature heterogeneous nucleosome clusters

Korolev

Zinchenko

Soman

et al. 2022

Sci Rep

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Large topologically associated domains (TADs) contain irregularly spaced nucleosome clutches, and interactions between such clutches are thought to aid the compaction of these domains. Here, we reconstituted TAD-sized chromatin fibers containing hundreds of nucleosomes on native source human and lambda-phage DNA and compared their mechanical properties at the single-molecule level with shorter ‘601’ arrays with various nucleosome repeat lengths. Fluorescent imaging showed increased compaction upon saturation of the DNA with histones and increasing magnesium concentration. Nucleosome clusters and their structural fluctuations were visualized in confined nanochannels. Force spectroscopy revealed not only similar mechanical properties of the TAD-sized fibers as shorter fibers but also large rupture events, consistent with breaking the interactions between distant clutches of nucleosomes. Though the arrays of native human DNA, lambda-phage and ‘601’ DNA featured minor differences in reconstitution yield and nucleosome stability, the fibers’ global structural and mechanical properties were similar, including the interactions between nucleosome clutches. These single-molecule experiments quantify the mechanical forces that stabilize large TAD-sized chromatin domains consisting of disordered, dynamically interacting nucleosome clutches and their effect on the condensation of large chromatin domains.

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Section: Resultssupporting

confidence: 87%

Section: Resultssupporting

confidence: 62%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Reconstituted TAD-size chromatin fibers feature heterogeneous nucleosome clusters

Korolev

Zinchenko

Soman

et al. 2022

Sci Rep

Self Cite

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“…More recently, Basak et al . (2020) investigated the compaction, dynamics, and internal motion of nanoconfined chromatin fibers. They used over-expressed, refolded, and purified histone octamers to reconstitute chromatin on T4-DNA, and nanofluidic devices with two different degrees of confinement (50 × 70 nm 2 and 120 × 130 nm 2 , respectively).…”

Section: Dna–protein Interactionsmentioning

confidence: 99%

DNA in nanochannels: theory and applications

Frykholm

Müller

et al. 2022

Quart. Rev. Biophys.

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Nanofluidic structures have over the last two decades emerged as a powerful platform for detailed analysis of DNA on the kilobase pair length scale. When DNA is confined to a nanochannel, the combination of excluded volume and DNA stiffness leads to the DNA being stretched to near its full contour length. Importantly, this stretching takes place at equilibrium, without any chemical modifications to the DNA. As a result, any DNA can be analyzed, such as DNA extracted from cells or circular DNA, and it is straight-forward to study reactions on the ends of linear DNA. In this comprehensive review, we first give a thorough description of the current understanding of the polymer physics of DNA and how that leads to stretching in nanochannels. We then describe how the versatility of nanofabrication can be used to design devices specifically tailored for the problem at hand, either by controlling the degree of confinement or enabling facile exchange of reagents to measure DNA-protein reaction kinetics. The remainder of the review focuses on two important applications of confining DNA in nanochannels. The first is optical DNA mapping, which provides the genomic sequence of intact DNA molecules in excess of 100 kilobase pairs in size, with kilobase pair resolution, through labeling strategies that are suitable for fluorescence microscopy. In this section, we highlight solutions to the technical aspects of genomic mapping, including the use of enzyme-based labeling and affinitybased labeling to produce the genomic maps, rather than recent applications in human genetics. The second is DNA-protein interactions, and several recent examples of such studies on DNA compaction, filamentous protein complexes, and reactions with DNA ends are presented. Taken together, these two applications demonstrate the power of DNA confinement and nanofluidics in genomics, molecular biology, and biophysics.

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