Constructing arrays of nucleosome positioning sequences using Gibson Assembly for single-molecule studies

Spakman, Dian; King, Graeme A.; Peterman, Erwin J.G.; Wuite, Gijs J. L.

doi:10.1038/s41598-020-66259-4

“…Our nucleosome unwrapping experiments reported a major ΔL c population at 72 ± 5 bp (center ± SD) (Fig. 1c), in agreement with the values reported in the literature for the two-step nucleosome unwrapping mechanism [14][15][16][17][18][19][20][21] . In addition, a smaller and wider peak was found at ~30 bp (Fig.…”

Section: Resultssupporting

confidence: 91%

Histone chaperones exhibit conserved functionality in nucleosome remodeling

Buzón

¹

,

Velázquez‐Cruz

²

,

González‐Arzola

³

et al. 2022

Preprint

0

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Chromatin homeostasis mediates some of the most fundamental processes in the eukaryotic cell. In this regard, histone chaperones have emerged as major regulatory factors during DNA replication, repair, and transcription. However, the dynamic nature of these processes has severely impeded their characterization at the molecular level. Here we apply single-molecule probing by fluorescence optical tweezers to follow histone chaperone dynamics in real-time. The molecular action of SET/template-activating factor-Iβ and nucleophosmin 1, representing the two most common histone chaperone folds, were examined using both nucleosomes and isolated core histones. We show that these chaperones present binding specificity for partially dismantled nucleosomes and are able to recognize and disrupt non-native histone-DNA interactions. Furthermore, we reveal that cytochrome c inhibition of histone chaperones is coupled to chaperone accumulation on DNA-bound histones. Our single-molecule approach shows that despite the drastically different structures of these chaperones, they present conserved modes of action mediating nucleosome remodeling.

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“…6). The measured step size confirms the wrapping of DNA around the histones to form nucleosome as previously reported 30 .…”

Section: Nucleosomes Hinder Condensin Compactionsupporting

confidence: 89%

Condensin Compacts DNA in 15nm steps and Requires FACT for Extrusion on Chromatin

Sudhakar

¹

,

Fisher

²

,

Escribano

³

et al. 2021

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0

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Condensin plays a central role in the organisation of chromosomes by compacting chromatin into loops during mitosis. Condensin achieves this through a loop extrusion mechanism that remains poorly understood. To identify the molecular steps of yeast condensin during loop formation, we used optical tweezers with fluorescence detection. We find that single yeast condensin complexes use ATP to extrude DNA through distinct 15 nm steps, thus advancing ~45 base pairs (bp) per step. Under increasing load, the condensin step size remains constant while step-dwell times increase, and stalls at forces >1 pN. We also show that nucleosome arrays hinder processive condensin extrusion and demonstrate that the histone chaperone FACT is required for compaction of nucleosomal arrays by condensin. Importantly, FACT-assisted compaction on nucleosomes also occurs through distinct 15 nm steps. Finally, we show that FACT is required for correct condensin localisation in vivo. Our results establish that loop extrusion by yeast condensin involves a 45 bp stroke that requires FACT for condensin function on chromatin

show abstract

“…6). The measured step size con rms the wrapping of DNA around the histones to form nucleosome as previously reported 30 .…”

Section: Nucleosomes Hinder Condensin Compactionsupporting

confidence: 81%

Condensin compacts DNA in 15 nm steps and requires FACT for extrusion on chromatin

Rueda

¹

,

Sudhakar

²

,

Fisher

³

et al. 2021

Preprint

0

View full text Add to dashboard Cite

Condensin plays a central role in the organisation of chromosomes by compacting chromatin into loops during mitosis. Condensin achieves this through a loop extrusion mechanism that remains poorly understood. To identify the molecular steps of yeast condensin during loop formation, we used optical tweezers with fluorescence detection. We find that single yeast condensin complexes use ATP to extrude DNA through distinct 15 nm steps, thus advancing ~45 base pairs (bp) per step. Under increasing load, the condensin step size remains constant while step-dwell times increase, and stalls at forces >1 pN. We also show that nucleosome arrays hinder processive condensin extrusion and demonstrate that the histone chaperone FACT is required for compaction of nucleosomal arrays by condensin. Importantly, FACT-assisted compaction on nucleosomes also occurs through distinct 15 nm steps. Finally, we show that FACT is required for correct condensin localisation in vivo. Our results establish that loop extrusion by yeast condensin involves a 45 bp stroke that requires FACT for condensin function on chromatin.

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Constructing arrays of nucleosome positioning sequences using Gibson Assembly for single-molecule studies

Cited by 28 publications

References 84 publications

Histone chaperones exhibit conserved functionality in nucleosome remodeling

Histone chaperones exhibit conserved functionality in nucleosome remodeling

Condensin Compacts DNA in 15nm steps and Requires FACT for Extrusion on Chromatin

Condensin compacts DNA in 15 nm steps and requires FACT for extrusion on chromatin

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