Structure and Scm3-mediated assembly of budding yeast centromeric nucleosomes

Dechassa, Mekonnen Lemma; Wyns, Katharina; Li, Ming; Hall, Michael; Wang, Michelle D.; Luger, Karolin

doi:10.1038/ncomms1320

Cited by 121 publications

(209 citation statements)

References 51 publications

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“…It has been previously shown that the Cse4 nucleosome wraps DNA in a right-handed orientation (6,7), consistent with in vivo observations of heterotypic tetrameric nucleosomes in Drosophila (8) and humans (9). However, several studies have shown that CenH3 nucleosomes are left-handed octamers in vitro (10)(11)(12)(13)(14).…”

mentioning

confidence: 71%

Tripartite organization of centromeric chromatin in budding yeast

Krassovsky

Henikoff

2011

Proc. Natl. Acad. Sci. U.S.A.

166

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The centromere is the genetic locus that organizes the proteinaceous kinetochore and is responsible for attachment of the chromosome to the spindle at mitosis and meiosis. In most eukaryotes, the centromere consists of highly repetitive DNA sequences that are occupied by nucleosomes containing the CenH3 histone variant, whereas in budding yeast, a ∼120-bp centromere DNA element (CDE) that is sufficient for centromere function is occupied by a single right-handed histone variant CenH3 (Cse4) nucleosome. However, these in vivo observations are inconsistent with in vitro evidence for left-handed octameric CenH3 nucleosomes. To help resolve these inconsistencies, we characterized yeast centromeric chromatin at single base-pair resolution. Intact particles containing both Cse4 and H2A are precisely protected from micrococcal nuclease over the entire CDE of all 16 yeast centromeres in both solubilized chromatin and the insoluble kinetochore. Small DNAbinding proteins protect CDEI and CDEIII and delimit the centromeric nucleosome to the ∼80-bp CDEII, only enough for a single DNA wrap. As expected for a tripartite organization of centromeric chromatin, loss of Cbf1 protein, which binds to CDEI, both reduces the size of the centromere-protected region and shifts its location toward CDEIII. Surprisingly, Cse4 overproduction caused genome-wide misincorporation of nonfunctional CenH3-containing nucleosomes that protect ∼135 base pairs and are preferentially enriched at sites of high nucleosome turnover. Our detection of two forms of CenH3 nucleosomes in the yeast genome, a singly wrapped particle at the functional centromere and octamer-sized particles on chromosome arms, reconcile seemingly conflicting in vivo and in vitro observations. Saccharomyces cerevisiae | chromatin immunoprecipitation | chromosome segregation

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mentioning

confidence: 71%

Tripartite organization of centromeric chromatin in budding yeast

Krassovsky

Henikoff

2011

Proc. Natl. Acad. Sci. U.S.A.

166

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“…The vast majority of unzipping traces (99%) showed at least one transfer event. For the first such transfer event on each unzipped template (Supplementary Table 2), 67% of traces showed a force signature consistent with that of a nucleosome [10][11][12][13][14][15][16] and 32% were consistent with that of a tetrasome 10,13 , though it is possible that some of these may have been hexasomes 17,18 . These results are in agreement with previous findings that parental H3/H4 tetrasomes generally remain intact after replication fork passage, while H2A/H2B dimers are more labile [19][20][21] .…”

Section: Resultsmentioning

confidence: 99%

“…The resulting unzipping force served as a sensitive detector for the presence of the nucleosome, with a force rise above the naked DNA baseline indicating both nucleosome location and composition (Fig. 1b) [10][11][12][13][14][15][16] . During unzipping, the force first followed that of naked DNA, until the fork reached the positioning sequence, and then a marked force rise characteristic of a canonical nucleosome 11 occurred, followed by a force drop.…”

Section: Resultsmentioning

confidence: 99%

“…Nucleosomes were identified as containing peaks separated by o65 bp, up to a maximum of three, in accordance with previous work 11 . Individual force peaks separated by more than 65 bp were considered to be tetrasomes 10,13 . Nucleosome transfer distances were measured as the distance from the first peak within the 601 nucleosome-positioning element to the first peak outside of the defined nucleosome force signature.…”

Section: Methodsmentioning

confidence: 99%

“…Aliquots of purified histones were stored in À 80°C at a final concentration of 2.7 mM. Nucleosomes were assembled on the Widom 601 nucleosome-positioning element 49 by salt dialysis [10][11][12][13]15,16,20,[50][51][52][53] . For the mechanical displacement assay, nucleosomes were assembled on a 764 bp template at a molar ratio of 1.25:1.00 of histone octamer to DNA.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

DNA Looping Mediates Nucleosome Transfer

Brennan

Forties²,

Patel

et al. 2017

Biophysical Journal

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Proper cell function requires preservation of the spatial organization of chromatin modifications. Maintenance of this epigenetic landscape necessitates the transfer of parental nucleosomes to newly replicated DNA, a process that is stringently regulated and intrinsically linked to replication fork dynamics. This creates a formidable setting from which to isolate the central mechanism of transfer. Here we utilized a minimal experimental system to track the fate of a single nucleosome following its displacement, and examined whether DNA mechanics itself, in the absence of any chaperones or assembly factors, may serve as a platform for the transfer process. We found that the nucleosome is passively transferred to available dsDNA as predicted by a simple physical model of DNA loop formation. These results demonstrate a fundamental role for DNA mechanics in mediating nucleosome transfer and preserving epigenetic integrity during replication.

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Variations on a nucleosome theme: The structural basis of centromere function

Moreno-Moreno

Torras-Llort²,

Azorı́n³

2017

BioEssays

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The centromere is a specialized chromosomal structure that dictates kinetochore assembly and, thus, is essential for accurate chromosome segregation. Centromere identity is determined epigenetically by the presence of a centromere-specific histone H3 variant, CENP-A, that replaces canonical H3 in centromeric chromatin. Here, we discuss recent work by Roulland et al. that identifies structural elements of the nucleosome as essential determinants of centromere function. In particular, CENP-A nucleosomes have flexible DNA ends due to the short αN helix of CENP-A. The higher flexibility of the DNA ends of centromeric nucleosomes impairs binding of linker histones H1, while it facilitates binding of other essential centromeric proteins, such as CENP-C, and is required for mitotic fidelity. This work extends previous observations indicating that the differential structural properties of CENP-A nucleosomes are on the basis of its contribution to centromere identity and function. Here, we discuss the implications of this work and the questions arising from it.

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Structure and Scm3-mediated assembly of budding yeast centromeric nucleosomes

Cited by 121 publications

References 51 publications

Tripartite organization of centromeric chromatin in budding yeast

Tripartite organization of centromeric chromatin in budding yeast

DNA Looping Mediates Nucleosome Transfer

Variations on a nucleosome theme: The structural basis of centromere function

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