Cargando…
Reconstitution of hemisomes on budding yeast centromeric DNA
The structure of nucleosomes that contain the cenH3 histone variant has been controversial. In budding yeast, a single right-handed cenH3/H4/H2A/H2B tetramer wraps the ∼80-bp Centromere DNA Element II (CDE II) sequence of each centromere into a ‘hemisome’. However, attempts to reconstitute cenH3 par...
Autores principales: | , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2013
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3675498/ https://www.ncbi.nlm.nih.gov/pubmed/23620291 http://dx.doi.org/10.1093/nar/gkt314 |
_version_ | 1782476103663747072 |
---|---|
author | Furuyama, Takehito Codomo, Christine A. Henikoff, Steven |
author_facet | Furuyama, Takehito Codomo, Christine A. Henikoff, Steven |
author_sort | Furuyama, Takehito |
collection | PubMed |
description | The structure of nucleosomes that contain the cenH3 histone variant has been controversial. In budding yeast, a single right-handed cenH3/H4/H2A/H2B tetramer wraps the ∼80-bp Centromere DNA Element II (CDE II) sequence of each centromere into a ‘hemisome’. However, attempts to reconstitute cenH3 particles in vitro have yielded exclusively ‘octasomes’, which are observed in vivo on chromosome arms only when Cse4 (yeast cenH3) is overproduced. Here, we show that Cse4 octamers remain intact under conditions of low salt and urea that dissociate H3 octamers. However, particles consisting of two DNA duplexes wrapped around a Cse4 octamer and separated by a gap efficiently split into hemisomes. Hemisome dimensions were confirmed using a calibrated gel-shift assay and atomic force microscopy, and their identity as tightly wrapped particles was demonstrated by gelFRET. Surprisingly, Cse4 hemisomes were stable in 4 M urea. Stable Cse4 hemisomes could be reconstituted using either full-length or tailless histones and with a 78-bp CDEII segment, which is predicted to be exceptionally stiff. We propose that CDEII DNA stiffness evolved to favor Cse4 hemisome over octasome formation. The precise correspondence between Cse4 hemisomes resident on CDEII in vivo and reconstituted on CDEII in vitro without any other factors implies that CDEII is sufficient for hemisome assembly. |
format | Online Article Text |
id | pubmed-3675498 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-36754982013-06-07 Reconstitution of hemisomes on budding yeast centromeric DNA Furuyama, Takehito Codomo, Christine A. Henikoff, Steven Nucleic Acids Res Gene Regulation, Chromatin and Epigenetics The structure of nucleosomes that contain the cenH3 histone variant has been controversial. In budding yeast, a single right-handed cenH3/H4/H2A/H2B tetramer wraps the ∼80-bp Centromere DNA Element II (CDE II) sequence of each centromere into a ‘hemisome’. However, attempts to reconstitute cenH3 particles in vitro have yielded exclusively ‘octasomes’, which are observed in vivo on chromosome arms only when Cse4 (yeast cenH3) is overproduced. Here, we show that Cse4 octamers remain intact under conditions of low salt and urea that dissociate H3 octamers. However, particles consisting of two DNA duplexes wrapped around a Cse4 octamer and separated by a gap efficiently split into hemisomes. Hemisome dimensions were confirmed using a calibrated gel-shift assay and atomic force microscopy, and their identity as tightly wrapped particles was demonstrated by gelFRET. Surprisingly, Cse4 hemisomes were stable in 4 M urea. Stable Cse4 hemisomes could be reconstituted using either full-length or tailless histones and with a 78-bp CDEII segment, which is predicted to be exceptionally stiff. We propose that CDEII DNA stiffness evolved to favor Cse4 hemisome over octasome formation. The precise correspondence between Cse4 hemisomes resident on CDEII in vivo and reconstituted on CDEII in vitro without any other factors implies that CDEII is sufficient for hemisome assembly. Oxford University Press 2013-06 2013-04-24 /pmc/articles/PMC3675498/ /pubmed/23620291 http://dx.doi.org/10.1093/nar/gkt314 Text en © The Author(s) 2013. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Gene Regulation, Chromatin and Epigenetics Furuyama, Takehito Codomo, Christine A. Henikoff, Steven Reconstitution of hemisomes on budding yeast centromeric DNA |
title | Reconstitution of hemisomes on budding yeast centromeric DNA |
title_full | Reconstitution of hemisomes on budding yeast centromeric DNA |
title_fullStr | Reconstitution of hemisomes on budding yeast centromeric DNA |
title_full_unstemmed | Reconstitution of hemisomes on budding yeast centromeric DNA |
title_short | Reconstitution of hemisomes on budding yeast centromeric DNA |
title_sort | reconstitution of hemisomes on budding yeast centromeric dna |
topic | Gene Regulation, Chromatin and Epigenetics |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3675498/ https://www.ncbi.nlm.nih.gov/pubmed/23620291 http://dx.doi.org/10.1093/nar/gkt314 |
work_keys_str_mv | AT furuyamatakehito reconstitutionofhemisomesonbuddingyeastcentromericdna AT codomochristinea reconstitutionofhemisomesonbuddingyeastcentromericdna AT henikoffsteven reconstitutionofhemisomesonbuddingyeastcentromericdna |