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Reconstitution of hemisomes on budding yeast centromeric DNA.

Furuyama T, Codomo CA, Henikoff S - Nucleic Acids Res. (2013)

Bottom Line: However, particles consisting of two DNA duplexes wrapped around a Cse4 octamer and separated by a gap efficiently split into hemisomes.Surprisingly, Cse4 hemisomes were stable in 4 M urea.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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

ABSTRACT
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.

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Pseudo-octasomes split in half during low-salt dialysis. (A) Gel-shift image (native 7% PAGE), AFM image and height distribution of control H3 octamers assembled with 147-bp Widom 601 duplex DNA. (B) Same as (A) except using Cse4/α62 particles (native 6% PAGE). (C) Same as (A) except using H3/α62 particles (native 6% PAGE). In (A–C), DNA standards are included only as a rough guide, as migration of nucleosomal particles varies depending on gel concentration and running conditions. (D) AFM image and height distribution of H3 particles produced by gradient dialysis from 2 to 0.25 M, followed by step dialysis to 0.25 mM, conditions that increase aggregation. Bar in each image = 100 nm. DNA duplexes (147-bp 601) added to each sample provided an internal height standard. (E and F) Gel-shifted bands contain all four histones. Bands from native 6% gels were excised as indicated and loaded onto an SDS–PAGE gel to determine the histone composition of the particles. Particles were assembled using (E) H3 or (F) Cse4.
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gkt314-F2: Pseudo-octasomes split in half during low-salt dialysis. (A) Gel-shift image (native 7% PAGE), AFM image and height distribution of control H3 octamers assembled with 147-bp Widom 601 duplex DNA. (B) Same as (A) except using Cse4/α62 particles (native 6% PAGE). (C) Same as (A) except using H3/α62 particles (native 6% PAGE). In (A–C), DNA standards are included only as a rough guide, as migration of nucleosomal particles varies depending on gel concentration and running conditions. (D) AFM image and height distribution of H3 particles produced by gradient dialysis from 2 to 0.25 M, followed by step dialysis to 0.25 mM, conditions that increase aggregation. Bar in each image = 100 nm. DNA duplexes (147-bp 601) added to each sample provided an internal height standard. (E and F) Gel-shifted bands contain all four histones. Bands from native 6% gels were excised as indicated and loaded onto an SDS–PAGE gel to determine the histone composition of the particles. Particles were assembled using (E) H3 or (F) Cse4.

Mentions: Nucleosome structures indicate that a 62-bp DNA duplex will fully wrap a hemisome, and therefore we used the 62-bp sequence derived from human α-satellite (α62), which corresponds to the first 62 bp of the inverted repeat that has been used in most structural studies of nucleosomes (30). To assay for assembly, we followed a native gel-shift protocol, which yields a homogeneous band for 147-bp control octasomes produced by conventional salt dialysis of a mixture containing H3 octamers and Widom 601 duplexes (Figure 2A, left). Under similar conditions, a mixture of Cse4 octamers and α62 duplexes yielded a single moderately sharp band migrating at 680 bp on a native 6% gel (Figure 2B, left), whereas a mixture of H3 octamers and α62 duplexes consistently yielded two major gel-shifted products (Figure 2C, left).Figure 2.


Reconstitution of hemisomes on budding yeast centromeric DNA.

Furuyama T, Codomo CA, Henikoff S - Nucleic Acids Res. (2013)

Pseudo-octasomes split in half during low-salt dialysis. (A) Gel-shift image (native 7% PAGE), AFM image and height distribution of control H3 octamers assembled with 147-bp Widom 601 duplex DNA. (B) Same as (A) except using Cse4/α62 particles (native 6% PAGE). (C) Same as (A) except using H3/α62 particles (native 6% PAGE). In (A–C), DNA standards are included only as a rough guide, as migration of nucleosomal particles varies depending on gel concentration and running conditions. (D) AFM image and height distribution of H3 particles produced by gradient dialysis from 2 to 0.25 M, followed by step dialysis to 0.25 mM, conditions that increase aggregation. Bar in each image = 100 nm. DNA duplexes (147-bp 601) added to each sample provided an internal height standard. (E and F) Gel-shifted bands contain all four histones. Bands from native 6% gels were excised as indicated and loaded onto an SDS–PAGE gel to determine the histone composition of the particles. Particles were assembled using (E) H3 or (F) Cse4.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675498&req=5

gkt314-F2: Pseudo-octasomes split in half during low-salt dialysis. (A) Gel-shift image (native 7% PAGE), AFM image and height distribution of control H3 octamers assembled with 147-bp Widom 601 duplex DNA. (B) Same as (A) except using Cse4/α62 particles (native 6% PAGE). (C) Same as (A) except using H3/α62 particles (native 6% PAGE). In (A–C), DNA standards are included only as a rough guide, as migration of nucleosomal particles varies depending on gel concentration and running conditions. (D) AFM image and height distribution of H3 particles produced by gradient dialysis from 2 to 0.25 M, followed by step dialysis to 0.25 mM, conditions that increase aggregation. Bar in each image = 100 nm. DNA duplexes (147-bp 601) added to each sample provided an internal height standard. (E and F) Gel-shifted bands contain all four histones. Bands from native 6% gels were excised as indicated and loaded onto an SDS–PAGE gel to determine the histone composition of the particles. Particles were assembled using (E) H3 or (F) Cse4.
Mentions: Nucleosome structures indicate that a 62-bp DNA duplex will fully wrap a hemisome, and therefore we used the 62-bp sequence derived from human α-satellite (α62), which corresponds to the first 62 bp of the inverted repeat that has been used in most structural studies of nucleosomes (30). To assay for assembly, we followed a native gel-shift protocol, which yields a homogeneous band for 147-bp control octasomes produced by conventional salt dialysis of a mixture containing H3 octamers and Widom 601 duplexes (Figure 2A, left). Under similar conditions, a mixture of Cse4 octamers and α62 duplexes yielded a single moderately sharp band migrating at 680 bp on a native 6% gel (Figure 2B, left), whereas a mixture of H3 octamers and α62 duplexes consistently yielded two major gel-shifted products (Figure 2C, left).Figure 2.

Bottom Line: However, particles consisting of two DNA duplexes wrapped around a Cse4 octamer and separated by a gap efficiently split into hemisomes.Surprisingly, Cse4 hemisomes were stable in 4 M urea.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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus