The study (Structural Analysis of the Hexasome, Lacking One Histone H2A/H2B Dimer from the Conventional Nucleosome) is aimed at unraveling the mystery behind the Hexasome`s structure. As the study recognizes, the Hexasome has been studied in different aspects i.e. chemically, functionally, biologically but its study structurally was limited. The reason behind this was that a method to obtain purified Hexasomes wasn`t known.
In this study though, Yasuhiro Arimura and the group of scientists involved were successful in obtaining purified Hexasomes in vitro which made the studies and objectives obtainable. The structural analyses of the Hexasome is important in the fact that one is able to understand nucleus based reactions e.g. transcription among others. One is also able to understand the mechanisms surrounding this Hexasome related processes e.g. Transcription
The objectives of this study are clear in the fact that they are analyzing the Hexasome structurally, in the process explaining its function in some of the reactions and also how on are able to obtain them in vitro while they are pure. The importance of the structural analysis is that once one understands the Hexasome structure he/she is able to comprehend the mechanism involved in the processes it takes part in. This also is important as one understands the role that the Hexasome plays in these processes.
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The summary includes the techniques followed to obtain purified Hexasomes, the relation of Hexasomes and various processes and the role played by the Hexasome. Hexasomes are presumed to be the intermediate that is usually formed during disassembly, re-assembly and remodeling of the nucleusome in transcription, DNA repair, recombination and replication. According to other studies the Hexasome is also said to take part in transcription elongation.
According to the study summary, the Hexasome is the Histone component once the Octasome loses the H2A/H2B dimer.
This process i.e. removal of the H2A/H2B dimer is observed when the RNA polymerase II passes through the nucleusome during the transcription process, the Histone chaperon FACT (Facilitate Chromatin Transcription) complex simulates the formation of the RNA polymerase II-dependant Hexasome. Also, the removal of H2A/H2B dimer can be influenced by the major Histone chaperone Nap 1.
To study the Hexasome members on this research team established a process by which one can obtain purified Hexasomes. This was done by obtaining purified human histones. The human histones are well expressed in Escherichia coli, hence they were produced as N-terminally His6-tagged proteins in Escherichia coli from resuspended insoluble fraction in 50ml of 50mm Tris-HCl buffer at the pH of 7.5, that contains 7M guanidine hydrochloride, 500mm NaCl and 5%glycerol. The His6-tagged histones were then purified by a three-step procedure, including nickel−nitrilotriacetic acid (Ni-NTA) agarose chromatography, thrombin protease (1 unit/mg of histones) treatment, and Mono S column chromatography. The purified histones are then dialyzed against water, they are then freeze dried and then stored at 40c.
As mentioned above the human Histone H2B, H2A, and H3 genes are well expressed in E.coli cells though the human H4 Histone was poorly expressed. Because of this a new Histone H4 gene with E.coli optimized codons. These recombinant human histones are expressed as His6-tagged proteins and are purified. The H2A/H2B dimer and the H3/H4 tetramer are refolded by dialysis against buffer without urea, and His6 -tags of the histones in the H2A/H2B dimer and the H3/H4 tetramer are removed by thrombin protease digestion.
After this the Preparation of the H2A/H2B, H2A.Z/H2B, H3/H4, and CENP-A/H4 Complexes follows. This then precedes Preparation of DNA Fragments for Hexasome Reconstitution. This is done by amplifying a 193-base-pair DNA fragment that contains the Widom 601 sequence by using the PCR. The formed 193-base-pair DNA fragments are ligated into promega and the Satellite112 plasmid containing the 16 tandem copies of the 58-base-pair sequence is obtained. The 54-base-pair and the 112-base-pair DNA fragments were also obtained through respective methods.
A technique also performed was the preparation o the Hexasome; The purified complexes H3/H4 and H2A/H2B complex are mixed with a 193-base-pair DNA fragment, this is done in a solution containing 2 Molar KCl. For the neucleosome CENP-A, instead of the H3/H4 complex the CENP-A/H4 was used and for the Nucleosome H2A.Z the H2A.z/H2B complex is used. These mixtures are dialyzed against a buffer at 40c. Using a peristaltic pump the Hexasome reconstitution is done by gradually decreasing the KCL concentration. This reconstituted Hexasomes are then incubated at 550c for one hour and separated from the free DNA and histones via electrophoresis. They are then dialyzed against 20mm Tris-HCl buffer at the pH of 7.5. The purified Hexasomes and Octasome undergo a treatment assay then dynamic light scattering(DLS) measurements analyzing the Octasome and Hexasomes. The small angle X-ray scattering (SAXC) with the scattering intensities being recorded and analyzed.
The results in the performed analyses indicate and confirm the formation of purified Octasome and Hexasome. Further the tests done i.e. the SAXC and the dynamic light scattering measurements explained about the structure of the Hexasome. First the assays i.e. MNase and Exo III exposed that the MNase- or ExoIII-susceptible DNA region of the Hexasome is actually larger than that of the Octasome, this shows that the DNA segments that are located at the entry or even exit site of the Nucleosome may be apart from the histone surface in the Hexasome hence the hydrodynamic radius of the Hexasome may be considered to be larger than that of the Octasome. This assay and the DLS indicate that the DNA segment is unwrapped at the entry or the exit of the Hexasome.
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The performance of the SAXC, with the 112 base pair DNA fragment and the Octasome containing a 146-base-pair DNA fragment that was reconstituted, provided that despite the differing lengths of DNA in the Hexasome and Octasome nearly identical curves were observed. This inferred that the structure of Hexasomes and Octasome are somehow similar though the difference in the radius of gyratin and the maximum diameter of the Hexasome were smaller than those of the Octasome hence local structural difference due to absence of H2A/H2B dimer and the Hexasome having a shorter DNA
From the Octasome crystal structure a model could now be made by removing one H2A/H2B dimer and the DNA segment that s located near this dimer. The crystal Hexasome model and the SAXC curve of the actual Hexasome according to the studies undertaken were similar.
The above figure represents the findings of the studies the group undertook. This are the results of the Small-angle X-ray scattering (SCAXC) showing the relation of the Octasome and the Hexasome. The relation between the Hexasome and the Octasome is clearly seen and the differences as the curve fall. The red dots represent the 112-base-paired Hexasome and the black dots the 146-base-paired Hexasome.