Nucleosome, formation

The association of histones to one or the other of the strands can be distinguished using electron microscopy of material from cells grown under conditions where protein (i.e. histone) synthesis is inhibited but DNA synthesis is not inhibited. 

Even the genes that code for proteins are more complex in vertebrates than in bacteria. Most, but not all, are expressed as long RNA molecules that are reduced in size by splicing together the coding segments. This yields a continuous template RNA that is sequentially decoded by protein-synthesizing enzymes. 

---

Figure 3. The stability of the nucleosome is affected by the length and the superhelicity of DNA. (a-b) The chromatin fibers were reconstituted from the purified plasmids and the histone octamers by a salt-dialysis method and observed under AFM. The 3 kb (a) or 106 kb (e) supercoiled circular plasmid was used as a template, (c) Relationship between the plasmid length and the frequency of nucleosome formation in the reconstitution process. The nucleosome frequency is represented as the number of base pairs per nucleosome and plotted against the length of the template DNA in supercoiled (filled circle) and linear (open circle) forms, (d) AFM image of the chromatin fiber reconstituted on the topoisomerase 1-treated plasmid, (e) Chromatin fiber reconstituted with Drosophila embryo extract. The chromatin fiber was reconstituted from plasmid DNA of 10kband the embryo extract of Drosophila, and was observed by AFM...

Ito T (2003) Nucleosome assembly and remodehng. Curr Top Microbiol Immunol 274 1-22 Ito T, Tyler JK, Kadonaga JT (1997) Chromatin assembly factors a dual function in nucleosome formation and mobilization Genes Cells 2(10) 593-600 Ivanovska I, Khandan T, Ito T, Orr-Weaver TL (2005) A histone code in meiosis the histone kinase, NHK-1, is required for proper chromosomal architecture in Drosophila oocytes. Genes Dev 19(21) 2571-2582... 

Even in 1978, it was realized the assembly of nucleosomes in vivo was likely to be a facilitated process. In fact, Laskey et al. [33] had discovered a factor, nucleo-plasmin that assisted in this assembly. Very recently. X-ray diffraction studies have revealed much about this protein, and its probable role in nucleosome assembly [34] or disassembly [35]. At the same time, single-molecule studies (see Zlatanova and Leuba, this volume, p. 369) have provided an insight into the dynamics and energetics of nucleosome formation. This appears likely to be an area in which rapid progress is possible. 

Fig. 1.41. The influence of the nucleosomes on the positioning of DNA-binding proteins. Example of a control region in which two regulatory DNA elements are separated by 60-90 bp but are brought near each other in 3D space via nucleosome formation. The super-helical arrangement of the DNA in the nucleosome brings the the two DNA elements close together. The DNA element-bound proteins PI and P2 are brought into closer contact with each other in this configuration than in a linear arrangement.

Although nucleosomes are distributed rather evenly along the DNA of a cell, there are some DNA sequences that favor nucleosome formation. The resulting positioned nucleosomes are often found in the vicinity of gene promoters, enhancers, and other... 

Eukaryotic DNA is replicated at a slower rate than prokaryotic DNA. One reason may be the requirement for the deposition of histone proteins on DNA (histone synthesis and DNA replication are coupled). Describe a model for the replication of eukaryotic DNA and nucleosome formation. 

DNA in eukaryotic chromosomes is complexed with histone proteins in complexes called nucleosomes. These DNA-protein complexes are disassembled directly in front of the replication fork. The nucleosome disassembly may be rate-limiting for the migration of the replication forks, as the rate of migration is slower in eukaryotes than prokaryotes. The length of Okazaki fragments is also similar to the size of the DNA between nucleosomes (about 200 bp). One model that would allow the synthesis of new eukaryotic DNA and nucleosome formation would be the disassembly of the histones in front of the replication fork and then the reassembly of the histones on the two duplex strands. Histone synthesis is closely coupled to DNA replication. 

Q.19.4 In experiments to investigate the protein structure of the nucleosome octamer, the isolated nucleosome is placed in a very high ionic strength solution. This step causes the dissociation of the DNA from the histones. Explain this in terms of the interactional energies of nucleosome formation. 

Hough, P. V. C., Mastrangelo, I. A., Wall, J. S., Hainfeld, J. F Simon, M. N., and Manley, J. L. (1982). DNA-protein complexes spread on N2-discharged carbon film and characterized by molecular weight and its projected distribution. Nucleosome formation in an in vitro transcription system. J. Mol. Biol. 160, 375-386. 

Nucleosome formation increases the packing ratio of the DNA to 7. However, this is far below the packing ratio required in vivo so that higher orders of packing are required. The next order level is a 10 nm diameter thin filament consisting of linear arrays of nucleosomes. A thicker (30 nm diameter) fibre appears to be... 

An immunoblot indicated that the level of the protein Bcl-2 was depressed when ascorbic acid was applied to tumor cells at a concentration of 70 p,M. Additional studies indicated that ascorbic acid at concentrations as low as 20 xM-1.25 p,M produced nucleosome formation (DNA fragmentation) of some of the tumor cells in a dose-dependent manner (Figs. 8 and 9). Subsequently, a determination of reduced levels of total mercaptans and intracellular levels of glutathione further suggested a prooxidant characteristic to the ascorbic acid treatment of the oral carcinoma cells (Figs. 10 and 11). 

FIGURE 8. Nucleosome formation in a human oral carcinoma cell line (SCC-25) was observed following treatment with ascorbic acid (20 xM, 3hr) (A), in comparison to untreated tumor cells (B). 

FIGURE 9. A comparison of the apoptotic response produced by ascorbic acid treatment compared to other antioxidant treatments. SCC-25 cells were stained for nucleosome formation as shown in Fig. 8. The relative number of apoptotic cells per 100 cells was counted. The treatment groups were 1) canthaxanthin, 2) p-carotene, 3) retinyl palmitate, 4) a-tocopherol acid succinate, 5) ascorbic acid, 6) glutathione (reduced), and 7) glutathione (oxidized). The treatment doses were 20, 10, and 1.25 fxM. 

In summary, the results of in vitro cancer cell studies demonstrated that ascorbic acid could exhibit a cytostatic or cytotoxic effect. This suppression of tumor cell growth was related to the loss of DNA repair and increased nucleosome formation and the development of programmed cell death or apoptosis. In animal studies, where ascorbic acid appeared to act as a cofactor or promoter, it was probable that the oxygenation state of the ascorbate radical was fundamentally changed so that it acted as a prooxidant rather than an antioxidant. 

Camerini-Otero, R. D., and Felsenfeld, G., 1977, Supercoiling energy and nucleosome formation The role of the arginine-rich histone kernel. Nucleic Acids Res. 4 1159. 

---