In this article we will discuss about:- 1. Coiled DNA Model 2. Nucleosome-Solenoid Model of Chromatin Fibres.
Coiled DNA Model:
This model of chromatin structure was proposed by DuPraw in 1965. As per this model, DNA molecule is associated with three classes of proteins:
(i) Lysine rich histories:
They are common to both A and B type fibres and are wrapped directly around the DNA double helix in its spiral grooves. Some of the histones may function to make the first order super coil, the type A fibril (10 nm).
(ii) Arginine rich histones:
These stabilize the second order supercoiling of A type fibrils into type B fibres (20-50 nm).
(iii) DNA dependent enzymes, e.g., RNA polymerase, function with type A fibrils.
Each histone molecule has a hypothetical wedge shaped structure and contains two DNA binding sites, (a) an amino-terminal site, and (b) a phosphoserine site. When both the sites are available, histones act to pack the DNA by supercoiling.
Template activity is prevented during the supercoiled condition of the fibres. Acetylation of N-terminal amino acid blocks one of the DNA binding sites; as a result, the histone molecules remain bounds to the DNA at the phosphoserine site. However, the supercoiling of the fibre is relieved making its DNA available for transcription. Histone phosphorylation blocks the second binding site (phosphoserine site).
Nucleosome-Solenoid Model of Chromatin Fibres:
This is the most widely accepted model; it was proposed by Kornberg and Thomas in 1974. According to this model, chromatin fibres consist of discrete particles called nucleosomes which generate a bead-like structure. The thread (string) is made up of the DNA molecules; wound around the beads but does not pass through the beads.
The nucleosome is the basic structural unit of chromatin fibre; it consists of DNA wound around an “octamer” of histone proteins. A bacterial enzyme, micro-coccal nuclease, has the ability to cut DNA between nucleosomes. However, this enzyme cannot cleave the DNA wound round the histone octamer.
Individual nucleosomes, therefore, can be obtained following digestion of chromatin with micro-coccal nuclease. The stretch of DNA) between two nucleosomes (this DNA is digested by micro-coccal nuclease) is called linker DNA or spacer DNA.
Composition of Nucleosomes:
A nucleosome consists of about 200 bp DNA associated with the histone octamer. The histone octamer of a nucleosome consists of two molecules each of the histones H2A, H2B, H3 and H4. The associated DNA and the histone octamer together constitute the core particle (Fig. 8.3).
Micro-coccal nuclease produces nucleosome monomers by cleaving the DNA between the core particles. This enzyme can further cleave the DNA of the nucleosomes, but at least 146 bp DNA remains associated with the protein octamer and is not available for the enzyme. Thus the core DNA (DNA bound to the core particle) is actually 146 bp in length.
The remaining part of the DNA is called linker DNA which joins neighbouring core particles of the chromatin fibres; linker DNA varies from 8 to 114 bp in different organisms and tissues. The histone HI is associated with the linker DNA.
The nucleosome is cylindrical in shape with a dimension of “11 nm (diameter) x 6 nm (length)” Fig. 8.3. Thus the circumference of a nucleosome is about 34 nm. The length of 146 bp DNA associated with the core particle is sufficient to wound around the particle about 1.8 turns (Fig. 8.3).
The width of DNA double helix is 2 nm; it thus occupies 4 nm of the cylinder length due to its two turns. One complete turn of the DNA double helix around the histone octamer involves 80 bp and therefore, the two points located 80 bp apart come close to one another on the nucleosome (Fig. 8.4).
Enzyme DNAase I and DNAase II can put nick on one strand of DNA. The nicks made by these enzymes on naked DNA are random. But nicks made by these enzymes on nucleosomal DNA is at regular intervals. They cut the DNA in the multiples of 10 bp.
The length of DNA double helix per turn is approximately 10 base pairs; it may be concluded that DNA double helix is tightly wound over the surface of the histone octamer and a small region of each turn of double helix is exposed.
DNAase I acts on this exposed region (Fig. 8.4). This type of analysis has indicated that there should be 10.5 bp per turn of double helical B-DNA. However, the average number of base pairs per turn in the nucleosome ranges from 10.2 to 10.4.
Organization of the Histone Octamer:
The core particle of nucleosome is an aggregate of four types of histones namely, H2A, H2B, H3 and H4; they are called core histones. The core particle contains two molecules of each type of histone and thus it is an “octamer”.
The histones H3 and H4 combine to form a “tetramer” (H32. H42). The other two histones (H2A and H2B) are present as dimers (H2A. H2B). Thus the core particle is composed of one tetramer (H32. H42) and two dimers 2(H2A. H2B).
The general structure of the core particle is formed by interaction among the histone molecules. The diameter of the “octamer” is decided by the dimension of the H32. H42 tetramer. The surface of the octamer makes a super-helical path for binding of the DNA.
The HI histone is associated with the linker DNA. Its location is adjacent to the core DNA. When micro-coccal nuclease cuts DNA between nucleosomes to produce nucleosome monomers, the HI is found associated with the nucleosome that has 160-170 bp of DNA.
On further action of this enzyme, the DNA outside the nucleosome core is digested away producing the nucleosome monomer with 146 bp. In this condition, the HI is lost from the core particle. HI can be removed from the chromatin without enzymatic action, leaving the DNA molecule intact. After removal of HI, the linker DNA is stretched out into a 10 nm fibre.
Thus HI histone is responsible for the formation of 30 nm chromatin fibre.
Nucleosome structure in the virus SV40:
The virus SV40 has double-stranded circular DNA which is organized in nucleosomes. The chromosome is called a minichromosome which is about 210 nm in length. The SV40 DNA is 5200 bp and about 1500 nm long. Thus the packing ratio in the minichromosome is about 7 (1500 nm/210 nm).
The chromosome is also supercoiled. When the supercoiling of the chromosome is relaxed, a circular structure is obtained. Removal of histone octamers yield naked DNA in which the number of supercoils can be estimated; this number is the same as the number of nucleosomes in this length of DNA.
When the histones form nucleosome, the DNA becomes wound around the octamer producing one negative supercoiled turn. Free DNA has 10.5 bp/turn whereas the DNA wound on nucleosome has 10.2 bp/turn of the helix.
Both the active and inactive chromatin have similar nucleosomal organization. The active form of chromatin contains non-histone proteins addition to histones. Nucleosome phasing refers to the nonrandom distribution and the specificity of distribution of DNA sequences on nucleosomes.
In phased condition, each site of DNA is always located at a particular position on the nucleosome. Thus every nucleosome lies at a unique position and the linker DNA consists of unique sites in phased organization of nucleosomes. In general, nucleosomes are in phase near the boundaries where non-histone proteins are bound to DNA. From the boundaries, a series of nucleosomes may be assembled sequentially.
The 10 nm fibre looks like a “string on beads”. The shape of nucleosome is more or less a flat cylinder and the 10 nm fibre is formed by stacking of the nucleosomes. The nucleosomes are tilted at an angle of 20° to the fibre axis. Coiling of the 10 nm fibre occurs in the presence of HI histone to give rise to a 30 nm fibre.
The number of nucleosomes per turn in the 30 nm fibre is 6 which have a radial organization (Fig. 8.5); this is called the solenoid model of chromatin structure. The coiling of nucleosomes is like a helix in which the angle of turn between the faces of nucleosomes is 60°.
Thus the hierarchy of organization of DNA associated with protein can be summarized as follows (Fig. 8.6):
(1) DNA double helix has a diameter of 2 nm; a single DNA molecule runs through the entire length of the chromosome. “solenoid”.
(2) DNA is associated with histones to form nucleosome particles. One nucleosome contains 200 bp DNA (67 nm long); it forms the fibre of 10 nm diameter. The diameter of nucleosome is 11 nm and thus the packing ratio of DNA is 67/11 or about 6.
(3) Coiling of 10 nm fibre occurs to form the chromatin fibre of about 30 nm diameter. It involves 6 nucleosomes per turn of the coil, thus making the DNA packing ratio of about 40.
(4) The folding of 30 nm fibre gives rise to a packing ratio of about 1000 in euchromatin.
(5) In mitotic metaphase chromosomes, the packing ratio becomes about 10,000. In other words, one pm length of the mitotic chromosome contains about 10,000 pm of DNA.
Nucleosome and replication of DNA:
It has been observed that nucleosomes reorganize immediately after DNA replication. In vitro assembly of nucleosomes has been studied using replication of SV40 DNA into the extract of human cells. The nucleosome assembly reactions occur on replicating DNA and it requires a factor CAF1. There are three questions regarding the nucleosome reorganization in replicating DNA.
(1) Whether the nucleosome is intact, the histone molecules are conserved in the octamer and newly synthesized histone form new octamers during DNA replication.
(2) Whether histone molecules separate from the core particle and new nucleosomes are organised in such a way that some old and some new histone molecules interact to form the histone octamer. In this case, there may be two situations:
(a) The old and new histone molecules assemble randomly to form an octamer.
(b) The tetramer H32.H42 of the older nucleosome associates with a new H2A. H2B dimer.
In vitro, nucleosomes can be assembled in two ways:
(i) The tetramer H32.H42 (kernel) binds to DNA and then H2A. H2B dimers are associated to form the octamer. Alternatively,
(ii) The octamer is formed first and then DNA binds to the octamer.
Experiments were conducted using heavy and light amino acids to understand the mechanisms of nucleosome assembly. Cells were grown in the medium containing heavy amino acids. Therefore, the histones synthesized contained heavy amino acids. Then these cells were allowed to grow in a medium containing light amino acids. DNA replication occurred and nucleosomes were formed.
The histone octamers from the chromatin were subjected to centrifugation.
Following two situations may occur:
(I) If old octamers are conserved and complete new histone octamers are formed, they will occupy two density positions, heavy and light, following centrifugation.
(II) If old octamers get dissociated into the constituent histone molecules, and then they reassemble into new octamers consisting of both old (heavy amino acids) and new (light amino acids) histone molecules; the octamer will have an intermediate density.
When the histone octamers were observed following density gradient centrifugation, there was only a single intermediate band between heavy and light densities. This indicates that the histone octamers are not conserved, but they are reassembled afresh and consist of both old and new histone molecules.
Certain short regions of chromatin are highly susceptible to DNAase I and other nucleases; these regions are called hypersensitive sites. Such sites lack the typical nucleosomal organization. The sensitivity of these regions to DNAase I may be two or more times greater than that of the rest of the chromatin.
Hypersensitive regions do not occur in inactive chromatin, but they are found associated with the genes which are being transcribed (expressed). Such a site consists of a sequence of 200 bp from which the nucleosomes are excluded. The hypersensitive sites are found at the origin of DNA replication, promoter and the centromeres.
The minichromosome of SV40 contains a region of about 350 bp which is free from nucleosome and is sensitive to nucleoses. This region consists of two hypersensitive sites for DNAase I and a “protected region”. Non-histone proteins are associated with the DNA to make a part of the region “protected” from nucleases.
Since the hypersensitive sites are found associated with promoters, origin of replication and centromeres, it may be considered that certain regulatory factors generate such sites. Several hypersensitive sites have been observed in the extra-chromosomal rDNA of Tetrahymena pyriformis.
The hypersensitive site in rDNA forms a boundary for a series of phased nucleosomes. In this rDNA, there are two points of transcription initiation from which the rDNA is transcribed in the opposite direction. Between these two points five phased nucleosomes containing 1000 base pairs of DNA are located.
Some hypersensitive sites may be susceptible to different kinds of nucleases. Such a hypersensitive site is located at the promoter of chicken (3-globin gene. This site extends from about – 70 to -270 bases (upstream) and is sensitive to several nucleases such as micro-coccal nuclease, MsP I, DNAase I and DNAase II. The point of action for the different nucleases seems to be quite different.