ORGANISATION OF CHROMOSOMES The human genome contains 3 x 109 bp.

If the DNA of all 46 chromosomes from one cell was linked together, it would measure one meter in length. However, in human as well as other eukaryotes, genomic DNA can be highly folded, constrained, and compacted by histone and non-histone proteins into chromatin and chromosome. This is accomplished by proteins that successively coil and fold the DNA into higher and higher levels of organization. Histones are a family of small, positively charged proteins termed H1, H2A, H2B, H3, and H4. DNA is negatively charged, due to the phosphate groups in its phosphate-sugar backbone, so histones bind with DNA very tightly. 1st Level of DNA Packing The basic structural unit of chromatin, the nucleosome, was described by Roger Kornberg in 1974. Two types of experiments led to Kornbergs proposal of the nucleosome model. First, partial digestion of chromatin with micrococcal nuclease (an enzyme that degrades DNA) was found to yield DNA fragments approximately 200 base pairs long. In contrast, a similar digestion of naked DNA (not associated with protein) yielded a continuous smear randomly sized fragments. These results suggest that the binding of proteins to DNA in chromatin protects the regions of DNA from nuclease digestion, so that enzyme can attack DNA only at sites separated by approximately 200 base pairs. Electron microscopy revealed that chromatin fibers have a beaded appearance, with the beads spaced at intervals of approximately 200 base pairs. Thus, both nuclease digestion and the electron microscopic studies suggest that chromatin is composed of repeating 200 base pair unit, which were called nucleosome. Individual nucleosome = beads on a string The nucleosome hypothesis was proposed by Don and Ada Olins in 1974 and Roger Kornberg. The majority of the DNA in eukaryotic cells is packaged into nucleosomes. The Nucleosomes are the basic unit of DNA packaging in eukaryotes, composed of a core of eight histone proteins and the DNA wrapped around them. The DNA between each nucleosome (the "string" in the "beads on a string") is called linker DNA. By assembling into nucleosomes, the DNA is compacted approximately six fold. This is

far short of the 1,000- to 10,000-fold DNA compaction observed in eukaryotic cells. The nucleosome contains ~200 bp of DNA associated with a histone octamer that consists of two copies each of H2A, H2B, H3, and H4. These are known as the core histones. This forms a chromatin subunit known as chromatosome. The association of DNA with the histone octamer forms a core particle containing 146 bp of DNA Core DNA has an invariant length of 146 bp, and is relatively resistant to digestion by nucleases. Linker DNA comprises the rest of the repeating unit. Its length varies from as little as 8 bp to as much as 114 bp per nucleosome. DNA is wrapped 1.65 times around the histone octamer. Histone H1 (linker) binds and compacts nucleosomes to form a 11/10nm fibre which represents the 1st level of organisation. Histones are by far the most abundant proteins associated with eukaryotic DNA. Eukaryotic cells commonly contain five abundant histones: H1, H2A, H2B, H3, and H4. Histones H2A, H2B, H3, and H4 are the core histones and form the protein core around which nucleosomal DNA is wrapped. Histone H1 is not part of the nucleosome core particle. Instead, it binds to the linker DNA and is referred to as a linker histone. The four core histones are present in equal amounts in the cell, whereas H1 is half as abundant as the other histones. 2nd Level of DNA Packing: 30 nm Fiber Once nucleosomes are formed, the next step in the packaging of DNA is the binding of histone H1. Like the core histories, H1 is a small, positively-charged protein. H1 interacts with the linker DNA between nucleosomes, further tightening the association of the DNA with the nucleosome resulting in the nucleosomal DNA forming a 30-nm fiber. This structure, which can also be observed in vivo, represents the next level of DNA compaction. DNA is further compacted when the DNA nucleosomes associate with one another to produce 30 nm chromatin Mechanism of compaction is not understood, but H1 plays a role (if

H1 is absent, then chromatin cannot be converted from 10 to 30 nm) DNA is condensed to 40th fold. There are two models for the structure of the 30-nm fiber. - In the solenoid model, the nucleosomal DNA forms a super helix containing approximately six nucleosomes per turn. - An alternative model for the 30-nm fiber is the "zigzag" model. - This model is based on the zigzag pattern of nucleosomes formed upon H1 addition. In this case, the 30-nm fiber is a compacted form of these zigzag nucleosome arrays. 3rd Level of DNA Packing: Chromsome Scaffold/300 nm Additional folding of the 30-nm fiber is required to compact the DNA further. Further compaction of DNA involves large loops of nucleosomal DNA. Although the exact nature of this folded structure remains unclear, one popular model proposes that the 30-nm fiber forms loops of 40-90 kb that are held together at their bases by a proteinacious structure referred to as the nuclear scaffold. A variety of methods have been developed to identify proteins that are part of this structure although the true nature of the nuclear scaffold remains mysterious. Two classes of proteins that contribute to the nuclear scaffold have been identified. One of these is topoisomerase II (Topo II) and the other one are the SMC (Structural Maintenance of Chromosomes) proteins - Structural non-histone protein could be involved. The scaffold itself appears to contain several proteins, notably large amounts of histone H1 (located in the interior of the fiber) and topoisomerase II. The presence of topoisomerase II further emphasizes the relationship between DNA underwinding and chromatin structure SARs (Scaffold Attachement Region) are very AT-rich fragments several hundred base pairs in length that were first identified as DNA fragments that are retained by nuclear scaffold/matrix preparations. They define the bases of the DNA loops that become visible as a halo around extracted nuclei and that can be traced in suitable electron micro-graphs of histone-depleted metaphase chromosomes. They are possibly best described as being composed of

numerous clustered, irregularly spaced runs of As and Ts. Metaphase chromosomes: 700nm fiber Formed by looping and coiling of condensed chromatin (Coiling of the 300 nm fiber) Associated with H1 phosphorylation phosphorylation (especially of histone H1) helps to pack nucleosomes together and thus tends to promote higher levels of chromatin compaction e.g. formation of metaphase chromosomes. ~10,000 fold compaction achieved