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Figures

Figure 1a: Courtesy Robert Saunders;

Figure 1c: Mark Hirst and David Shuker/ Open University;

Figure 1d: Dr Thomas Broker;

Figure 1e: Dr Georg
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References

Boyle, S., Gilchrist, S., Bridger, J. M., Mahy, N. L., Ellis, J. A. and Bickmore, W. A. (2001) The spatial organization of human chromosomes within the nuclei of normal and emerin-mutant cells, Human Molecular Genetics, 10(3), pp. 211–219.
Bulyk, M. L., Huang, X., Choo, Y. and Church, G. M.. (2001) Exploring the DNA-binding specificities of zinc fingers with DNA micro-arrays, Proceedings of the National A
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End of of unit questions

Question 1

What effect does ethidium bromide intercalation have on supercoiled DNA?

Answer

When ethidium bromide intercalates into duplex DNA, it results in an unwin
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Summary of Section 8

  1. Eukaryotic DNA is compacted through a hierarchical series of events, from nucleosomes to 10nm and 30 nm fibres, then through further stages to the chromomena fibres, which are attached to scaffolds to form loops. In preparation for mitosis, these fibres are further compacted to prevent physical damage.

  2. Chromosomes within the interphase nucleus occupy defined regions.

  3. DNA within the eukaryotic interphase nucleus is in a dyn
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8.4 The organisation of the mitotic chromosome

In order to prepare the chromosome for mitosis, a process in which DNA molecules become physically separated, an additional stage of compaction occurs to reach the highest level. The processes involved in this final stage of compaction are not clearly understood, but can be represented diagrammatically as in Figure 43. The process probably involves the coiling together of scaffolds into the higher-order structure.


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8.3 Chromosome distribution within the nucleus

DNA from any one particular chromosome is a single chain, many millions of bases long, and this chain is attached to a scaffold structure. It is not surprising then, that if we examine the interphase nucleus, each chromosome is seen to fill a localised area. This localised distribution of individual chromosomes is illustrated in Figure 42 with an examination of human chromosomes within the interphase nucleus. In these examples, special DNA probes have been used to detect the location of the e
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8.2 Chromosome scaffolds

Most of the chromosomal DNA chains within the interphase nucleus are believed to be held on a scaffold or backbone structure made from various proteins, with loops of between 20 and 200 kb extruding from attachment sites. This chromosome structure is shown schematically in Figure 40. The scaffold, as well as permitting further compaction, serves to bring the DNA together in organised regions. There are many different protein components of these scaffolds, amongst them DNA topoisomerases.


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8.1 Introduction

The average human cell has around two metres of DNA within its nucleus. In the interphase nucleus, in which transcription and replication are going on, this DNA is packaged into nucleosomes that are variably compacted, through association with H1, into larger 30nm fibres. In fact, the average nucleus most likely contains DNA with a continuum of chromatin configurations, ranging from highly open 10 nm fibres, through to 30 nm fibres and fibres that are even more tightly packed together, call
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Summary of Section 7

  1. Packaging of DNA serves to protect against damage, to compact the DNA helix into a suitable size within the cell, and to act as both a platform for and an intrinsic part of the structural and regulatory machinery involved in DNA metabolism.

  2. DNA compaction in prokaryotes achieved through a combination of supercoiling and interactions with proteins that aid DNA bending.

  3. Compaction of the eubacterial chromosome is facilitated
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Core histone tail modification regulates DNA compaction

SAQ 34

What effect would neutralising the positive charges on the octamer N-terminal tails have upon the compaction of DNA by H1?

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Nucleosomal DNA packaging into a 30 nm fibre: the role of histone H1

When chromatin is isolated from the nucleus and examined under the electron microscope, it can be seen as a 30 nm fibre. This fibre is formed through the action of the histone H1 on the nucleosomal DNA in the 10 nm fibre. In contrast to the other histone proteins, H1 does not contain the histone fold motif.

Compaction of the 10 nm fibre to give the 30 nm fibre is achieved by interaction of the H1 protein with both the linker DNA and the histone octamers, as shown in Figure 31
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The histone fold and formation of the nucleosome

We have seen how in the eubacterial chromosome, bending DNA serves to facilitate its compaction. A similar process occurs in eukaryotic cells in that DNA is bent and wrapped around a protein unit. In this case, the core unit is a protein–DNA complex termed a nucleosome. The nucleosome comprises the core histone proteins H2A, H2B, H3 and H4 arranged in a structure known as the core histone octamer, with an associated length of DNA. In order to understand how the nucleosome is a
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The histone proteins

The genes for the histone proteins are very highly conserved across eukaryotes, reflecting their importance in DNA packaging. The histone family consists of five groups of proteins, histones H1, H2A, H2B, H3 and H4. An examination of their amino acid content gives us clues as to how the histones fulfil their role in DNA packaging. Rather like the polyamines in bacteria, these proteins are highly positively charged, with up to 20% of their amino acids being lysine or arginine, the charged side
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7.3 The eukaryotic chromosome

Whilst the bulk of eukaryotic DNA is packaged by proteins different from those in the eubacterial chromosome, the principles of bending DNA and neutralising the negative charges in its backbone are shared. Eukaryotic cells have considerably larger genomes than do prokaryotes (in most cases over 1000 times the size of the E. coli genome – see Author(s): The Open University

The DPS protein compacts the eubacterial chromosome during stress

When an E. coli cell enters into stationary phase, transcription and cell division cease completely. In such cells, the normal chromatin components, such as those described above, are replaced by a negatively charged protein called DPS. The interaction between DPS and DNA appears to be a specialised bacterial adaptation to survive starvation. In normal conditions of growth, the DNA within the bacterial cell is distributed evenly throughout the entire cytoplasm. In stationary cells, how
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DNA supercoiling and protein binding in the E. coli chromosome

As discussed earlier, the DNA of the E. coli chromosome is highly negatively supercoiled due to the action of the DNA gyrase enzyme (Section 3.2). This negative supercoiling serves to assist in compaction of the DNA, with the repulsive forces of the sugar-phosphate backbones being counteracted by polyamines. Many of the proteins
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7.2 The eubacterial chromosome

Some of the diverse roles of chromatin components can be illustrated by examining the E. coli chromosome. Like most prokaryotes, E. coli has a single chromosome consisting of a single double-stranded circular DNA molecule. There is no nucleus present, but the E. coli DNA is within a discrete entity in the cytoplasm called the nucleoid. The nucleoid contains a multitude of proteins and is in close proximity to the ribosomes, where translation occurs. In addition to
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7.1 Introduction

Until now, we have discussed DNA primarily as a double helix, but in its natural state within the cell it is found packaged as a complex mixture with many different proteins and other components. You have already seen examples of proteins with specific roles to play, such as topoisomerases and the proteins with various DNA binding domains, but in this section we will turn our attention to the proteins that serve to pack and organise the DNA into what we call chromatin.

The packaging of
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Summary of Section 6

  1. Sequence-specific protein–DNA interactions are achieved through the formation of non-covalent bonds between amino acid side-chains in the protein and bases in the major groove of the DNA.

  2. Non-sequence-specific protein–DNA interactions are achieved primarily through electrostatic interactions between positively charged amino acid side-chains and the negatively charged DNA backbone.

  3. Protein and DNA conformation can be alt
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6.4 Non-specific DNA-protein interactions

As we saw above, most sequence-specific DNA binding proteins recognise and bind to their target DNA sequence with a high affinity by utilising structural domains that make sequence-specific contacts with the DNA bases in the major groove. These contacts utilise extensive non-covalent bonding and hydrophobic interactions. In contrast, non-specific protein–DNA interactions occur with much lower affinity. The reason for this low binding affinity is that most non-sequence-specific interactions
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