Chromatin Structure

Chromosomes represent the largest and most visible physical structures involved in the transfer of genetic information. Surprisingly, our understanding of chromosome organization is most complete for the smallest and most fundamental structural units. These units are the nucleosomes which contain both DNA and histones. Long folded arrays of nucleosomes comprise the vast majority of chromatin. In this section I discuss the structural features of DNA and histones, how they assemble into nucleosomes and how nucleosomes fold into chromatin fibres. Finally, I describe what we know about the organization of the chromatin fibre into a chromosome and how this can be modified in various ways.

2.1 DNA AND HISTONES

The most striking property of a chromosome is the length of each molecule of DNA incorporated and folded into it. The human genome of 3 × 109 bp would extend over a metre if unravelled; however, this is compacted into a nucleus of only 10­5 m in diameter. It is an astonishing feat of engineering to organize the long linear DNA molecule within ordered structures that can reversibly fold and unfold within the chromosome. Not surprisingly, many aspects of chromosome structure reflect the impediments and constraints imposed by having to bend and distort DNA.

2.1.1 DNA structure

DNA has an elegant and simple structure around which the chromosome is assembled. The DNA molecule exists as a long unbranched double helix consisting of two antiparallel polynucleotide chains. DNA always contains an equivalent amount of the deoxyribonucleotide containing the base adenine (A) to that with the base thymine (T), and likewise of the deoxyribonucleotide containing the base guanine (G) to that with the base cytosine (C) (Fig. 2.1). Each base is linked to the pentose sugar ring (2-deoxyribose) and a phosphate group. The 5′ position of one pentose ring is connected to the 3′ position of the next pentose ring via the phosphate group (a 5′-3′ linkage) to create the polynucleotide chain (Fig. 2.2). The two antiparallel polynucleotide chains are attached to each other by hydrogen bonding between the bases. G is always base paired to C, and A is always base paired to T. In addition to the stability imparted by hydrogen bonding, hydrophobic base stacking interactions occur along the middle of the double helix (Fig. 2.3) (see Calladine and Drew, 1997 or Sinden, 1994 for details).

Physical studies using X-ray diffraction indicate that under conditions of physiological ionic strength, DNA is a regular helix, making a complete turn every 3.4 nm with a diameter of 2 nm. This particular DNA structure is known as B-DNA and has approximately 10.5 bp/turn of the helix. This means that every base pair is rotated approximately 34° around the axis of the helix relative to the next base pair. This results in a twisting of the two polynucleotide strands around each other. A double helix is formed that has a minor groove (approximately 1.2 nm across) and a major groove (approximately 2.2 nm across). The geometry of the major and minor grooves of DNA will be seen later to be crucial in determining the interaction of proteins with the DNA backbone. The double helix is right handed (Fig. 2.4).

Beyond this basic description, DNA structure is exceedingly plastic. Crystallization of various oligonucleotides indicates that a variety of DNA sequences will yield recognizable B-form DNA structures (Privé et al., 1991; Yanagi et al., 1991). More severe alterations in the conditions under which DNA is examined do, however, generate distinct conformations. Dehydrating the fibre will cause the double helix to take up a structure known as A-DNA (11 bp/turn); or placing DNA with a defined sequence of alternating G and C bases in solutions of high ionic strength will lead to the formation of a left-handed helix known as Z-DNA (12 bp/turn). The existence of either of these extreme structures in the eukaryotic nucleus under normal physiological conditions is controversial. However, their formation indicates the gross morphological changes that DNA can be forced to undergo (Drew et al., 1988; Calladine and Drew, 1997).

How do we know what structure populations of DNA molecules have in solution? Two experimental methodologies have been commonly used. The first employs DNA cleavage reagents and a flat crystal surface (Rhodes and Klug, 1980). When DNA is absorbed from solution on to a flat calcium phosphate surface and cut with DNase I, the enzyme cuts DNA most readily where it is exposed away from the surface. The average spacing between the sites of cleavage gives the approximate number of base pairs per turn of DNA (Fig. 2.5). This is determined by the electrophoresis of denatured molecules through a polyacrylamide gel. A better reagent for this purpose is the hydroxyl radical. Hydroxyl radicals are generated by the Fenton reaction in which an Fe(II) EDTA complex reduces hydrogen peroxide to a hydroxide anion and a hydroxyl radical.

EDTA2–+H2O2→FeEDTA1–+OH–+OH

The radical is about the size of a water molecule and has little sequence specificity in cleaving DNA. This it does by breaking the pentose sugar rings of individual deoxyribonucleotides. In contrast, DNase I is a large enzyme which has considerable sequence preferences. In both instances, the number of base pairs per turn of a large population of different DNA sequences bound to a crystal surface is found to be 10.5 (Tullius and Dombroski, 1985). This result is consistent with DNA having a B-form configuration as determined by X-ray studies.

The second method to examine DNA structure in solution reaches the similar conclusion that DNA has a B-form conformation at physiological ionic strength; however, a completely different strategy is used. It is generally found that a population of closed circular DNA molecules, identical in length and sequence, contains different numbers of superhelical turns. Superhelical turns can be simply defined by the following description: a single superhelical turn is introduced into a closed circular DNA molecule if the molecule is broken, one end of the molecule is then fixed, the other is rotated once and the two ends then rejoined. Supercoils can be positive or negative depending on which way the free DNA end is rotated. Closed circular molecules of the same length and sequence with different numbers of superhelical turns are known as topoisomers. Each population of small closed circular DNA molecules that differ in length by a few base pairs will exist as a distribution of topoisomers. These can be resolved by electrophoresis through an agarose gel matrix. A molecule which has a length corresponding to an integral number of helical turns will exist predominantly as a single topoisomer whereas a molecule which deviates from this by half a helical turn will be equally likely to exist with the superhelical turn in a positive or negative sense. The number of DNA molecules with a particular mobility in the agarose gel will be reduced by half since the molecules exist as an equal mixture of topoisomers. Examining the relationship between DNA length and the distribution of topoisomers allows the number of base pairs per turn of DNA to be calculated. The result of 10.5 bp/turn is close to that derived from crystal binding studies (Horowitz and Wang, 1984). Finally, theoretical calculations of the most stable configuration of DNA, which actually preceded much of the experimental work, suggested a value of 10.6 bp/turn (Levitt, 1978). The range of values around 10.5 bp/turn, obtained both experimentally and theoretically, provides a sound basis for considering alterations in this structure based on DNA sequence content and histone-DNA interaction.

Aside from the dramatic changes in DNA structure seen on formation of A- or Z-DNA, local variations in DNA sequence can significantly influence DNA conformation and properties of the helix. Our most extensive knowledge of the local changes in B-form DNA structure due to sequence content comes from studying AT-rich DNAs. For example, oligo(dA).oligo(dT) tracts are found experimentally, using both spectroscopic techniques and DNA cleavage reagents such as the hydroxyl radical, to be straight and rigid with a constant narrow minor groove width (Nelson et al., 1987; Hayes et al., 1991a). This is believed to be a consequence of...