3 Structural aspects of DNA
3.1 The helical structure of DNA
Having outlined the general principles of nucleic acid structures, we will now focus on how these principles influence the formation of specific structures found in DNA.
The helical structure of DNA arises because of the specific interactions between bases and the non-specific hydrophobic effects described earlier. Its structure is also determined through its active synthesis; that is, duplex DNA is synthesised by specialist polymerases upon a template strand. Within the helix, the two complementary DNA chains form what is called an antiparallel helix, where strands have opposite 5′ to 3′ polarity.
We outlined earlier the general principle of base pairing. Now we will go into more detail about why these base pairs arise within a duplex DNA. The specificity of Watson-Crick base pairing results from both the hydrogen-bonding factors we described earlier and steric restrictions imposed by the two deoxyribose–phosphate backbones. If we consider the steric restrictions first, we can start by asking how much ‘room’ there is between the two backbones and which bases will fit within this space to make suitable pairs.
First, the ‘gap’ across the helix between each of the deoxyribose–base glycosidic bonds is approximately 10.8 Å and a purine–pyrimidine base pair fits perfectly within this space, whereas two pyrimidine bases would be too far away from each other to allow hydrogen bonding. Thus spatial considerations limit each base pair to being between a purine and a pyrimidine. Note that mispairings, such as that between two purines, do occur during replication, but such mispairings distort the helix and are readily detected and corrected. Apart from the spatial considerations, specific requirements apply for the formation of hydrogen bonds between the bases in helical DNA, and the final positions of the hydrogen atoms within each base pair will be influenced by the positions of the bases after stacking interactions have occurred. Consequently, each base pair has a well defined position.
Look back at Figure 5a and try to visualise pairing between A and C bases. Describe the result.
There is no potential for hydrogen bonding between these bases.
Similarly, there is only potential for formation of a single hydrogen bond between G and T bases.
To satisfy steric restrictions of base pairing and to maximise the hydrophobic interactions between successive base pairs, the two polynucleotide chains in DNA are coiled around a common axis. In the structure of DNA proposed by Crick and Watson in 1953, known as B-DNA, adjacent bases are separated by 3.4 Å along the helix axis and the helix rotates 36° between each base.
How many base pairs are present within one helical turn of B-DNA?
Since the helix turns by 36° for each base, it will take 10 base pairs to rotate through one helical turn (360°).
The structure of the B-DNA helix is shown in Figure 9a. In this figure, the bases extend horizontally across the helical plane, and as described earlier, base stacking results in the backbone spiralling around at an angle of approximately 30°. If you take a closer look at the sugar–phosphate backbone in B-DNA, you can see that it spirals around the core. In the case of B-DNA, the helix is described as being ‘right-handed’, because if you look at the helix end-on, the 5′–3′ strand corkscrews away from you in a right-handed or clockwise spiral.
As can also be seen from Figure 9a, B-DNA has two obvious ‘grooves’ in its structure; the larger is known as the major groove, the narrower as the minor groove. These grooves result from the geometry of the sugar-base structure and base-pair interaction, as shown in Figure 9b. Within the major groove, a large portion of the base is exposed and it will perhaps not surprise you to learn that this is where most protein–DNA interactions occur that depend upon the specific recognition of individual bases within the DNA. Such interactions depend upon the formation of hydrogen bonds between amino acid side-chains in the protein and atoms in the bases that are not involved in base pairing; these atoms are identified in Figure 9c. You will see later in this unit how the accessibility of bases within the major groove permits protein–DNA interactions without interfering with base pairing.
Look back at the structure of the modified nucleoside 5-methylcytidine in Figure 4b. What will be the position of the methyl group in a B-form helix?
The methyl group of 5-methylcytidine will project into the major groove.
The position of the methyl group on 5-methylcytidine in DNA is significant. Projecting into the major groove as it does, this group can potentially interfere with protein–DNA interactions. We will see the importance of this interference when we consider the influence of DNA cytidine methylation in the regulation of transcription, where the methyl group can directly interfere with the binding of transcription factors.
Although Watson and Crick described the right-handed helical B form of DNA, X-ray diffraction studies have identified other forms of DNA structure. These different structures are dependent upon crystallisation conditions or base composition and two forms, A–DNA and Z-DNA, are shown for comparison in Figure 10.
Like B-DNA, A–DNA is a right-handed helix. In A–DNA, the major and minor grooves are of similar dimensions and there are 11 base pairs per helical turn compared with 10 in B-DNA. In Z-DNA, which has 12 base pairs per helical turn, the sugar-phosphate backbone forms a ‘zig-zag’ conformation, after which this structure is named.
Examine the structure of Z-DNA shown in Figure 10 in more detail. Compared with the A and B forms, can you describe the nature of the ‘handedness’ of the helix and its grooves?
You will notice that, when compared to the A and B forms, Z-DNA is left-handed; that is, the backbone spirals the opposite way round the helical axis from that seen in the A and B forms. Due to the kinking of the backbone, the nucleotides themselves bulge out more, leaving only one groove which is equivalent to the minor groove in B-DNA.
These different DNA secondary structures have been demonstrated from crystal structures, but what do we know about the structure of DNA in vivo, in the cell? The B form is the lowest-energy state for the DNA duplex. In its native duplex state, when not denatured for transcription, replication or repair, the helical secondary structure of DNA in the cell is generally believed to be the B form. However, there is a degree of fluidity in the structure adopted by DNA within an active cell, and other secondary structures such as A– and Z-DNA could exist. Z-DNA is known to form in vitro within particular stretches of DNA with alternating purines and pyrimidines, such as with 5′-(GC)n-3′. It is difficult to demonstrate the occurrence of Z-DNA in vivo, as it is believed to form only transiently within genomic DNA, though it is thought to occur in association with transcription. Note that the A-form duplex is predominantly found in double-stranded RNA, which cannot form the B-helix structure due to steric conflict involving the 2’ hydroxyl groups of the ribose component. Furthermore, hybrid duplexes, consisting of one strand each of RNA and DNA, are also thought to adopt an A-form structure. Such hybrids must occur in the transcription of RNA from a DNA template.
It should be noted that DNA is capable of adopting other higher-order structures, particularly in vitro, and we will now discuss some of these structures that may have biological roles to play. In the cell, DNA is, of course, found complexed with a variety of cellular proteins, many of which contribute to higher-order structures where the basic helix is folded into more condensed states. DNA can loop around itself and around proteins specific for this role. A number of different conformations and structures adopted by this versatile molecule have been identified and characterised, many of which have a particular purpose within the cell.