Chapter 2 Section 4 NUCLEIC ACIDS
2.4.2. STRUCTURE OF DNA
The nucleic acids are long linear polymers. Frederick Meischer first isolated DNA in 1865, obtaining it from pus scraped from surgical bandages. Early in the 20th century, the bases of DNA were identified as the purines adenine (A) and guanine (G), and the pyrimidines cytosine (C) and thymine (T). DNA occupies a central and critical role in the cell as the genetic information in which all the information required to duplicate and maintain the organism resides. Thus all information necessary to maintain and propagate life is contained within the linear array of the four bases. The base composition of DNA is very variable amongst the species and the adenine- thymine content ranges from 27 – 75% in prokaryotes to about 45 – 53% in mammals.
As in proteins, different levels of structure in nucleic acids can be described. The primary structure of nucleic acids is the order of bases in the polynucleotide sequence, whereas the secondary structure is the three-dimensional conformation of the backbone. The way this structure is supercoiled in space can be described as the tertiary structure and the way the nucleic acids interacts with proteins is regarded as the tertiary structure.
Though nucleic acids are made of four monomeric units whereas proteins have twenty, sequencing of the nucleic acids was a much more difficult task because distinctive sequences were more difficult to identify and there are apparently fewer specific sites for selective cleavage. The discovery of restriction endonucleases which are able to cleave DNA at specific oligonucleotide sites, and the perfection of the technique of polyacrylamide gel electrophoresis separation methods to resolve nucleic acid fragments generated by restrictriction endonuclease treatment, effectively simplified the procedure such that, at present, the task of sequencing nucleic acids is simpler than deciphering the sequence of polypeptides. The whole process has also been automated to the extent that identifying 104 bases per day has become routine. The development of such automated procedures has been largely responsible for the deciphering of the almost 2.9 billion base pairs of the human genome.
5’ to 3’ direction of DNA sequences
Fig. 2.4.5. The chemical structure of the nucleotide adenosine triphosphate (ATP).
The numbering for the ring carbon and nitrogen atoms is shown in the above figure. The six- membered rings of the bases are numbered in a counterclockwise direction, starting with a nitrogen. In order to distinguish numbering of the sugar carbon atoms from that of the bases, the sugar carbons are numbered with a prime (’), starting with the atom which is connected to the base, and continuing around the sugar ring, away from the oxygen atom. It can also be seen from the figure that for the deoxyribose sugars, there are phosphate atoms connected to the oxygen atoms adjacent to the 3’ and 5’ carbon. If one looks at the two ends of a polynucleotide chain (see fig. 2.4.6), one can see that one end of the chain has a 5’ phosphate on it, and this is called the 5’
end, while the other has a 3’ OH on it, and is called the 3’ end. By convention the nucleic acid sequence is always read or numbered from the 5’ end; for example in the figure the sequence would be read as 5’ TGCA 3’. The DNA and RNA polymerases, enzymes in cells which copy the DNA, also always synthesize in the 5’ to 3’ direction.
Primary Structure of DNA
The polymerization of nucleotides gives rise to the nucleic acids. The nucleotide monomers are linked together by the formation of two ester bonds by phosphoric acid. The hydroxyl groups to which the phosphoric acid is esterified are those bonded to the 3’ and 5’ carbons on adjacent residues. The resulting repeated linkage in a polynucleotide is a 3’, 5’-phosphodiester bond.
Figure 2.4.6 represents the structure of a fragment of DNA chain. The sugar-phosphate backbone repeats itself down the length of the chain. The specific sequence of bases in the DNA molecule carries the information necessary for making the numerous proteins in the cell. In other words, the blue-print for every living organism is contained in its DNA.
Fig. 2.4.6. A fragment of a DNA chain. (Source: Garrett, R. H. and Grisham, C.M., 1999, p 336 fig.11.17)
Double Helical Secondary Structure of DNA
Polynucleotides usually occur in the form of thermodynamically favourable helical structures and not in their extended form. The helical structure results from stacking of bases along the helix axis with the bases perpendicularly oriented along the helix and touching one another. This arrangement, however, leaves no free space between two successive bases. Such stacked single- stranded helices, therefore, are not commonly encountered in living cells. Instead, polynucleotide sequences tend to associate with each other to form double helices.
Although some forms of DNA exist as single-stranded structures, the most widespread DNA structure is the double helix. This structure was first proposed in 1953 by Watson and Crick, partly based on previously available X-ray diffraction studies and forms a landmark in the history of science. Most of the advances in molecular biology has directly stemmed from this structure.
One purely hypothetical conformational possibility for a two-stranded arrangement would be a ladder-like structure in which the base pairs are fixed at 0.6 nm apart because this is the distance between adjacent sugars in the DNA backbone. Because H2O molecules would be accessible to the spaces between the hydrophobic surfaces of the bases, this conformation is energetically
The double helix can thought of as resulting from the interwinding of two right-handed helical polynucleotide chains around a common axis. Hydrogen bonds form between opposing bases on the two chains. Such hydrogen bonding is possible only if the two chains are antiparallel. The two types of pairing which results through hydrogen bonding are always adenine-thymine and guanine-cytosine (fig. 2.4.7).
Fig. 2.4.7. Base pairs of DNA: The bases must be in this orientation to form base pairs. The dotted lines indicate hydrogen bonds between the bases. Although the hydrogen bonds hold the bases and thus the two DNA strands
together, they are weaker than covalent bonds and allow the DNA strands to separate during replication and transcription. (Source: Smith et al, 2004, p 211, fig. 12.6)
This relationship between bases in the double helix is described as complementary because every base on one strand is complementary to its matched base on the other strand. This also stems from the experimental finding that the mole percentages of adenine and thymine in DNA are equal and the same being the case for guanine and cytosine. An adenine-thymine base pair has two hydrogen bonds between the bases and a guanine-cytosine base pair has three. Several factors account for the stability of the double-helical structure of DNA. First, both internal and external hydrogen bonds stabilize the double helix. Second, the negatively charged phosphate groups are all situated on the exterior surface of the helix in such a way that they have minimal effect on one another and are free to interact electrostatically with cations in solution such as Mg2+. Third, the core of the helix consists of the base pairs, which, in addition to being H-bonded, stack together through hydrophobic interactions and van der Waals forces that contribute significantly to the overall stabilizing energy.
The sugar-phosphate backbone is the outer part of the helix, with the chains running in antiparallel directions, one 3’ to 5’ and the other 5’ to 3’ (Figure 2.4.8). This is a result of the stereochemical consequence of the way A:T and C:G base pairs form leading to the sugars of the respective nucleotides having opposite orientations.
The sugar-phosphate backbones of the helix are not equally spaced along the helix axis. This leads to the formation of empty spaces, called grooves, between the atoms of the two chains. There is a large major groove and a smaller minor groove in a double helix (Figure 2.4.9). As each phosphate group of the backbone carries a negative charge at the physiological pH, positively charged DNA-binding proteins, such as histones and drugs, bind to DNA in these grooves.
Fig.2.4.8 Antiparallel strands of DNA. For the strand on the left, the 5’-carbon of each sugar is above the 3’-carbon, so it runs 5’ to 3’. For the strand on the right, the 3’-carbon of each sugar is above the 5’-carbon, so it runs 3’ to 5’.
(Source: Smith et al, 2004, p 212, fig.12.8).
Fig. 2.4.9. The minor and major grooves formed in the cylindrical column formed by the double helix. The major groove is shown in orange and the minor groove in yellow. The carbon atoms of the backbone are in white. (Berg et
al, 2002, fig. 27.8)
Types of DNA
The double helix exists in various forms designated as A, B, and Z (See Figure 2.4.10 below). B-
right dimensions to fit the inside diameter of the double helix. The outside diameter of the helix is 2 nm and the length of one complete turn of the helix along its axis is 3.4 nm containing 10 base pairs.
Fig. 2.4.10. Z, B, and A forms of DNA. The solid black lines connect one phosphate group to the next. (Source:
Smith et al, 2004, p 213, fig 12.10)
DNA also occurs naturally in another form called A-DNA. This form has 11 base pairs for each turn of the helix and the distance required to complete one helical turn is 2.46 nm instead of 3.4 nm. These base pairs do not lie perpendicular to the helix axis but at an angle of about 20 degrees to the perpendicular like the blades of a propeller. The A form is a short squat form compared to the B form which is longer and thinner. One similarity between the A and the B forms is that both are right-handed or clockwise helices.
A variant form of DNA discovered more recently (1979), called Z-DNA, is left-handed and its geometric characteristics are quite different from the more conventional forms. Z-DNA was the first crystal structure of a DNA molecule to be solved. The phosphodiester backbone in Z-DNA has a “zigzag” arrangement of the phosphate and sugar backbone rather than the smooth conformation that characterizes the other double-stranded forms. It is also longer and thinner than B-DNA and one turn of its helix has 12 base pairs rather than the 10 in a B-DNA. It lacks a major groove. This unique type of DNA forms under sequence-dependent conditions that require an alternating purine-pyrimidine sequence. Other chemical environmental factors such as high salt, the presence of some cations, and DNA supercoiling, also favor the formation of Z-DNA. The biological function of Z-DNA is not known definitely. It may play a role in the regulation of gene expression.
Intercalating Agents
In solution certain hydrophobic molecules with fused heterocyclic rings, such as ethidium bromide, acridine orange and actinomysin D can insert between the stacked base pairs of DNA causing it to partially unwind. The ease with which such intercalating agents insert into the DNA helix indicates that the van der Waals bonds they form with the bases are more favourable than the bonds between the bases themselves. Furthermore, the fact that these agents slip in suggests that
the double helix must temporarily unwind and present gaps for these agents to occupy. This indicates the dynamic nature of the DNA double helix in solution and makes it clear that the structure of B-DNA in solution is not a rigid, linear rod but instead behaves like a dynamic, flexible molecule.
DNA Denaturation
DNA does not form a rigid linear structure incapable of change of form. The double helix is disrupted during any transformation of the molecule such as DNA replication, transcription, repair and recombination. Such transformations cause the very forces that hold the two strands together to allow separation of the strands, but as soon as the relatively few base pairs separate they close up again and the adjacent base pairs then unwind. Thus, disruptions of double strandedness are propagated along the length of the helix, though at any particular moment, the large majority of bases remain hydrogen bonded. All bases in a double stranded form can therefore pass through the single-stranded state, a few at a time.
Changes in pH, temperature or ionic strength disrupt the hydrogen bonds and the two strands separate and acquire a random-coil conformation. This process is described as denaturation or, more appropriately, helix-to-coil transition. The process is associated with a number of major physical changes, such as an increase in buoyant density, reduction in intrinsic viscosity, and a change in the ability to rotate polarized light. If temperature is the denaturing agent, the course of the denaturation can be followed spectrophotometrically because the relative absorbance of the DNA solution at 260 nm increases as much as 40% as the bases unstack. This increase in absorbancy is known as a hyperchromic shift. The rise in absorbance coincides with strand separation, and the midpoint of the absorbance increase is termed the melting temperature, Tm. Different types of DNA differ in their Tm values as they differ in their relative C + G content. The higher the G + C content of a DNA, the higher its melting temperature because G : C pairs are held by three H bonds whereas A : T pairs have only two. Tm is also dependent on the ionic strength of the solution; the lower the ionic strength, the lower the melting temperature. Ions suppress the electrostatic repulsion between the negatively charged phosphate groups in the complementary strands of the helix, thereby stabilizing it.
At pH values greater than 10, extensive deprotonation of the bases occurs, destroying their hydrogen bonding potential and denaturing the DNA duplex. Similarly, extensive protonation of the bases below pH 2.3 disrupts base pairing. Small solutes that readily form H bonds are also DNA denaturants at temperatures below Tm if present in sufficiently high concentrations to compete effectively with the H-bonding between the base pairs. Examples include formamide and urea.
Thus, the generation of single stranded DNA in the cell was considered to be a transitory state.
However, the discovery by Sinsheimer in 1959 of the bacteriophage ØX 174 genome which was found to be a single-stranded circular DNA, caused renewed interest in the biological properties of single-stranded DNA. Today there is much greater understanding of the physical and chemical
DNA Renaturation
Denatured DNA strands can be reformed into a double helix if the denaturing conditions are removed and appropriate physiological temperature and salt conditions are achieved. This is called renaturation or reannealing (Figure 2.4.11) and requires the reassociation of the strands into a double helix. The initial process of reassociation or nucleation is slow as it depends on the sequence alignment of the two strands but thereafter, once the sequences are aligned, the strands zipper up quickly. If the denaturation is incomplete, the renaturation is rapid. Annealing is possible even if the complementary strands have been completely separated, though under these conditions it is a slow process as many of the realignments are imperfect and have to be broken down for proper pairings to form. The process occurs more quickly if the temperature is warm enough to promote diffusion of the large DNA molecules but not so warm as to cause melting.
Fig. 2.4.11. Thermal denaturation and renaturation of DNA. The nucleation phase progresses slowly, but once the complementary sequences have been aligned, they quickly form a double helix. (Source: Garrett, R. H. and
Grisham, C.M., 1999, p 273 fig.12.19)
Nucleic Acid Hybridization
The property of complementary strands self-associating into a duplex had led to the development of the technique of hybridization. If DNA from two different species are mixed, denatured, and allowed to cool slowly so that reannealing can occur, artificial hybrid duplexes may form, provided the DNA from one species is similar in nucleotide sequence to the DNA of the other.
The degree of hybridization is a measure of the sequence similarity or relatedness between the two species. Depending on the conditions of the experiment, about 25% of the DNA from a human forms hybrids with mouse DNA, implying that some of the nucleotide sequences (genes) in humans are very similar to those in mice. Hybrid molecules consisting of a DNA strand and a cognate messenger RNA or a DNA strand and a complementary DNA (cDNA) can be formed.
The technique of hybridization is commonly used in molecular biology. When combined with gel electrophoresis techniques that separate hybrid molecules by size and radioactive labeling to
provide a detectable signal, the resulting analytic techniques are called Southern (DNA/cDNA) and Northern blotting (DNA/RNA), respectively. Hybridization also gives us the power to identify specific genes from a vast pool of genetic material. An appropriately labeled oligo- or polynucleotide, referred to as a probe, is constructed so that its sequence is complementary to a target gene. The probe specifically base pairs with the target gene, allowing identification and subsequent isolation of the gene.
Tertiary Structure of DNA
Since the DNA molecule is considerably longer than its width, it can fold back on itself similar to the way proteins fold back on themselves giving rise to a tertiary structure. The relaxed state of the double helix has no other twists in it other than the double helical twists. Further twisting and coiling (supercoiling) of the double helix is therefore possible.
Fig. 2.4.12. Bacterial plasmid DNA in the relaxed (left) and supercoiled (right) state.
(Source: Nelson and Cox, 2004, p 932, fig 24.13)
Prokaryote chromosomes are closed circular DNA duplexes, as are almost all plasmid DNAs.
Circular DNA is formed if the free terminals of a linear double helix are brought together and joined by a phosphodiester bond. This circular DNA will be relaxed if no other manipulations are induced, i.e., it will have the thermodynamically favoured structure of the linear double helix which has 10 base pairs per turn of the helix. However, if the linear double helix is unwound slightly before the ends are joined to form a circle, a strain is introduced in the molecular structure. This strained structure, characterized by a deficit of turns, is known as negative superhelical DNA. The underwinding results in the participation of more base pairs per helical turn, which produces a decrease in the angle of twist between adjacent base pairs. The strain produced by the deficit of turns results in the disruption of hydrogen bonds and the opening of the double helix over a small region of the double helix. To compensate for the unwinding, the DNA assumes a new conformation. If the unwinding leads to an extra left-handed helical twist to a right-handed double helix, the circular DNA is said to be negatively supercoiled. If, instead of underwinding, there is an overwinding of the closed-circle double helix, a right-handed or
rewind it. Topoisomerases relieve tortional stress that develops in cellular DNA molecules during replication or other processes. They hydrolyze a phosphodiester linkage in one strand of the double helix, relax the supercoiling by rotating one strand around the other, and then reseal the break. DNA gyrases induces negative supercoiling in relaxed, closed-circular DNA.