By the early 1950s, interest in DNA as a candidate for the genetic material had reached a high level of excitement. Critical experiments, described in Box 4.1 and Box4.2, had strongly indicated this to be the long-sought genetic substance. Scientists still had no idea about DNA’s structure, although there were already some provocative clues. For example, it had been shown that a peculiar relationship existed between the stoichiometry of bases in natural DNA: the amount of A was always about the same as the amount of T, with a similar result for G and C. These general rules concerning the base composition of any DNA were deduced by Edwin Chargaff from analysis of the nucleotide composition of DNA samples from different organisms (Table 1). The first of Chargaff’s rules states the equal representation of A and T and of C and G in any DNA molecule; in other words, the sum of purines, A + G, equals the sum of pyrimidines, C + T. The second rule concerns the overall composition of DNA in different organisms: it varies from one species to another. Any pro posed structure must explain Chargaff’s rules and clarify the even more mysterious problem of how the genetic material could be accurately replicated when cells divide or organisms reproduce.
Table1. Base composition of DNA (mole %) and ratios of bases that led to the formulation of Chargaff’s rules. The slight deviations in the A/T, G/C, and purines/pyrimidines ratios from 1.00 reflect uncertainties in experimental measurements. Bacteriophage φX174 genome is single-stranded and thus does not follow Chargaff’s rules. [Data reported in Sober HR (ed.) (1970) Handbook of Biochemistry: Selected Data for Molecular Biology, 2nd ed. With permission from CRC Press.]
It is now widely known that the solution came from the brilliant insights of James Watson and Francis Crick, guided by the experimental work of others, including that of Chargaff and the crystallographic results of Rosalind Franklin and Maurice Wilkins (Box 3). The right-handed double-helix model proposed by Watson and Crick (Figures 1 and 2) fitted all the requirements of a genetic material so perfectly that it just had to be correct. First, the A-T and G-C base pairings allowed strong hydrogen-bonding between the two DNA strands (Figure 2) and accounted for the peculiar base stoichiometry, A = T and G = C, noted above. This structure also put the 1′-carbons in either pair exactly the same distance apart, 1.08 nm, allowing for a uniform, unstrained double helix. Finally, and most importantly, the model provided a strong hint, noted by Watson and Crick, as to how DNA could be replicated. Note that the two strands in the double helix, which are antiparallel, meaning they run in opposite directions, are exact complements of one another. If the strands were separated and each was used as a template to specify a new strand, two new double stranded molecules would be obtained, each an exact copy of the original duplex (Figure 3). This mode of copying, called semiconservative replication, was demonstrated to be correct by Matthew Meselson and Frank Stahl.
Fig1. Francis Crick, James Watson, and their model of the double-helical DNA structure. [Barrington Brown, © Gonville & Caius College / Coloured by Science Photo Library.]
Fig2. Schematic of the double-helix structure of B-form DNA. Base pairing occurs between a purine on one strand and the corresponding pyrimidine on the other strand; the pairing between these complementary bases allows the distances between the C-1′ on the sugar moieties to be exactly the same for both adenine–thymine and guanine–cytosine base pairings. Hydrogen-bonding between the complementary bases and stacking interactions between successive pairs are both important in stabilizing the helix. The original Watson Crick model closely resembles this but with 10.0 base pairs/turn. [Adapted, courtesy of the U.S. National Library of Medicine.]
Fig3. The double-helical structure of DNA allows for a simple semiconservative mechanism of DNA replication. The unwinding of the two strands of the parental duplex, shown in brown, is accompanied by synthesis of two complementary daughter strands, shown in red, each using one parental strand as a template. This model of replication of the genetic material was envisaged by Watson and Crick in 1953, as soon as they deciphered the structure of DNA.
The original Watson–Crick model was an inspired guess, based on fiber X-ray diffraction patterns. Such a pattern does not provide the kind of unambiguous structure that can be obtained from X-ray diffraction patterns from single crystals. Many years passed before true crystals of small DNA molecules were obtained and studied by this technique. The results turned out to be remarkably close to those predicted by the Watson–Crick model (Figure 4). There are in fact nearly, but not exactly, 10 base pairs for each turn of the helix, and the bases are almost, but not exactly, perpendicular to the helix axis (Table 2).
Fig4. Structure of B-DNA as derived from crystallographic studies. (A) Ball-and stick model. (B) Space-filling model. The base stacking is especially clear in this model, in which each atom is represented by a sphere of its van der Waals radius.
Table2. Structural parameters of the three forms of double-helical DNA.
The fascination with the double helical DNA structure and the realization of its ability to be the carrier of genetic information that could be propagated from cell generation to cell generation was overwhelming. This fascination can be seen in numerous sculptures of the structure erected all over the world, as illustrated in Figure 5.
Fig5. (A) DNA double helix sculpture by Charles Jencks. (B) A 50-foot-tall sculpture of DNA, “Portrait of a DNA Sequence,” hangs in the main stairwell of the Life Sciences Building, administrative home of the College of Biological Sciences at the University of California Davis. The sculpture was created in 1997 by artist Roger Berry to be both artistically expressive and scientifically accurate. It con sists of two stainless-steel backbones forming the double helix with 200 base-pairs that link the strands and encode information displayed in colored glass. The bases change color depending on the angle of view. (C) Art and science collide in “What’s in Your DNA?” Cancer Research UK teamed up with 21 designers to raise funds for an advanced research facility in the heart of London. 21 double helix sculptures were dotted around London, in the “London Art Trail,” as a fund-raising collaboration between designers and Cancer Research UK. [A, Simona Flamigni/Shutterstock. B, Copyright © (2018) The Regents of the University of California, Davis campus. All Rights Reserved. Used with permission. C. Ron Ellis/Shutterstock.]
At elevated temperature, DNA will denature in the sense that the duplex will dissociate into single strands (Figure 6). This process often occurs at a sharply defined temperature and is therefore also called melting. The melting point is increased by high G/C content or high salt, facts that are often useful in experimental proto cols. Denatured DNA can reconstitute double-stranded base-paired duplexes upon slow cooling or annealing. The ability of single strands of DNA of complementary base sequence to form DNA duplexes forms the basis for a number of experimental hybridization techniques for labeling DNA fragments.
Fig6. Melting of double-stranded DNA. Absorbance at 260 nm is usually measured in high-salt buffers, 100 mM Na+, as the temperature of the solution is slowly raised at a rate of 1°C/min. The melting temperature is defined as the temperature at which 50% of the DNA is still in the form of a duplex. The three different curves illustrate the dependence of melting behavior on the base composition of the DNA. Poly[d(AT)] is represented by the red line, naturally occurring DNA by green, and poly[d(GC)] by blue. The melting temperature, Tm , is indicated by the corresponding dotted lines. (Inset) Electron micrograph of partially denatured DNA. [Inset, from Liu Y-Y, Wang P-Y, Dou S-X et al. (2005) Sci. Technol. Adv. Mater. 6: 842–847. With permission from Elsevier.]
