The X-ray contribution to the History of Molecular Genetics

In 1953, James Watson and Francis Crick discovered the structure of DNA. This is generally viewed as the definitive event in the history of molecular genetics, resulting in a mass of knowledge about the function of the nucleic acids, and thus the unearthing of exactly how genes replicate and carry information. Garland E. Allen attributes the work leading to this ‘remarkable achievement’ to the union of three separate areas of study: the structurists, the biochemists and the informationists. [Allen, pp. 191-2] The latter two groups had, respectively, shown a connection between genes and the enzymes related to metabolic pathways, and discovered that the nucleic acids carried hereditary information in some groups of bacteria and viruses. Meanwhile, the apparatus and techniques refined by Astbury and the X-ray crystallographers in the study of proteins were being adopted by the structurists in studying the molecular structure of DNA.

Before such studies could take place, however, the nucleic acids had to be recognised as the principle substances of heredity. Astbury himself had, in 1938, taken an imperfect fibre diagram of nucleic acid, and attempted (incorrectly) to interpret its structure. However, at this time neither he, nor any other scientist, were aware of the importance of DNA; indeed Astbury had not even sought out a DNA sample to photograph, one had instead been sent to him (as an authority in the X-ray field). The events which led to the consensus of DNA’s role in heredity, culminating with the work the 1953 publication of Hershey and Chase’s work on bacteriophages, did not involve the crystallographers. However, once this role had been confirmed, and the need to determine structure and function thus highlighted, the X-ray crystallographers, and their photographs of DNA, became important again. When Watson and Crick began their work on the structure of DNA, Maurice Wilkins and Rosalind Franklin, at King’s College, London, had been using X-ray techniques to analyse the structure of nucleic acids for some time.

Watson and Crick would thus seek a model of DNA which complimented this X-ray diffraction data. The information from Franklin’s photograph provided them with three limiting measurements: the width of the double helix; the height between the stacked parallel bases; and the height of one complete turn of the helix. They were then able to ascertain that the same pattern recurs periodically around the fibre axis.

However, Watson and Crick’s model also had to account for results uncovered by the informationist school: the dual functions of auto-catalysis (where a molecule catalyzes the some of the early steps in its own formation) and heterocatalysis (where a molecule catalyzes the formation of other different molecules). Furthermore, there were the mathematical results of John Griffith, who had calculated the theoretical attractive forces between like bases in a DNA molecule, discovering that unlike bases attracted each other. This was followed by an encounter with the biochemist Erwin Chargaff, who introduced Watson and Crick to the published literature which, supporting Griffith’s calculations, found 1:1 base ratios. However, it was later revealed that Griffith’s and Chargaff’s results were ignored, and only taken notice of again when they appeared to be supported by the model.

Watson and Crick now had to, taking the limiting factors into account, use a process of trial and error to ascertain the correct structure of DNA. They used a method introduced by Linus Pauling, who had been studying the alpha-helix of protein at Caltech. Pauling built models, based on theoretical considerations, and then tested these models against X-ray diffraction results. Thus Watson and Crick used scale models of the purine and pyrimidine bases, constructing models out of these which supported the requirements of hydrogen bonding and Chargaff’s ratios. Various possible models were built, and then tested against the X-ray diffraction patterns of DNA.

Watson and Crick narrowed the possibilities down to a small selection: a two- or three-chain molecule with the bases pointing inward and the sugar-phosphate backbone outward, or the bases pointing outward and the sugar-phosphate backbones inward. New X-ray data was continually being produced and analysed at King’s College, and soon it was discovered that (contrary to earlier belief) a two-stranded structure was indeed a possibility, and that the sugar-phosphate backbone was most likely on the outside. Problems arose due to the fact that neither Watson nor Crick realised that they had been using the wrong forms of the bases. This was pointed out to them by the American chemist Jerry Donohue, and soon afterwards a solution was found: adenine-thymine and cytosine-guanine pairs, held together by hydrogen bonds. The final structure consisted of ‘two helices wound around each other, like a kind of spiral staircase with steps composed of paired bases, and with the sugar-phosphate backbone running along the outside’. (Allen, p. 219)

Data produced by the X-ray crystallographers contributed to this discovery by providing, alongside chemical and mathematical considerations, limiting measurements. Each of these considerations helped to paint a partial picture, but none of them could take full responsibility for the outcome. This is clear when it is remembered that Astbury himself had an X-ray photograph of DNA as early as 1938 (although it was not as successful as Franklin’s) and yet did not come close to a correct structure. Olby suggests that, had an attempt been made to uncover the DNA structure purely through a crystallographic approach, the wrong pairing scheme would have been used. (Olby, p. 438) Crystal studies at the time suggested a ‘Hoogsteen’ like-with-like pairing, and thus crystallographic deduction would certainly not have been successful without external influences. However, once the correct base parings had been postulated, Watson and Crick returned to the X-ray data, spending nearly a week building models and testing them against every major angle of rotation. The diffraction patterns were used as an authority, against which all theories were tested and verified. As Olby writes, ‘A structure is said to be proven when it can be shown to follow unambiguously from the X-ray data’ (Olby, p. 437)

In James Watson’s popular account of the discovery of the double helix, he places a considerable amount of emphasis on the role of X-ray crystallography. In this account, the unearthing of the correct structure is catalyzed by Watson’s sighting of Rosalind Franklin’s X-ray photograph of the B-form of DNA. However, Watson also partially attributes his success to the help received from Jerry Donohue, a chemist with whom he and Crick shared an office. He notes that, without this chemical help, Wilkins and Franklin had no chance of finding the correct structure before Crick and himself (although this was also due to delays in DNA research resulting from irreconcilable differences between Wilkins and Franklin). However once, with the acknowledged help of a chemist, the structure had been found, Watson writes that he immediately returned to the experimental X-ray data for confirmation. Furthermore, he relates the enthusiasm expressed by Sir Lawrence Bragg upon discovering that such a result had been obtained using ‘the X-ray method he had developed forty years before’ (Watson, p. 171). Presumably Bragg was referring to his fundamental law of diffraction; clearly the cumulative work produced by the X-ray crystallographers from Bragg onwards contributed in some part to the discovery of the double helix.

The structure of DNA was important because it provided a great deal of insight into function, explaining autocatalysis and heterocatalysis, and thus suggesting a mechanism for how DNA stores genetic information. Detailed structure was crucial and so it comes as no surprise that the X-ray crystallographers, working in a field concerned purely with molecular structure, would play a vital role in this aspect of the history of genetics. The diffraction data produced by Rosalind Franklin was continually used by Watson and Crick, both in formulating their ideas, and verifying their finished model. It was the agreement of their results with crystallographic evidence which led to the double helix being accepted by the scientific world.

by Imogen Clarke