The discovery of the double helix

The discovery of the double helix, by James Watson and Francis Crick of Cambridge University, UK, in 1953, was the most important breakthrough in twentieth-century biology. The nature of the double helix revealed how genes can replicate, a puzzle that had seemed almost impossible to solve just a few years earlier. According to Watson in his book The Double Helix, the work was a desperate race against the famous American biochemist Linus Pauling, who initially thought that DNA was a triple helix. This mistake
gave Watson and Crick the time they needed to complete their description of the double-helix structure.

When Watson and Crick began their work, the structures of the nucleotides, and the way these are linked together to form a polynucleotide were already known. What was not known was the actual structure of DNA in a living cell. Was it a single polynucleotide,
perhaps folded up in some way? Or were there two or more polynucleotides in a DNA molecule?

To solve the structure of DNA, Watson and Crick used model building—they built a scale model of what they thought a DNA molecule must look like. The model had to obey the laws of chemistry, which meant that if a polynucleotide was coiled in any way then its atoms must not be placed too close together. It was equally vital that the model takes account of the results of other investigations into DNA structure. One of these studies was carried out by Erwin Chargaff at Columbia University in New York, the other, by Rosalind Franklin at King’s College, London.

 

Untitled
figure 1 Paper Chromatography

Chargaff’s base ratios paved the way for the correct structure

Erwin Chargaff became interested in DNA in the 1940s when scientists first realized that DNA might be the genetic material. He decided to use a new technique, called paper chromatography, to measure the amounts of each of the four nucleotides in DNA from different tissues and organisms. In paper chromatography, a mixture of compounds is placed at one end of a paper strip, and an organic solvent, such as n-butanol, is then allowed to soak along the strip. As the solvent moves it carries the compounds with it,
but at different rates depending on how strongly each one absorbs into the paper matrix (Figure 1).

Chargaff purified DNA from different sources and treated each sample with acid to break the molecules into their component nucleotides (Figure 2). He then used paper chromatography to separate the nucleotides in each mixture so their concentrations could be measured. The results were quite startling. They revealed a simple relationship between the proportions of the nucleotides in any one sample of DNA. The relationship is that the number of adenines equals the number of thymines, and the number of guanines equals the number of cytosines. In other words, A = T and G = C.

Chargaff did not speculate to any great extent, at least not in his publications, about the relevance of these base ratios to the structure of DNA. In fact, Watson and Crick appear to have been unaware of his results until they met Chargaff when he visited Cambridge
University. But once they became aware of the A = T and G = C relationship, they knew this had to be accounted for in their model of DNA.

Chargaff_s experiments
figure 2 Chargaff’s experiment

X-ray diffraction analysis indicates that DNA is a helical molecule

The second piece of evidence available to Watson and Crick was the X-ray diffraction pattern obtained when a DNA fiber is bombarded with X-rays. X-rays have very short wavelengths—between 0.01 and 10 nm—comparable with the spacings between atoms
in chemical structures. When a beam of X-rays is directed onto a DNA fiber some of the X-rays pass straight through, but others are diffracted and emerge at a different angle (Figure 3). As the fiber is made up of many DNA molecules, all positioned in a regular
array, the individual X-rays are diffracted in similar ways, resulting in overlapping circles of diffracted waves which interfere with one another. An X-ray– sensitive photographic film placed across the beam reveals a series of spots and smears, called the X-ray
diffraction pattern.

X-ray diffraction pictures of DNA fibers were made by Rosalind Franklin, during 1952, using techniques previously developed by Maurice Wilkins (Figure 4). The pictures immediately showed that DNA is a helix, and mathematical calculations based on them revealed that the helix has two regular periodicities of 0.34 nm and 3.4 nm. But how do these deductions relate to Chargaff’s base ratios and to the actual structure of
DNA?

 

X-ray diffraction analysis
figure 3 X-ray diffraction analysis

 

Pulling together the evidence

Watson and Crick put together all the experimental data concerning DNA and decided that the only structure that fitted all the facts was the double helix shown in Figure 2.6. The main difficulty was deciding how many polynucleotides were present in a single molecule. This could be estimated from the density of the DNA in a fiber. Several measurements of DNA fiber density had been reported, and they did not agree. Some suggested that there were three polynucleotides in a single molecule, others suggested two. Pauling thought the first set of measurements were correct and devised a triple-helix structure that was completely wrong. Watson and Crick decided it was more likely to be two.

Once two polynucleotides had been decided on, it became clear that the sugar–phosphate backbone had to be on the outside of the molecule. This was the only way the various atoms could be spaced out appropriately within the models that Watson and Crick built.
The models also indicated that the two strands had to be antiparallel and the helix right-handed. The X-ray diffraction data enabled the dimensions of the helix to be set. The periodicity of 0.34 nm indicated the spacing between individual base pairs, and that of 3.4 nm gave the distance needed for a complete turn of the helix.

What about Chargaff’s base ratios? These were the key to solving the structure. Watson realized, on the morning of Saturday, March 7, 1953, that the pairs formed by adenine–thymine and guanine–cytosine have almost identical shapes (see Figure 2.7). These
pairs would fit neatly inside the double helix, giving a regular spiral with no bulges. And if these were the only pairs that were allowed, then the amount of A would equal the amount of T, and G would number the same as C. Everything fell into place, and the greatest mystery of biology—how genes can replicate—had been solved.

 

Figure 4 Franklin’s “photo 51” showing the diffraction pattern obtained with a fiber of DNA. The cross shape indicates that DNA has a helical structure, and the relative positions of the various dots and smears enable the periodicities within the molecule to be calculated. (From R. Franklin and R. G. Gosling, Nature 171: 740–741, 1953. With permission from Macmillan Publishers Ltd.)

resut of diffraction
figure 4 Franklin’s “photo 51” showing the diffraction pattern obtained with a fiber of DNA. the cross shape indicates that DNA has a helical structure, and the relative position of the various dots and smears enable the periodicities within the molecule to be calculated.

 

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