Nitrogenous Bases Term Analysis

The moment was thus appropriate to think seriously about some curious regularities in DNA chemistry first observed at Columbia by the Austrian-born biochemist Erwin Chargaff. Since the war, Chargaff and his students had been painstakingly analyzing various DNA samples for the relative proportions of their purine and pyrimidine bases. In all their DNA preparations the number of adenine (A) molecules was very similar to the number of thymine (T) molecules, while the number of guanine (G) molecules was very close to the number of cytosine (C) molecules. Moreover, the proportion of adenine and thymine groups varied with their biological origin. The DNA of some organisms had an excess of A and T, while in other forms of life there was an excess of G and C.

I realized that the phosphate groups in Linus’ model were not ionized, but that each group contained a bound hydrogen atom and so had no net charge. Pauling’s nucleic acid in a sense was not an acid at all. Moreover, the uncharged phosphate groups were not incidental features. The hydrogens were part of the hydrogen bonds that held together the three intertwined chains.

Without the hydrogen atoms, the chains would immediately fly apart and the structure vanish.

Everything I knew about nucleic-acid chemistry indicated that phosphate groups never contained bound hydrogen atoms. No one had ever questioned that DNA was a moderately strong acid. Thus, under physiological conditions, there would always be positively charged ions like sodium or magnesium lying nearby to neutralize the negatively charged phosphate groups. All our speculations about whether divalent ions held the chains together would have made no sense if there were hydrogen atoms firmly bound to the phosphates. Yet somehow Linus, unquestionably the world’s most astute chemist, had come to the opposite conclusion.

Interrupting her harangue, I asserted that the simplest form for any regular polymeric molecule was a helix. Knowing that she might counter with the fact that the sequence of bases was unlikely to be regular, I went on with the argument that, since DNA molecules form crystals, the nucleotide order must not affect the general structure. Rosy by then was hardly able to control her temper, and her voice rose as she told me that the stupidity of my remarks would be obvious if I would stop blubbering and look at her X-ray evidence.

[…]

Without further hesitation I implied that she was incompetent in interpreting X-ray pictures. If only she would learn some theory, she would understand how her supposed antihelical features arose from the minor distortions needed to pack regular helices into a crystalline lattice.

The instant I saw the picture my mouth fell open and my pulse began to race. The pattern was unbelievably simpler than those obtained previously (“A” form). Moreover, the black cross of reflections which dominated the picture could arise only from a helical structure. […] The real problem was the absence of any structural hypothesis which would allow them to pack the bases regularly in the inside of the helix. Of course this presumed that Rosy had hit it right in wanting the bases in the center and the backbone outside. Though Maurice told me he was now quite convinced she was correct, I remained skeptical, for her evidence was still out of the reach of Francis and me.

Though I kept insisting that we should keep the backbone in the center, I knew none of my reasons held water. Finally over coffee I admitted that my reluctance to place the bases inside partially arose from the suspicion that it would be possible to build an almost infinite number of models of this type. Then we would have the impossible task of deciding whether one was right. But the real stumbling block was the bases. As long as they were outside, we did not have to consider them. If they were pushed inside, the frightful problem existed of how to pack together two or more chains with irregular sequences of bases. Here Francis had to admit that he saw not the slightest ray of light.

My aim was somehow to arrange the centrally located bases in such a way that the backbones on the outside were completely regular—that is, giving the sugar-phosphate groups of each nucleotide identical three-dimensional configurations. But each time I tried to come up with a solution I ran into the obstacle that the four bases each had a quite different shape. Moreover, there were many reasons to believe that the sequences of the bases of a given polynucleotide chain were very irregular. Thus, unless some very special trick existed, randomly twisting two polynucleotide chains around one another should result in a mess. In some places the bigger bases must touch each other, while in other regions, where the smaller bases would lie opposite each other, there must exist a gap or else their backbone regions must buckle in.

Despite the messy backbone, my pulse began to race. If this was DNA, I should create a bombshell by announcing its discovery. The existence of two intertwined chains with identical base sequences could not be a chance matter. Instead it would strongly suggest that one chain in each molecule had at some earlier stage served as the template for the synthesis of the other chain. Under this scheme, gene replication starts with the separation of its two identical chains.

As the clock went past midnight I was becoming more and more pleased. There had been far too many days when Francis and I worried that the DNA structure might turn out to be superficially very dull, suggesting nothing about either its replication or its function in controlling cell biochemistry. But now, to my delight and amazement, the answer was turning out to be profoundly interesting. For over two hours I happily lay awake with pairs of adenine residues whirling in front of my closed eyes. Only for brief moments did the fear shoot through me that an idea this good could be wrong.

Suddenly I became aware that an adenine-thymine pair held together by two hydrogen bonds was identical in shape to a guanine-cytosine pair held together by at least two hydrogen bonds. All the hydrogen bonds seemed to form naturally; no fudging was required to make the two types of base pairs identical in shape.

[…]

The hydrogen-bonding requirement meant that adenine would always pair with thymine, while guanine could pair only with cytosine. Chargaff’s rules then suddenly stood out as a consequence of a double-helical structure for DNA. Even more exciting, this type of double helix suggested a replication scheme much more satisfactory than my briefly considered like-with-like pairing.

Rosy’s instant acceptance of our model at first amazed me. I had feared that her sharp, stubborn mind, caught in her self-made antihelical trap, might dig up irrelevant results that would foster uncertainty about the correctness of the double helix. Nonetheless, like almost everyone else, she saw the appeal of the base pairs and accepted the fact that the structure was too pretty not to be true. Moreover, even before she learned of our proposal, the X-ray evidence had been forcing her more than she cared to admit toward a helical structure. The positioning of the backbone on the outside of the molecule was demanded by her evidence and, given the necessity to hydrogen-bond the bases together, the uniqueness of the A-T and G-C pairs was a fact she saw no reason to argue about.

Fortunately, by the time my letter reached Cal Tech the base pairs had fallen out. If they had not, I would have been in the dreadful position of having to inform Delbrück and Pauling that I had impetuously written of an idea which was only twelve hours old and lived only twenty-four before it was dead.