Gene, 135 (1993) 15-18 0 1993 Eisevler Science Publishers B.V. All rights reserved. 0378-1119/93/$06.00

GENE 07436

15

Looking backwards: a birthday card for the double helix*

(X-ray diffraction patterns; genetic code; replication: messenger RNA; recombinant DNA; base pairing; transformation; complementary structures)

Francis Crick

The Salk Institute. La Jolla. CA 92037, USA

Received by G. Bernardi: 19 May 1993; Accepted: 3 June 1993; Recewed at publishers: 20 July 1993

SUMMARY

This ‘birthday’ paper outlines very briefly the history of the discovery of the DNA double helix, the way it was

received and how it was confirmed. The paper also discusses why, at that time, we foresaw so little of the rapid progress produced by the techniques of recombinant DNA. The key feature of the nucleic acids - discovered by Jim Watson -

is their ability to form specific base pairs.

About forty years ago, in the spring of 1953, Jim

Watson and I were lucky enough to hit on the double helical structure of DNA, with the crucial feature of the specific base-pairing between the two chains. The story has been told often enough, not only in Jim’s breezy ac- count of the discovery, The Double Helix, but also in more scholarly works, such as The Path to the Doggie

Helix by Robert Olby and The Eighth Day of Creation

by Horace Freeland Judson. Jim, twelve years younger than I, was then a post-dot, while I was still a graduate student in the process of writing a thesis on the 3D struc- ture of proteins and polypeptides. We had done none of the experimental work other than model building. The X-ray diffraction patterns given by fibers of DNA were produced by Rosalind Franklin, assisted by Ray Gosling, following the exploratory work of Maurice Wilkins and, before that, of Bill Astbury.

Why was the structure so striking? To understand this,

it helps to go back in time and recall what was known

C~~~es~~~e~ce to. Dr. F.H.C. Crick, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA. Tel. (1-619) 453-4100, Fax (l-619) 550-9959. *Presented at the COGENE Symposium, ‘From (he Double Helix to the Human Genome: 40 Years of Molecular Genetics’, UNESCO, Paris, 21-23 Apnl 1993.

Abbreviations: aa, ammo acid(s); lD, uni-dimensional 3D, three- dimenszona1.

just before the double helix was discovered. In those days, classical Mendelian genetics was fairly well developed. It was known that most genes were located in the chromo- somes, both from linkage studies and from the study of the giant salivary chromosomes of Drosophila melanogas-

ter. Chromosomes were known to contain both proteins and DNA (and perhaps some RNA), though what little

was known about those proteins - mainly histones - was largely incorrect.

Avery, MacLeod and McCarty had demonstrated that the ‘transforming factor’ of pneumococcus was DNA, and although this had been disputed, by the early Fifties it

was well established. Avery had realized that the trans- forming factor ‘may be a gene’ though he had not said

so in public. Notice that this evidence did not demon- strate that the gene was sorely made of DNA. For all that was known then, some of the genetic specificity could

have been carried by protein. The idea that DNA was an important part of genes was further suggested by the Hershey and Chase experiment on the infection of ~scher~chia co/i by phage T4. This was, at the time, a somewhat dirty experiment, but it made transforming- factor DNA seem less of an isolated freak.

Lederberg and Tatum had shown that bacteria did indeed possess genes (which had not been widely accepted before their work). Beadle and Tatum had put forward

16

the ‘one gene one enzyme’ hypothesis. based on their

work on ~~~z/~~sp(~~~i CIYISSCI. This idea was thought to be

very suggestive, though not yet firmly established.

In 1945, DNA was believed to be a rather short mole-

cule, possibly with equal amounts of the four bases. Even

its general chemical structure was not properly estab-

lished. By early 1953, it was realized that DNA molecules,

if carefully prepared (by avoiding too much shearing),

could be fairly long. Chargaff had discovered (and Wyatt

had confirmed f the base ratios (A = T. G = C). though he

had not clearly seen their significance. Todd and his co-

workers had cleaned up the general chemistry of DNA.

Even in the twenties Muller (and later Haldane) had

seen the importance of discovering the chemical nature

of the gene. By 1953, there were several rather crude esti-

mates of the typical size of a gene. They suggested that

it could be a macromolecule (consider how many genes

have to be packed into the very restricted volume of the

head of a sperm). Schroedinger’s little book. M’~LQ is Lifr?,

drew attention to the problem and put forward the semi-

nal idea that the gene was an ‘aperiodic crystal’ (I doubt

if, being a physicist, he had ever heard of organic

polymers).

So what was the difficulty? The basic problem, or so

it seemed to me, was how to copy a 3D structure. It was

known that proteins were the key machine tools of the

cell; that they were often globular in shape. and that their

chemical specificity (such as enzymatic activity) could

often be destroyed by mild heating. X-ray diffraction pat-

terns of a few proteins suggested that each had a well-

defined 31) structure. Thus, it seemed highly probable

that the correct 3D structure was necessary for their spec-

ificity. But how could this possibly be copied? To copy a

structure, one makes a complementary structure - a mold _ and then a complementary structure to that. This pro-

cess can copy the pattern of the outside of an ob,ject. but

how does one copy the inside?

It was surmised that each protein was made of one (or

a few) polypeptide chain, which had to be folded in the

correct way to produce the required 3D structure. The

obvious hypothesis, or so it seemed to me. was that the

gene determined the aa sequence of its protein, and that

the protein then folded itself up into the required 3D

shape. This idea transformed an intractable 3D problem

into a much easier ID problem. All the gene had to do

was to see that the aa were assembled in the correct order

in the polypeptide chain.

I had arrived at these ideas before the discovery of the

double helix. Jim and I saw immediately that the specific

base-pairing of our DNA model suggested not only how

it was replicated, but also how it could direct protein

synthesis. The linear sequence of bases must be a bio-

chemical code for the aa sequence of the protein whose

synthesis it controlled. We beltrled this h~~pp~n~d mainly.

if not exclusively. in the cytoplasm and was directed. not

by DNA molecules themselves, but by RNA copies of

parts of them. Hence Jim’s slogan: DNA makes RNA

makes protein.

Now let us return to the discovery of the double helix.

Why was the structure not immediately obvious?

Rosalind’s beautiful X-ray pictures of the A-form had

well-defined spots, not just smears. In addition. the gene-

ral chemical formula of DNA had been est~~blished by

the chemists. Unfortunately, both sources of mformatlon

the X-ray data and the chemical formula ~ were incom-

plete. The X-ray pictures gave only half the information

needed to reconstruct the electron density of the struc-

ture, since it provided the amplitudes of the Fourier com-

ponents, but not their phases. The chemical formula.

together with general information about bond lengths

and angles, was not enough to precisely define the 3D

structure, because of the somewhat free rotation about

the many single chemical bonds, especially in the

backbone.

This meant that there was no automatic way of going

from the experimental data to the structure. By contrast,

if the structure could be guessed, it was merely a problem

in computation to derive the X-ray pattern it should give.

This put a high premium on a successful guess.

The X-ray data showed clearly that the unit cell of the

crystallites in the fibers was very large - the repeat in the

direction of the fiber axis was in the region of 30 A, while

the diameter of the structure was about 20 r\. It was a

pl~~usibte assumption that the structure was a regular

helix, and indeed the X-ray patterns hinted at this. This

meant that the ‘asymmetric unit’ of the structure was

probably much smaller than the unit cell. so that not

many free parameters would be needed to define the

structure. As it turned out, the (pseudo) asymmetric unit

was half a base pair.

Unfortunately, Rosalind thought she had X-ray evi-

dence that the A-form was not helical. She eventually got

an improved X-ray picture of the B-form (the wetter of

the two) which clearly showed helical features, but she

put this aside while she continued to concentrate on the

A-form, since this gave much better X-ray pictures.

We had reaiized earlier that Chargaff’s results (that the

amount of A equaled the amount of T, while that of G

equaled the amount of C) suggested that the bases were

paired together. but we did not see how they did this

until Jerry Donahue put us right on the correct tauto-

merit forms of the bases. The actual pairing was found

by Jim while he was playing with paper cut-outs of the

bases. The pairs immediately looked correct. because of

their dyad symmetry, since this fitted with the idea that

the two chains ran in opposite directions. I had earlier

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deduced this from Rosalind’s description of the space group of the A-form.

After that it was fairly plane sailing. We posted off our manuscript on about April lst, 1953 and it was published in Nature, together with the papers by Franklin and Gosling, and by Wilkins, Stokes and Wilson, on April 25th. A month or so later, Jim and I published a second paper in Nature which discussed the genetic implications of the structure.

There are many other aspects of the story - the incor- rect three-chain structure suggested by Linus Pauling, for example - but these have been well covered by earlier accounts, so I will not enlarge on them here.

Rosalind Franklin moved to Bernal’s group at Birkbeck College to work on tobacco mosaic virus, while Maurice Wilkins started on his extensive X-ray studies of both the A- and the B-forms. He and his collaborators were able to show that chemically satisfactory models of a base-paired double helix had X-ray diffraction patterns that agreed fairly well with the experimental X-ray patterns.

How was the double helix received? There was rather little about it in the newspapers, if only because there was much less scientific journalism then. The reactions of the scientific community were mixed. The X-ray evi- dence supporting the double helix was, at that time, only suggestive. It was not enough to establish the structure beyond a reasonable doubt. To some, the whole idea seemed too simplistic. It certainly lacked biochemical support. In spite of this, most of the members of the phage group were enthusiastic about our ideas, especially as Max Delbrtick liked both the structure and its implica- tions. Others were not impressed. Some years later, Jean Brachet, the Belgian biochemist and embryologist, told me that when he read our papers, he thought ours was another silly theoretical idea, better ignored. Arthur Kornberg also thought nothing of it (perhaps because DNA did not appear to be an enzyme) and dismissed it as mere speculation. The reactions of many biochemists, such as Joseph Fruton, ranged from coolness to muted hostility. They had long considered the biochemistry of the gene to be based on proteins, not nucleic acids, and thought the problem far too difficult to tackle in the im- mediate future. It did not help that the structure had been put forward by two people who were obviously not card- carrying biochemists.

Two new approaches finally established the general correctness of the double helix. When it became possible to synthesize, in reasonable amounts, short, defined se- quences of DNA, one could obtain single crystals of each chosen molecular species. Moreover, heavy atoms could be added to such crystals. This meant that one could obtain the 3D structure, using the method of isomor-

phous replacements, without making any assumptions about what the structure was like. In addition, the spatial

resolution was usually better than that obtained with DNA fibers, if only because the material in the latter was always a mixture of sequences.

The first results produced a surprise - a new form of DNA, the so-called Z form - but other, less restricted DNA sequences showed the classical double helix struc- ture, though with subtle variations due to the interactions between neighboring bases. There is now much work aimed at understanding the finer points of the structure.

The second method that showed that the two DNA chains are truly intertwined, and not side-by-side (as sug- gested by two groups of workers) was mainly due to Keller and to Wang. They were able to estimate the link- ing number of circular DNA, showing that there was one complete turn every ten residues or so. This effectively disposed of all simple side-by-side models.

It thus took almost thirty years for the double helix to be established beyond all reasonable doubt, as opposed to being merely plausible (as it was at first) or fairly prob- ably correct, as it was after all the studies on DNA fibers.

I am often asked how much did we foresee, in 1953, about what has happened since. The double helix was so suggestive that we did see, in broad outline. much that happened up to about 1966, when the ‘universal’ genetic code was almost completely established. We made one bloomer, in thinking that the ribosomal RNA was the messenger RNA. Fortunately, we saw the light in a memo- rable episode on Good Friday, 1960, while Francois Jacob was telling us about the latest version of the so- called PaJaMo experiment. This experiment suggested that mRNA had a limited life-time, and thus could not be the ribosomal RNA. Sydney Brenner realized that, in a phage-infected cell, the mRNA for the infecting virus was the RNA described some years earlier by Volkin and Astrachan. There is a dramatic account of this episode in Horace Judson’s book. Jim Watson and Francois Gros independently arrived at the same conclusion.

I doubt if there is any other case in which a simple molecular structure suggested so much, and so much of fundamental importance. Equally remarkable is the speed with which the broad outlines of classical molecular bio- logy were established. It was little more than twelve years from the double helix to the genetic code. For the first few years, progress was rather slow, but after 1960 it accelerated dramatically. I do not think this rate of pro- gress was entirely an accident. We could easily have picked the wrong approach. For example, it could have been argued that what mattered most was the intact 3D structure of any protein, and that the key problem was not how the polypeptide chain was synthesized, but how it folded itself up. If we had chosen this as our main

1x

interest. we would have got nowhere since, even today. we have no general answer to this problem. Or we might have dissipated all our efforts in simplistic theoretlcal ideas about coding, such as the comma-free code. AlterIiati~~ely, we could have tried to solve all the prob- lems using only genetic methods.

Although we realized the importance of a biochemical approach, our group in Cambridge did not make as big a contribution as we might have hoped, but our constant propaganda about the importance of the coding problem stimulated others to work on it. In those days, not every biochen~ist realized the import~~nce of genetics. Fred Sanger is alleged to have said about me: ‘That fellow Crick ~ very keen on genes’. but before long Brenner, Benzer and I were giving lectures to Sanger and his group on the elements of genetics.

What we did not foresee was recombinant DNA and rapid DNA sequencing. Consequently, if I had been asked if it would ever be possible to sequence the entire human genome, I would have predicted that this would take at least another hundred years.

Why were we so blind? It is interesting to review the basis of the techniques used in recombinant DNA work. There is, first, the use of sophisticated physical-chemical separation techniques (such as gel electrophoresis), radio- active tracers, and detection by ultra-violet absorption. We were already familiar with earlier forms of these meth- ods. Then there is the use of enzymes as chemical tools, an approach strongly disapproved of, at that time, by organic chemists, who thought that using an enzyme was a form of cheating. We had no such reservations, but 1 do not think we clearly realized just how specific many enzymes could be nor how many useful enzymes would be discovered. The third useful type of technique depends on the pairing of the bases. This is what makes it possible to devise simple but powerful methods of molecular re- cognition using nucleic acids rather than proteins. We were only too aware of the importance of base-pairing,

but even so we did not appreciate how easily It could be used to select complementary chains. It is this key process

that IS essential for almost all the techniques used In re- combinant DNA.

The other general technique is biological magnifica- tion: the growth of bilhons of bacteria or viruses from a single individual. This we were familiar with, but it is of limited use unless one can also manipulate defined bits of nucleic acid.

Above all it is the c~~nhin~tion of all these techniques that has produced such fantastic results - who would have believed that site-specific mutagenesis would be so easy. But as far as I know, nobody in the Fifties and early Sixties foresaw how rapid progress would be.

The other speakers in this symposium will describe just how far we have come now. Jim, at the end. will tell us where he thinks we are going. There is still a long way to travel before we shall have laid bare all the secrets of developmental biology and neurobiology, to say nothing of coming to close grips with the manifold problems of biological evolution. But there is little doubt that, after 40 years, everyone in the field is racing ahead at an increasing speed. And this we owe mainly to the nature of DNA (and RNA) and in particular to the specific pair- ing of the bases. So 1 feel I can truthfully say: ‘Happy Fortieth Birthday, Double Helix.’

REFERENCES

See any good textbook of Molecular Biology. The books referred to are Watson, J.D.: The Double Helix. a Personal Account of the Dtscovery

of the Structure of DNA. In: Stent, G. (Ed t. A Norton Critical EdItion: Text. Commentary, Reviews, Original Papers. W.W. Norton, New York. London. 1980.

Olby. R.: The Path to the Double Hehx. The MacMillan Press. London. 1974.

Judson, H.F : The Eighth Day of Creation: the Makers of the Revolution m Biology. Snnon and Schuster. New York, 1979.