786 Motecutar Biotopy The double helix a personal view Xoture Vol. %jS April 96 1974 Fr;lncis Crick hIrdica1 Rc+xearch Council Lahortttov for Molecular Biology. Hills Road, Cambridge, UK Francis Crick review the papers prblished .?I years ago on the structure of DNA and the reaction to them. FOR this anniversary I thought it might. be appropriate to look back, in a rather informal way, at the original papers on the structure of DNA to see how they appear today in the light of 21 years of research. . During the spring and summer of 1953 ,Ji Watson and I wrote four papers on the structure and function of DNA. The first appeared in Nature on April 25 accompanied by two pnprs from King's College London, the first by Wilkins, Stokes and Wilson, the other by Franklin and Gosling. Five weeks later we published a second paper in Nature, this time on the genetic implications of the structure. A general dia- cusaion was included in the volume that came from that year's Cold Spring Harbor Symposium, the subject of which was viruses. We also published a detailed technical account of the structure, with rough coordinates, in an obscure jour- nal* in the middle of 19s. The first Nature paper was both brief `and restrained. Apart fmm the structure itself the only fenture of the paper which has excited' comment was the short sentence: "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mech- anism for the genetic material". This has been described as `coy', a word t.hat few would normally rssociate with rithrr of the authors, at least in their wientific work. In fact it was a compromise, reflecting a difference of opinion. I was keen that the paper should discusa the genetic impli- cntions. W&on wss ngainet it. He su&ed from periodic fears that the structure might be wrong and that he had made an ass of himself. I yielded to his point of view but insisted that something be put in the paper, otherwise some- one else would certainIy write to make the suggestion, as- suming we had been too blind to .w! it. In short, it was a claim to priority. Why, then, did we change onr minds and, within only a few weeks, tite the more speculntivc paper of Zvlay 301 The main reason was that when we sent the first dnft of our initial paper to King's College we had not yet seen their own papers. Consequently we had little idea of how strongly their X-ray evidence supported our structure. The famous `helical' X-ray picture of the B form, reproduced by Franklin nnd Gosling in their paper, hnd been shown to Watson, but he certninlp had not remembered enough details to construct the arguments about Bessel functions and distances which the experimentalit gave. Z myself, at that time, had not seen the picture nt all. Consequently we were mildly surprised to discover that they had got so far and delighted to see how well their evidence supported our idea. Thus emboldened, Watson was easily persuaded thnt we should write a second paw. The papers in Nature The two experimental papera of April 25 overlap to a con- siderable extent. Rosalind Franklin's paper mentions the crystnllinc A structurr, but only brieflv, except for the claim that the Patterson superposition fun&on (which was in the press at the time) supported two chains rather than three. Roth papers stress that there must be more than one chain in the structure. Indeed Naurice WiIkins had personally told Chargaff that a year or so earlier. Both present the nrgu- ment that the positions of the intensity m&ma ruled out two (parallel) chains related by a dpd parallel to the fibre axis. Neither gave the neat argument, due to Watson, that their own density measurement, together with the observed change in length between the two fomw,, supported two chains rather than three. Fmnklin noted that if there were several chains they could not bc cqunily spaced and that `equivalence' favoured two rather than thmr. It was not e.xplicitlp stated, however, that equivalence implies dyad 3xes perpendicular to the fibin axis and that therefore the tnv chains must run in opposite directions. Nor did she reahse that the monoclinic unit cell of the A form also suggested this, although we had deduced this from her own experimental data. Both p3pe1-s correctly concluded from the intensity posi- tions that the phosphat+sugar backbone was on the outside of the structure and that the bases were stacked on the in- side. Franklin repeated the argument, which she had made to us verbally a year earlier, that the phosphates would be hydrated (in which she wns perfectly right) and therefore that they nould probably be on the outside of the molecule. In short, both the groups nt King's College had obtained a fairly general idea of the structure but they had done no proper model building. Mainly because of this they had missed the pairing of the bases and they had completely overlooked the significnnce of Chnrgaff's rule. The omissions in t,he paper by Watson nnd myseIf are nlso striking. The structure is produced like a rabbit out of a hat, with no indication as to how we arrived at it. No dimen- sions are given (let alone coordinates) except that the base pairs were 3.4 A apart nnd that the structure had IO base pairs in ita repent. The exact nnture of the base pairing was not immcdirtely obvious; nor even unambiguous since at that timo there wcm two systems for numbering pyrimi- dine rings. Most of this information was provided in the subsequent papers. However the general nature of the structure was clear enough, though the tone of the paper ("it must be regarded as unproved until it has been checked ngainst more exact results") was, apart from the short firat pnragmph, rather muted. THE DOUBLE HELIX: A PERSONAL VIEW (F. Crick) . . MOLECULAR BASIS OF BIOLOGICAL SPECIFICITY (L. PlUlhl@ . . . . . . . . . . * * . . . MOLECULAR BIOLOGY IN A LIVING CELL L1. B. Gurdon) BUILDING THE TOWER OF BABBLE (E. Chrmd') . . . MOLECUUR BIOLOGY AN0 METAPHYSICS (0. S. Stmt) DNA BBFORE WATBON.CRICK (R. Olby) . . . -. . . NEW DIRECTIONS IN MOLECUIAR BIOLOQY (B. Bnnnw) AOBALIND FRANKLIN AND THE DOUBLE HELIX (A. Klup) MOLECUIAR BlOLOQlBlB COME OF AQE IN ARIES (0. A. WIndSor) . . , . . . . . . . . . . . 758 766 769 772 776 779 702 7a5 787 Natwe Vol. .9.#7 April 26 1974 Molecular Bioiogy 767 Although a casual reader could easily have overlooked the significance of the first set of papers, especially as they were full of obscure crystallographic jargon, hc could hnrdly miss the impact of our second one. The biologically im- portant features of the proposed structure were explicitly described. The base pairs were listed with the minimum of hedging nbout tautomerism and were illustrated in scale diagrams. The proposed duplication mechanism was spelt out in simple terms, unmarred by any trnce of algebra In spite of the discussion of the difficulties of unwinding, the list of unsolved problems and the reservations about the unproved nature of the structure, the final paragraph leaves little doubt that the authors thought they had a good idea. I How do they stand today? How have these early papers stood the test of time? It cnn now be taken as firmly established that DNA usually con- sists of two chains, wound together and running in opposite directions. The evidence for this statement is so extensive that it would take too long to quote it all here. The fact that normally A pairs with T, and G with C, is also well established but the details were less certain until recently. The G:C pair was never in serious doubt. Watson and I drew this with only two hydrogen bonds but mentioned in our technical paper' that three was also a possibility. This was made almost certain by the theoretical arguments of Pauling and Corey? and was confirmed by X-ray structure determinations of single crystals of base pairs. The same technique showed that the A:T (or A:U) pair in single crystals usually did not have the configuration Watson and I suggmted. The matter was only finally resolved about a year ago when Rich and his colleagues published two crystal structures; that of GpC paired with itselfa and ApU paired with itself* (the backbone in each case was ribose). both to about 0.9 A: They show not only the expected configurrrc tions for the base pairs but also make is highly likely that, as we claimed, nucleic acid helices are right handed, In 1953 it was uncert.ain whether RNA could form a double helix. Watson and I stated that we thought we could not build our model for the B form of DNA with an RNA backbone. The discoverv of double-stranded RNA viruses proved, however, thnt biological RNA too could form a double helix. thouah with sliahtlv different Dammeters. The detailed coo&at& we had (iencatively) suggested for DNA were soon shown to be incorrect (we had put the backbone at too big a radius) and much more accurate coordinates were provided by Wilkins and his colleagues, using fairly sophisticated methods of handling their much improved X-ray data. The general correctness of this work haa been strongly supported recently by the single-crystal studies, mentioned above, of Rich and his coworkers. Recently, Brams haa put forward evidence that the pnram- eters of a DNA double helix may vary somewhat with base composition, though whether this is a trivial variation or has deep biological implications is at present uncertain. Watson and I were so impressed with the apparent uniformity of the double helix from diierent biological sources and the rsgn- larity of the backbone of our model that we had no hesita- tion in saying that it "seems likely that the precise sequence of the bases is the code that carries the genetic information", an idea which gave me plenty to think about in the next 10 or 12 years. Nothing wns said &out the possibility that the two chains might be melted apart and then annealed together again, correctly lined up. The discovery of this by Marmur and Dotv has provided one of the essential tools of molecular biology. I can still remember the excitement I felt when Paul Doty told me about it at breakfast one day in New York in a hotel overlooking Central Park. But- in other respects we were almcet too far sighted, as witness our remark that recombination would probably depend upon base pairing. We struggled for several years to produce neat models for this, all to no avail; partly because we accepted copy choice too easily but also because we were trying to invent a mechani.mr which did not need additional hnzymes. This showed a gap in our ovemll grasp of molecular biology, which CM also be glimpsed in our tentative suggestion that DNA synthesis might not need an ensyme, a remark I should certainly not make today except perhaps in the con- text of the ongn of life. As to DNA replication, our earliest description was mainly schematic. We realised that plain nucleotides were not likely to be the immediate precursor but missed the rather obvious idea that they were nucleoside triphosphates, again a lack of insight into biochemistry. We did suggest the so-c&d Y mechanism (in the Cold Spring Harbor paper) but did not mention the difficulties due to the direction of synthesis of antiparallel chains, though I frequently emphasised it a few years later. Looking back, I think we deserve some credit for not being inhibited by the difficulty of unwinding which wo clenrly rccognised and for our forthright stand against pamnemic (aa opposed to plectonemic) coiling. In this in- stance our grasp of X-ray diffraction was invaluable. The functions of DNA It is, of course, somewhat a matter for surprise that DNA synthesis is not fully understood even today. It would take too much space to discuss the complex and rnpidly moving field here. Semiconservative replication in many instances is firmly established. The process certainly occurs as if base pairing were taking place, but I have often asked myself what evidence would make it certain that bsae pairing really occurs rather than some elaborate allosteric mechanism. even though the latter seems unlikely. Perhaps only .an X-ray determination of the structure of the nolvmerase will 6nallv answer the question. Meanwhile the topi& of Okazaki fr& men@ rolling circle models, RNA primers and the exact roles of the various @olymerases will keep many people busy. Even at that early period we did at least ask wbether the-DNA of a chromosomi was in one long molecule, though the idea of circular DNA never occurred to us. Nor did we suggest thnt a virus might -have single-stranded DNA. There is however one remark which may turn out to be perspicacious `I. . . we suspect that the most reasonable way to avoid tangling is to have the DNA fold up into a compact bundle as it is formed". As we struggle with the structure of the E. cdi chromosome and the even more formidable problem of the structure of the chromosomes of higher organisms-probably the major unsolved problem of molecular biology today-it might be worth remembering this tentative suggestion from the distant past. The other topic we touched on was mutation. This was of the base-substitution type-there is no hint of frameshift mutants. We totnlly missed the possible role of enzymes in repair although, due to Claud Rupert's earIy vey; elegant work on photoreactivation, I later came to realise that DNA is so precious that probably many distinct repair meeh- anisms would exist. Nowadays one could hardlv discusa mutation without considering repair at the same time. There is no hint in these early papers that nucleic acid might form a complex three-dimensional structure such as we now find in transfer RNA nor even the idea of the hypothetical Gierer loops. Our message was that DNA was simple and alone carried the genetic information. We saw no reason to complicate it till we had to. For the same ma- son although we must have drawn a G:U pair we attached no importance to it. "Wobble" was still far in the future, but these, it seems to me, are forgivable oversights. Reactions to the structure It is really for the historian of science to decide how our structure was received. This is not an easy question to 708 Mdecdar Bidogy Notve VoLP.@ApdP819?~ amwcr IJSC~IHO tborc was na,turaUp a spectrum of opinion whiib changed with time. There it 110 doubt, however. that it had 8 comd&nbk and immediate impact on an influential group of active &n&us. Mainly due to Max Deibriick. copies of the initial three papers were distributed to dl those attending the 1963 Cold Spring Hubor Symposium and Wat- son's talk wu added to the programme. A little later I gsve a lecture at the Rockefeller which I nm told produced con- siderable interest, partly I think because I mined an en- tbuaiutic pmsentatim of our ideas with a fairly cool assess- ment of the exuerimental evidence, roughly on the lines of the article whit& appearvxi in S&n@k Amen&~ in October, 19!U Svdnev Brenner. rho had just finished his PhD. at Oxford under Ffioshelwood, appointed himself, in the s&n- wr of 19!M. as Our Representative rt Cold Spring Harbor nnd took some psios to get the idess over to Demerec. It wss about this time that Jfatt >Imhon, just moving into bidogy from physical chemistry, grasped the importance of inventing s new method to tsckle the problem of semicon- servative replication, a theoretical snaly& which led to density gmdiiot centrifugation. But not everyone was con- vincrd. Barry Commoner insisted. with some force. that ph>sieists oversimplified bide, in which he wss not com- pletely wrong. Chargaff, when I visited him in the winter of 1953-S+ tdd me (with hi customary insight) that while our first paper in N&UC we interesting, our second psper on the g&e&c implications was no good st all. I was mildly surorised to find. when. some \-ears later. in 1959. I tlllked witi Fritz Lipm&n who had &ranged that I should give a series of lectures at the Rockefeller, tht he had not really grasped our scheme of Dx.4 replication. (It emerged thst he had heen talking to ChargaB.) By the end of the lectures. however, when he summed up, he gave a remarkably clear outline of our ideas Arthur Komberg hss told me thst when he bn work on DKA replication he did not believe in our mechanism, but hi own brillisnt experiments soon made him J convert, though slwa!w a careful and critical one. It was hii work which produced the first good evidence that the two chains run in opposite directions. AU in all it seems to me that wc got a very fsir hearing, better than AveT and rertainlr s lot better than 3fendel. Sot that `it wss all plain wiling. We were naturally de- lighted with the work of JIeselson and Stahl. and of Her- bert Taylor, on semiconwrvative replicstioo, though I have never thought this the ewence of our idess which lies nther in the bass Iruiring. Seymour Benser's genetic nnalysis of the rll locus of &age-T4 hncouraged us greatly. But we had to live through the claims of Mar&k that there wss no DNA in At&&eggs and of 8 Canadian group that the amount of DSA synthesis in one cell cycle was twice the expected amount. At a later stage Cavalieri claimed that. the b&c DS.4 structure had four chains, mt.her than two, an idea which cropped up agin more recently, On the crystallographic side Donohue, whose advice had been crucinl to our under: standing of base pairing, was s persistent critic of the validitv of the later X-ray work, but in recent years he csrried it too far, refusing, for example, to admit as evidence the great sccumulation of data showing that the two chsins are sntipamllel. (In 1956, he hnd r&r published. with Stern. a ouite erroneous structure having iike-with-like pair- ing.) -1 hope the recent psprrs by Rich. referred to above. hnvc to some extent reduced his doubts, which st times had some justifirrtion. Who might have discovered it? Then there is the question, whnt would have happened if Watson and I had not put forward tho DNA structure? This is `i5y' history which I am told is not in good repute with historians, though if a historian cnnnot give plausible auswers to such questions I do not see whst historical rnalysis is about. If Watson had heen killed by a tennis ball I am reasonably sure I would not have solved the structure done, but who would? Olbp has recently addressed hiidf to this question. Watson and I always thought that Linus Pauliag would he hound to to have another shot at the stntc- ture once he had seen the %IK'S College X-ray data, but he has recently stated that even though he immediately liked our structure it took him a littie time to decide fin& that his own wss wrong. Without our model he might never hsve done so. Rosalind Franklin wss only two steps away from the solution. She needed to reslii that the two chains must run in opposite directions and that the bases, in their cor- rect tautomeric forms, were paired together. She was, how- ever. on the point of leaving King's College and DNA, to work instead on T&IV with Remal. Maurice WiUdns had announced to us, just before he hew of our structure, that he was going to work full time on the problem. Our persistent propaganda for model building had also had its e&et (we had previously lent them our jigs to build models but they had not used them) and he was proposing to give it a try. I doubt myself whether the dimvery of t.he structure could have been de&xl for more than two or three years. There is a more general argument, however, recently pro- posti by Gunther Stent and supported by such o sophisti- rated thinker S.I Medawnr. This is that if Vatson and I had not discovered the st.ructure, instead of being revealed wit.h 3 flourish it wouId have trickled out nnd that its impact vmuld have been far less. For this sort of resson Stent had argued that a scientific discovery is more skin to r work of art than is generally admitted. Style, he ,argues, is ns important as content. I am not completely convinced by this nrgument, at least in this case. Rather than believe that Watson and Crick made the DKA structure, I would rather stress that the &UC- ture made Watson and Crick. After sll, I wss almost totally unknown at the time and Watson was regarded, in most circles. as too bright to he really sound. But what I think is overlooked in -,zx~ch srgumeuts is the intrinsic beauty of the Dx.4 double helix. It. is the molecule which has style. quite as much as the scientists. The genetic code wss hot revesled all in one go but it did not lack for impact once it had been pieced together. I doubt if it mnde all that dif- ference thst it was Columbus who discovered Americn. Wht mattered much more was that people and money were avail- able to exploit the discovery when it. was made. It is this aspect of the history of the DNA st.mcture which I think demands nttention, mther than the personal dements in the net of discovery, however interrsting they rnsy bs as r.~ object lesson (good or bsd) to other workers. My own reactions I have sometimes been asked whether I had ever contem- plated writing my own account of the discovery. In the 1950s I did give a lecture on this subject to R group of historians of science at timbridge and to a similar group at Oxford. I wm sble to be nth& more srholnrlv th&t Watson could show himself in The Doub?e IIplir. which is better regsrded ss r rather +-id fragment of his autobiography, written for n lay audience. As to a book I confess I did get ns far ns composing R title (The Loose Screw) and what I hoped w-35 3 catchy opening ("Jim wss always clumsy with his hands. One had only to see him peel an orange. . .") but I found I hsd no stomach to go on. Recently we made R fJm together about it for undergmduates. Much had to be left out when the film came to be cut but it does to some extent supplement Jim's book. Since Olby's detailed and scholarlv account. will soon he availnble I doubt if there is now much more I can usefully add. Finally one should perhaps ask the personal question- am I gI.sd thrt it happend RS it did? I con only answer that .Voture Vol. .?JS .4pril 26 l!Z/, . Molecular Biology 769 I enjoyed every moment of it, the downs aa well as the ups. ' Crick, F. H. C., and Wntson, J. D.. Proc. R. SW.. ASI, 1 t certainly helped me in my subsequent propa2anda for the genetic code. But to couvcy my own feelings, I cannot do ? p.%??.(?!%nd Corey, R. U., Arche Bioclrem. Biophfp, SS, bct.ter than quote from a bnlhant nnd perceptive lecture 164-181 (1056). a Dw, R D. Seeman, N., Rosenberg. J., and Rich, A.. Prnc. I heard years ago in Cambridge by the painter John Minton ml)t. Ad. Sci. USA., 70,849-8&i (19W). (he later committed suicide) in which he said of his own ' Rooenberz. J.. Seeman. S., Kim, J. J.. Suddath. F., Si~:holap, . artist.ic creations "the important thin2 is to be there when J-J.. nnd Rich. A., iVafrcn. 23, X&M f12f3). the pict,ure is painted". And this. it seems to me, is partly J Rmm, S.. and Tougsrd, P., N&are ,tetp Bin!.. 229, 122-131, ( 1972). :L matter of luck and partly pond judgement, inspiration and * Oh. R. C.. The Pnlh lo the Dnctble lleliz (Mnrmitlnn. Lon- persistent nppliralion. don. 1974).