The DNA Double Helix Fifty Years On

Computational Biology and Chemistry 27 (2003) 461–467

Commentary The DNA double helix ﬁfty years on

Robert B. Macgregor Jr.∗, Gregory M.K. Poon

Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ontario, Canada M5S 2S2 Received 16 June 2003; received in revised form 8 August 2003; accepted 12 August 2003

Abstract

This year marks the 50th anniversary of the proposal of a double helical structure for DNA by James Watson and Francis Crick. The place of this proposal in the history and development of molecular biology is discussed. Several other discoveries that occurred in the middle of the twentieth century were perhaps equally important to our understanding of cellular processes; however, none of these captured the attention and imagination of the public to the same extent as the double helix. The existence of multiple forms of DNA and the uses of DNA in biological technologies is presented. DNA is also ﬁnding increasing use as a material due to its rather unusual structural and physical characteristics as well as its ready availability. © 2003 Published by Elsevier Ltd.

Keywords: DNA; Double helix; Molecular biology; Nanotechnology

1. Introduction netic transmission of characteristics. At about the same time as Mendel was carrying out his studies, DNA was discovered 1953 witnessed the birth of a science icon with the pub- by the Swiss physiologist Friedrich Miescher. For the first lication of James Watson and Francis Crick’s proposal of a several decades after its discovery the role of DNA in cellular double helix structure of DNA in the journal Nature (Watson processes remained unexplained and for the most part unin- and Crick, 1953a). In their original publication they pro- vestigated. During the early part of the 20th century, various posed that DNA consists of two molecules that are wound biochemical pathways and principles were brought to light; around each other to form a right-handed helix that is sta- however, the question remained, which cellular molecule bilized by hydrogen bonding interactions between comple- provides the basis for Mendellian genetics? The different mentary base pairs between the two molecules. They pointed disciplines interested in cellular processes, biochemistry, out that this proposed structure provided a hint as to how genetics, and microbiology took very different approaches DNA could be self-replicating (Watson and Crick, 1953b). in their investigations of this question; however, none fo- The proposal rationalized and accommodated a great deal cused on DNA. Proteins and enzymes were known to be of current experimental information and pointed the way to the molecular phenotype of cells; however, the link between other experiments that could verify it. The process of veri- the gene-carrying molecule and these proteins was anything fication provided additional revelations about the molecular but clear. DNA was generally considered to be chemically, mechanisms of cellular processes, and it provided a model and thus structurally, too simple to contain the presumably on which many other ideas could be based. complex information necessary for heredity. This view changed over the course of about 20 years starting in 1928 when Fred Griffith found that a benign 2. The path to molecular biology strain of pneumococcal bacteria could be transformed into a pathogenic strain by exposure to a cell-free extract of In the 19th century, the Moravian monk Gregor Mendel the pathogenic strain. In the mid 1930s Oswald Avery and had given a solid quantitative grounding to the idea of ge- coworkers set out to purify this transforming factor. Chemi- cal analysis of the extract showed that DNA had transformed ∗ the pneumococcal bacteria and in 1944 he published the first Corresponding author. Tel.: +1-416-978-7332; fax: +1-416-978-8511. results demonstrating that DNA was the genetic material E-mail address: [email protected] (R.B. Macgregor Jr.). (Avery et al., 1944). A few years later, the results of Hershey

1476-9271/$ – see front matter © 2003 Published by Elsevier Ltd. doi:10.1016/j.compbiolchem.2003.08.001 462 R.B. Macgregor Jr., G.M.K. Poon / Computational Biology and Chemistry 27 (2003) 461–467 and Chase (1952) reinforced Avery’s finding by demonstrat- give rise to the proteins for which they code. At that time ing that DNA mediated the production of progeny virus there was no known molecule or mechanism that would link in bacteriophage-infected Escherichia coli bacteria. During these two structures. A proposal by Mahlon Hoagland sug- this time, George Beadle and Edward Tatum had shown in gesting the formation of a complementary structure between the early 1940s that enzyme synthesis in cells was con- DNA and RNA appeared in the magazine Scientific Ameri- trolled by genes and that there was one gene for each en- can in 1959. The next year experimental data from the labo- zyme (Beadle and Tatum, 1941). ratory of Sol Spiegelman (Nomura et al., 1960) showed the Taken together these were extremely important discover- involvement of a DNA–RNA hybrid molecule that was cru- ies putting an end to nearly a century of speculation about the cial to protein synthesis; this molecule was messenger RNA chemical nature of genes. It was interesting to know that ge- (mRNA). Oddly, although RNA was known to form heli- netic information somehow resided in DNA; however, there cal structures similar to DNA, the necessity or likelihood was no way to incorporate it into the current understanding of formation of a transient DNA–RNA hybrid had not been of cellular mechanisms. It was not clear how the genes on considered. With the discovery of mRNA the modern cen- DNA could be transformed into proteins; several pieces of tral dogma of molecular biology, DNA makes RNA makes the puzzle were still missing. This was the state of knowl- protein, was established. edge in biology during which the work on the structure of DNA began. In the early 1950s several prominent scientists had turned 3. The success of the Watson–Crick model their efforts toward elucidating the structure of DNA, the perceived importance and prestige of these studies was Synthesis, verifiability, and extrapolation are hallmarks enhanced by the fact that Linus Pauling, the chemist who of great scientific ideas. Many other experiments in biology received the Nobel Prize for his descriptions of chemi- that occurred after the proposal of the double helix are con- cal bonding, was working on the structure of biological sidered to be direct consequences of the Watson and Crick molecules, including DNA. Studies of the structure of double helix. This may be overstating the case somewhat; molecules had been made possible by the advent of X-ray many seminal discoveries about the molecular biology of diffraction, a technique that had been recently developed the cell had already been made and would have been made by physicists. And although X-ray diffraction worked well with or without a working model for the structure of DNA. for small molecules, work on large biological molecules However, it is clear that their proposal is a major landmark like proteins or DNA was slow for a number of reasons. In in the progression of understanding of biological systems the case of analyzing the structure of DNA the difficulties that began in the 19th century. There have been many other were exacerbated by the fact that there were no crystals of scientific milestones but none have so consistently captured DNA available meaning that the diffraction images were the imagination of the public and scientific community. acquired using oriented fibers of DNA. The diffraction In purely structural terms, the DNA double helix was the patterns obtained using fibers is poor and only gross de- first model for a major macromolecular cellular component tails of the structure can be determined. For example, it and it remains the greatest success of structural biology was known that DNA was helical and that the bases were to date. Although there are currently thousands of protein oriented perpendicular to the axis of the helix. However, structures known and the three dimensional structures of neither the relative orientation of the molecular groups nor the other cellular components have also been elucidated, the handedness of the helix could be ascertained. this was not the case in the 1950s. A working model for any When Watson and Crick combined the known data about large cellular molecule was a great novelty at the time; the DNA, the results of the X-ray diffraction analysis, and a first protein structure was still about ten years away. DNA great deal of imagination they struck upon a structure that is much simpler than any protein and Watson and Crick’s satisfied all of the requirements. Because of the earlier work model was consistent with all the known information about of Avery, Hershey and Chase, and Beadle and Tatum, when the composition and properties of DNA. Although detailed Watson and Crick published their proposed structure the information concerning protein structure is often useful in scientific world was ready. Their two publications in 1953 guiding further experimentation about a particular protein, offered an entirely new way of understanding the molec- such knowledge does not have the generality, and thus, ular mechanisms underlying many cellular processes, such does not have the impact of the originally proposed double as cell division, genetic inheritance, and protein synthesis. helical structure for DNA. This is true because it is very dif- However, despite the fact that the advances between 1927 ficult to extract general principles from most protein data, and 1953 had revolutionized the understanding of the cell a the details are important. In contrast to this the details of number of very large questions had also been raised. the structure of the double helix are not as important as the In the mid- to late-1950s, after the publication of the concepts that arose from it and the challenges it presented. Watson and Crick structure the biggest difficulty posed by The Watson–Crick model was a simple and elegant the results of the previous twenty-five years was how the model that facilitated the subsequent understanding of genes present in the double helical structure of the DNA many biological processes such as DNA replication, gene R.B. Macgregor Jr., G.M.K. Poon / Computational Biology and Chemistry 27 (2003) 461–467 463 recombination, transcription, mutation, and in turn impor- oneers in this new science made important discoveries about tant developments such as chemo- and antisense therapies. the nature of information and noise. For the first time, in- Nonetheless, it is possible to imagine a world in which formation and its quality could be expressed in quantitative molecular biology exists with little or no knowledge of the terms. The increasing use and power of digital computers structural nature of DNA. The validity of this assertion is accompanied these theoretical insights. We are not aware of seen in the fact that the first x-ray crystal structure of a any contact between information scientists and Watson or DNA molecule was not published until more than 25 years Crick, nor did computers play any direct role in the genesis after the first appearance of Watson and Crick’s double he- of their idea. However, Watson and Crick’s description of lix (Wing et al., 1980). The intervening years were hardly DNA as a double helix and the subsequent deciphering of fallow for our understanding of the cell. A great deal of bi- the genetic code by other researchers began to move biology, ology had been done and would have been possible without or molecular biology, to a level with these more quantitative a model of the structure. sciences. The cell was, and in many ways remains, a black As an example, after the discovery that DNA carried the box, but for the first time there was at least the possibility to genetic message it was clear that the message had to be treat biology as a branch of knowledge that at least some- encoded somehow. A logical exercise, with or without the day could be understood in mathematical terms. It provided double helical model DNA, would lead to the same conclu- an arrow, a directionality to the processing of information sion: a minimum of three bases are necessary to encode the in the cell: DNA makes RNA makes protein. And although twenty amino acids that constitute proteins. Chargaff had it is not digital it is quite clearly quantized. shown that adenosine and thymine bases always occurred in the same proportions as do guanines and cytosines. With this knowledge, one can conjecture that it would have been 5. Alternatives to the Watson–Crick proposal possible to propose a working model of DNA, consistent with all of the data, without the double helix model. Other alternative structures have expanded the conforma- Two other technological breakthroughs played equally im- tional repertoire of DNA. Indeed, shortly after the publica- portant roles in the development of molecular biology. The tion of the double-helix structure, it was apparent that un- first of these was the ability to rapidly sequence DNA. The winding the two strands in order to replicate the DNA prior methods published by Sverdlow et al. (1973), Maxam and to cell division would pose a very significant topological Gilbert (1977), and Sanger et al. (1977) led to the discov- problem. The so-called side-by-side model essentially elim- ery of the complexity of eukaryotic genomes and eventually inates the topological linking of the two strands of DNA to the sequencing of the genomes of several organisms, in- by allowing the base pair interactions to occur with the cluding humans. The second significant technological feat backbones of the two molecules alternating between left- was the emergence of a rapid, inexpensive, method of syn- and right-handed helices (Millane and Rodley, 1981). An- thesis of short strands of DNA (Gait and Sheppard, 1977; other topologically unlinked model of DNA has also been Hutchinson et al., 1978). Many other scientific and practical proposed by Biegeleisen (2002). Although these alternative uses of DNA have arisen as a consequence of these two inno- models rectify certain difficulties inherent in the double he- vations. Automated, rapid sequencing, the polymerase chain lical structure of DNA, they are inconsistent with great deal reaction, forensic and archeological uses of DNA, are some of experimental evidence that is described by Watson and of the examples of the uses of DNA that were unimaginable Crick’s model. It should be pointed out that the structure of a short time ago but were made possible by the convergence DNA inside of a cell remains a mystery and it is entirely of the knowledge of several different scientific disciplines possible that DNA adopts conformations in the cell that have in Watson and Crick’s proposal of a structure of DNA. not or cannot be observed in a test tube. A few years after the proposal of the double helix, an alternative type of interaction between the complementary bases 4. The rise of information science and the double helix was put forward by Hoogsteen. The alternative base pairing interactions apparently is found only in unusual DNA It is also interesting to consider the era in which Watson structures. Several of these unusual, or non-canonical, struc- and Crick lived. The middle of the twentieth century saw the tures were discovered in the 1950s as increasing numbers rise of two other factors that provided an interesting back- of biochemists and physical chemists turned their attention drop to their work, namely, the concomitant rise in infor- to DNA. The most easily interpretable experiments inves- mation and computer science. Physicists, mathematicians, tigating the structure of DNA relied on the use of simple and engineers were interested in the information content of synthetic polymers consisting of repeats of one base for different types of signals. Their interest was driven in part the entire length of the polymer, for example polyadenosine by certain consequences of quantum mechanics, discovered (poly(A)) or polyuracil (poly(U)). As chance would have it, earlier in the century and in part because of the increasing these simple polymers, exhibit some very interesting behav- amount and value of information. This led to the rise of infor- ior; titrations of complementary strands with each other, for mation science, Claude Shannon and Alan Turing, early pi- example poly(A) and poly(U) showed that the stoichiom- 464 R.B. Macgregor Jr., G.M.K. Poon / Computational Biology and Chemistry 27 (2003) 461–467 etry of the interacting strands could be either 1:1, the ex- At first these non-standard conformations were consid- pected proportion, or 1:2. Another interesting example is ered biophysical chemical oddities with little or no biologi- the self-association of polyguanine which was found to as- cal relevance. However, it would now appear that most, if sociate into four-stranded structures. not all of the structures listed in Table 1 can and do exist Although the stoichiometry of these molecules differed in cells. An interesting footnote in the progress towards from that of the double helix, i.e. three or four individ- the structure of DNA was provided by the publication ual DNA strands instead of two, the Watson–Crick double of the first high-resolution x-ray structure by the group helix was used as a starting point for interpreting the re- of Alex Rich in 1979. Much to the surprise of everyone, sults. Thus, the unusual stoichiometry between poly(A) the DNA in their study, the hexanucleotide, d(CGCGCG), and poly(U) was assumed to arise from the formation of crystallized as a left-handed helix (Wang et al., 1979). It a three-stranded helix. In this particular case, model build- has since been shown that this particular sequence mo- ing and then crystallographic results showed that the bases tif, alternating CG residues, will adopt left-handed he- interacted via two different types of hydrogen bonding pat- licity in the appropriate solvent conditions and that, in terns: which are now known as Watson–Crick binding and general DNA exists as a right-handed helix for other Hoogsteen hydrogen binding. sequences.

6. One molecule, many different helices 7. DNA as a material

These early unusual structures provided only a hint of the Although it may be argued that much of molecular biology conformational plasticity of nucleic acids. In the majority of might have existed in something resembling its present form cases, the novel structures are based upon the double helix without Watson and Crick’s proposed structure, the fact that as originally proposed; a partial list of the types of struc- DNA is indeed a double stranded structure has had impli- tures is given in Table 1. In many cases the different types of cations in areas of science that probably were not expected structures result from relatively minor alterations in the he- 50 years ago. The structural linearity of DNA, its ability to lix structures, e.g. the difference between the A and B con- form predicable complementary interactions between two formation (Fig. 1). Other changes are much more dramatic molecules, and the advent of solid-phase DNA synthesis and differ radically from the double helix structure proposed have made DNA one of the most attractive building blocks by Watson and Crick. Perhaps the two best examples of this for the synthesis of what is now called nanostructures. class of structures are Z-DNA and parallel-stranded DNA. Nanostructures are molecular species with dimensions on In Z-DNA the helical sense of the molecule is reversed, the nanometer scale that are synthesized from the “bottom resulting in a left-handed helix (Fig. 1). In parallel DNA, up.” That is, these structures are made starting with smaller the relative orientation of the two complementary strands of molecules that are allowed to associate in a controlled DNA is parallel as opposed to the more standard antiparallel manner in order to construct the ﬁnal, nanometer-scale arrangement observed in most other structures. structure. Several laboratories have exploited the base

Table 1 A sample of the different types of DNA structures. Most of the entries in the Table have sub-classes and not all of the structures necessarily have any known biological relevance. All of the known structures involve some form of a helix although it may differ radically from Watson and Crick’s proposal. Structure Description

A-DNA Duplex isoform found in dehydrated environments. First ﬁbre defraction by Roselind Franklin was of A-DNA. Only isoform accessible to RNA and RNA/DNA duplexes. B-DNA Duplex isoform found in most biological environments. Original structure proposed by Watson and Crick. Not adopted by structures containing RNA. Z-DNA Left-handed duplex favored by GC-rich sequence under high salt. Discovered in ﬁrst single-molecule crystallographic structure of DNA (Wang et al., 1979) M-DNA Putative duplex isoform in which WC base pairs coordinate divalent ions (e.g., Zn2+,Ni2+ and Co2+) at alkaline pH. (Aich et al., 1999). Noted for electrical conductive properties. P-DNA Once postulated structure of DNA by Linus Pauling, with an interwound phosphate backbone and exposed bases (Pauling and Corey, 1953). Observed in positively supercoiled B-DNA duplex under a stretching force (Allemand et al., 1998). Triplex Three-stranded isoform formed by binding of a third strand to the major groove of a B-DNA duplex. Formed by two homopyramidine and one homopurine strands and favored by low pH or high ionic strength. H-DNA Intramolecular triplex formed composed of a hairpin duplex by one strand and Hoogsteen base paring by another. Sequences exhibit mirror symmetry along the strands. G-quartet Four-stranded isoform supported by Hoogsteen base pairing among four guanine residues, coordinating a monovalent ion (e.g., K+, + + Na ,NH4 ). Contacts may be intra-strand (as in telomeric ends of chromosomal DNA) or propagate linearly (G-wire). Frayed wires Multistranded superstructures formed by oligonucleotides with long consecutive guanine runs (Protozanova and Macgregor, 1998). Non-guanine nucleotides branch out and may be functionalized. R.B. Macgregor Jr., G.M.K. Poon / Computational Biology and Chemistry 27 (2003) 461–467 465

Fig. 1. A world of helices. From left to right, the crystal structures of three families of DNA structure: A-DNA, B-DNA, and Z-DNA. The A and B forms are right handed double helices that differ in the details of the parameters such as the orientation of the base pairs relative to the helix axis and the number of base pairs per turn of the helix. The Z form is a left handed helix. Watson and Crick had arrived at the structure that today is called the B form from model building based their data. Although their data could not discriminate between the two different helix senses, they chose the right-handed orientation because of they found it impossible to build a left handed double helix that obeyed the known structural constraints. (These structures are taken from the Protein Data Base, ID Numbers 440D, 5DNB, and 1DJ6.) pairing capability of DNA for this purpose. Many cre- Watson and Crick’s proposal of a double helical structure ative structures have been synthesized using only DNA for DNA double helix facilitated and greatly accelerated by the laboratory of Nadrian Seeman. Among the ac- the subsequent scientific discoveries by providing a simple complishments of his group is the synthesis of a DNA accurate model. The discoveries during the 30 years between molecule with the connectivity of a cube (Fig. 2), and roughly 1930 and 1960 set the ground work for the scientific “nanomechanical device” based upon the B-Z confor- and technological advances of the past 40 years. It was one mational change (Fig. 3). Other DNA-based molecular of the most important steps in the development of molecular machines have also been created (Fig. 4)(Yurke et al., biology in the latter half of the twentieth century. It would 2000). be difficult to say that the advent of the double helix was In 1994, Leonard Adleman took advantage of the par- the single most important of the discoveries; the discovery allel nature and specificity of DNA basepairing to obtain must be considered in the context in which it was made. a DNA-based solution to the Travelling Salesman Problem On the other hand, it is abundantly clear that Watson and (Adleman, 1994). Although this problem can be solved much Crick’s proposal remains the most widely popularized of more quickly and efficiently by electronic digital comput- the giant steps in biology that occurred in the middle of the ers, the specificity of DNA basepairing and the very large last century. Much of modern molecular biology would have number of parallel interactions that occur in a double helix been possible perhaps without it but many key experiments make DNA (or RNA) an interesting tool for solving certain central to our knowledge would have been much slower in very large combinatorial problems. coming. The structure originally proposed has now been verified and it remains one of the greatest successes of the elucidation of function by consideration of the structure. The standard Watson–Crick structure also inspired a number of developments that authors probably had not foreseen; perhaps the most intriguing of these is the use of DNA as a material for the synthesis of molecule-sized devices and machines. Note: There are several excellent books describing the history of molecular biology from different perspectives. These include: A History of Molecular Biology, by M. Morange, Harvard University Press, Cambridge,1998; Life Science in Fig. 2. DNA as a material: a schematic of the backbone of a DNA molecule the Twentieth Century, by G. E. Allen, Wiley, New York, whose helix axes have the connectivity of a cube. These nanostructures 1975; The Path to the Double Helix, by R. Olby, Macmillan, were synthesized in the laboratory of N. Seeman (from Seeman, 1998). London, 1974. 466 R.B. Macgregor Jr., G.M.K. Poon / Computational Biology and Chemistry 27 (2003) 461–467

Fig. 3. A DNA-based nanomechanical device. Top: A molecular model of a molecule constructed entirely from right-handed B-DNA. At the center of the connecting helix is a 20-nucleotide region (yellow) that can adopt either the left-handed Z conformation or the right-handed B conformation. When the B-Z transition takes place, this same yellow portion becomes left-handed Z-DNA (bottom). The large green and red circles indicate ﬂuorescent dyes covalently attached to the DNA that change their relative positions, when the B-Z transition is induced. The change in the position of the dyes is reversible and may be cycled (from Mao et al., 1999).

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