Cold Spring Harb Symp Quant Biol-1953-Watson-SQB ... - Biology

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Downloaded from symposium.cshlp.org on March 26, 2009 - Published by Cold Spring Harbor Laboratory Press STRUCTURE OF DNA 127 spaced equally along the fiber axis, but are probably displaced from each other by about threeeighths of the fiber axis period, an inference again in qualitative agreement with our model. The interpretation of the X-ray pattern of Structure A (the crystalline form) is less obvious. This form does not give a meridional reflexion at 3.4 A, but instead (Figure 2) gives a series of reflexions around 25 ~ off the meridian at spacings between 3 A and 4 A. This suggests to us that in this form the bases are no longer perpendicular to the fiber axis, but are tilted about 25 ~ from the perpendicular position in a way that allows the fiber to contract 30 per cent and reduces the longitudinal translation of each nucleotide to about 2.5 A. It should be noted that the X-ray pattern of Structure A is much more detailed than that of Structure B and so if correctly interpreted, can yield more precise information about DNA. Any proposed model for DNA must be capable of forming either Structure A or Structure B and so it remains imperative for our very tentative interpretation of Structure A to be confirmed. (2) The anomolous titration curves of undegraded DNA with acids and bases strongly suggests that hydrogen bond formation is a characteristic aspect of DNA structure. When a solution of DNA is initially treated with acids or bases, no groups are titratable at first between pH 5 and pH 11.0, but outside these limits a rapid ionization occurs (Gulland and Jordan, 1947; Jordan, 1951). On back titration, however, either with acid from pH 12 or with alkali from pH 21,6, a different titration curve is obtained indicating that the titratable groups are more accessible to acids and bases than is the untreated solution. Accompanying the initial release of groups at pH 11.5 and in the range pH 3.5 to pH 4.5 is a marked fall in the viscosity and the disappearance of strong flow birefringence. While this decrease was originaIly thought to be caused by a reversible depolymerization (Vilbrandt and Tennent, 1943), it has been shown by Gulland, Jordan and Taylor (1947) that this is unlikely as no increase was observed in the amount of secondary phosphoryl groups. Instead these authors suggested that some of the groups of the bases formed hydrogen bonds between different bases. They were unable to decide whether the hydrogen bonds linked bases in the same or in adjacent structural units. The fact that most of the ionizable groups are originally inaccessible to acids and bases is more easily explained if the hydrogen bonds are between bases within the same structural unit. This point would definitely be established if it were shown that the shape of the initial titration curve was the same at very low DNA concentrations, when the interaction between neighboring structural units is small. (3) The analytical data on the relative proportion of the various bases show that the amount of adenine is close to that of thymine, and the amount .of guanine close to the amount of cytosine + 5- methyl cytosine, although the ratio of adenine to guanine can vary from one source to another (Chargaff, 1951; Wyatt, 1952). In fact as the techniques for estimation of the bases improve, the ratios of adenine to thymine, and guanine to cytosine q- 5-methyl cytosine appear to grow very close to unity., This is a most striking result, especially as the sequence of bases on a given chain is likely to be irregular, and suggests a structure involving paired bases. In fact, we believe the analytical data offer the most important evidence so far available in support of our model, since they specifically support the biologically interesting feature, the presence of complementary chains. We thus believe that the present experimental evidence justifies the working hypothesis that the essential features of our model are correct and allows us to consider its genetic possibilities. V. GENETICAL IMPLICATIONS OF THE COMPLEMENTARY MODEL As a preliminary we should state that the DNA fibers from which the X-ray diffraction patterns were obtained are not artifacts arising in the method of preparation. In the first place, Wilkins and his co-workers (see Wilkins et a/., 1953) have shown that X-ray patterns similar to those from the isolated fibers can be obtained from certain intact biological materials such as sperm head and bacteriophage particles. Secondly, our postulated model is so extremely specific that we find it impossible to believe that it could be formed during the isolation from living cells. A genetic material must in some way fulfil two functions. It must duplicate itself, and it must exert a highly specific influence on the cell. Our model for DNA suggests a simple mechanism for the first process, but at the moment we cannot see how it carries out the second one. We believe, however, that its specificity is expressed by the precise sequence of the pairs of bases. The backbone of our model is highly regular, and the sequence is the only feature which can carry the genetical information. It should not be thought that because in our structure the bases are on the "inside," they would be unable to come into contact with other molecules. Owing to the open nature of our structure they are in fact fairly accessible. A MECHANISM FOR DNA REPLICATION The complementary nature of our structure, suggests how it duplicates itself. It is difficult to imagine how like attracts like, and it has been suggested (see Pauling and Delbriick, 1940; Friedrich-Freksa, 1940; and Muller, 1947) that self duolication may involve the union of each part with an opposite or complementary part. In these discussions it has generally been suggested that protein and nucleic acid are complementary to each other and t~at self replication involves the alternate
Downloaded from symposium.cshlp.org on March 26, 2009 - Published by Cold Spring Harbor Laboratory Press 128 I. D. WATSON AND F. H. C. CRICK syntheses of these two components. We should like to propose instead that the specificity of DNA self replication is accomplished without recourse to specific protein synthesis and that each of our complementary DNA chains serves as a template or mould for the formation onto itself of a new companion chain. For this to occur the hydrogen bonds linking the complementary chains must break and the two chains unwind and separate. It seems likely that the single chain (or the relevant part of it) might itself assume the helical form and serve as a mould onto which free nucleotides (strictly polynucleotide precursors) can attach themselves by forming hydrogen bonds. We propose that polymerization of the precursors to form a new chain only occurs if the resulting chain forms the proposed structure. This is plausible because steric reasons would not allow monomers "crystallized" onto the first chain to approach one another in such a way that they could be joined together in a new chain, unless they were those monomers which could fit into our structure. It is not obvious to us whether a special enzyme would be required to carry out the polymerization or whether the existing single helical chain could act effectively as an enzyme. DIFFICULTIES IN THE REPLICATION SCHEME While this scheme appears intriguing, it nevertheless raises a number of difficulties, none of which, however, do we regard as insuperable. The first difficulty is that our structure does not differentiate between cytosine and 5-methyl cytosine, and therefore during replication the specificity in sequence involving these bases would not be perpetuated. The amount of 5-methyl cytosine varies considerably from one species to another, though it is usually rather small or absent. The present experimental results (Wyatt, 1952) suggest that each species has a characteristic amount. They also show that the sum of the two cytosines is more nearly equal to the amount of guanine than is the amount of cytosine by itself. It may well be that the difference between the two cytosines is not functionally significant. This interpretation would be considerably strengthened if it proved possible to change the amount of 5-methyl cytosine in the DNA of an organism without altering its genetical make-up. The occurrence of 5-hydroxy-methyl-cytosine in the T even phages (Wyatt and Cohen, 1952) presents no such difficulty, since it completely replaces cytosine, and its amount in the DNA is close to that of guanine. The second main objection to our scheme is that it completely ignores the role of the basic protamines and histories, proteins known to be combined with DNA in most living organisms. This was done for two reasons. Firstly, we can formulate a scheme of DNA reproduction involving it alone and so from the viewpoint of simplicity it seems better to believe (at least at present) that the genetic specificity is never passed through a protein intermediary. Secondly, we know almost nothing about the structural features of protamines and histories. Our only clue is the finding of Astbury (1947) and of Wilkins and Randall (1953) that the X-ray pattern of nucleoprotamine is very similar to that of DNA alone. This suggests that the protein component, or at least some of it, also assumes a helical form and in view of the very open nature of our model, we suspect that protein forms a third helical chain between the pair of polynucleotide chains (see Figure 4). As yet nothing is known about the function of the protein; perhaps it controls the coiling and uncoiling and perhaps it assists in holding the single polynucleotide chains in a helical configuration. The third difficulty involves the necessity for the two complementary chains to unwind in order to serve as a template for a new chain. This is a very fundamental difficulty when the two chains are interlaced as in our model. The two main ways in which a pair of helices can be coiled together have been called plectonemic coiling and paranemic coiling. These terms have been used by cytologists to describe the coiling of chromosomes (Huskins, 1941; for a review see Manton, 1950). The type of coiling found in our model (see Figure 4) is called plectonemic. Paranemic coiling is found when two separate helices are brought to lie side by side and then pushed together so that their axes roughly coincide. Though one may start with two regular helices the process of pushing them together neces. sarily distorts them. It is impossible to have para. nemic coiling with two regular simple helices going round the same axis. This point can only be clearly grasped by studying models. There is of course no difficulty in "unwinding" a single chain of DNA coiled into a helix, since a polynucleotide chain has so many single bonds about which rotation is possible. The difficulty occurs when one has a pair of simple helices with a common axis. The difficulty is a topological one and cannot be surmounted by simple manipulation. Apart from breaking the chains there are only two sorts of ways to separate two chains coiled plectonemically. In the first, one takes hold of one end of one chain, and the other end of the other, and simply pulls in the axial direction. The two chains slip over each other, and finish up separate and end to end. It seems to us highly unlikely that this occurs in this case, and we shall not consider it further. In the second way the two chains must be directly untwisted. When this has been done they are separate and side by side. The number of turns necessary to untwist them completely is equal to the number of turns of one of the chains round the common axis. For our structure this comes to one turn every 34 A, and thus about 150 turns per million molecular weight of DNA, that is per 5000
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Cold Spring Harb Symp Quant Biol-1953-Watson-SQB ... - Biology

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