Three Experiments Which Demonstrate That The Strands of Form I DNA are Topologically Non-Linked


By Ken Biegeleisen, M.D., Ph.D.

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      The following three experiments collectively cast grave doubt upon the double-helical structure for DNA. It would be difficult to imagine the mind which, after reading this, still maintains that the structure of DNA "requires no further investigation".


Robert Chambers' Experiment


In 1978, at which time I was an M.D.-Ph.D. student in the Biochemistry Department of the New York University School of Medicine, Dr. R.W. Chambers reported to me personally the spontaneous appearance of Form I DNA in preparations of complementary single-stranded circular DNA which had been created from fX174 replicative form DNA (i.e., fX174 RF).

Dr. Chambers had known, for several years, that I believed the strands of native circular duplex DNA to be topologically non-linked ("TN"). Although he clearly did not consider the problem worth spending time thinking about, he nevertheless had a strong opinion about it: He strongly disagreed with me.

In fact, the work he was doing had the goal of proving the very opposite of what I was saying. Chambers was trying to actually create TN DNA to prove that it was NOT Form I, but something entirely different. He was working on this problem at the same time as Charles Weissmann, a former New York University School of Medicine professor who had moved to Switzerland, and was one of the founders of Genentech, one of the first commercial gene engineering companies.

They were both attempting to create what has come to be known as "Form V", an anomalous duplex structure which results from the annealing of separated complementary circles of single-stranded DNA under rather odd conditions. Weissmann published the result first. The serious deficiencies in Weissmann's work are discussed elsewhere in this web site, and I won't repeat that discussion here. The important point is that Chambers, upon seeing Weissmann's publication, ceased work on the project, and retired his own complementary circles of single-stranded DNA to the refrigerator.

Three months later, he thought of another use for them, and subjected them to analytical cesium chloride density gradient centrifugation to ascertain whether or not they were still a relatively pure population of single-stranded circles. He found, quite to his surprise, that a significant amount of Form I had inexplicably appeared in his DNA.

That's no problem if DNA is topologically non-linked (which I shall refer to as "TN"). Then, all that's happening is this:

BUT what if native circular DNA is a right-handed Watson-Crick helix, as traditional theory requires? Then in order to create Form I from single-stranded circles, three things must happen: (1) one of the circles must break open (which is entirely possible), (2) it must wrap itself around an intact complementary circle (also entirely possible), and (3) it must "spontaneously" seal itself (entirely IMPOSSIBLE!).

Here's a crude sketch of these three things:

The first two steps are non-problematical, but the third step, the sealing of a nicked strand, is exceedingly unlikely to happen without an enzyme. Since Chambers had no enzymes in his DNA, my conclusion -- the obvious conclusion -- is that circular duplex DNA is not a helix.

Because Chambers knew that I believed in the theory of TN DNA, which predicts that separated single-stranded circles can indeed come together to make Form I, he communicated to me his observation that this is exactly what happened in his test tube of complementary single-stranded circles. "Doesn't this prove that DNA is NOT a helix?", I asked him. To my astonishment, he said "No!". He then took me into a classroom and drew a "reaction mechanism" on the blackboard, wherein the three steps alluded to above were all carried out and facilitated by the "enzyme", water!!

Now, wait a minute! There's something wrong with the thermodynamics here. Sure, DNA can be nicked. Sure, single-stranded DNA can re-anneal to form a duplex structure. But how did that nick get sealed?

Every biochemistry graduate student has lived through the nightmare of waking up to find that his/her Form I DNA has spontaneously degraded into Form II. But what graduate student has ever awakened to find that his nicked Form II has magically re-sealed itself into Form I? It's never happened!

In other words, the system within which DNA spontaneously becomes nicked cannot also be the very same system within which nicked DNA spontaneously becomes re-sealed. That simply cannot happen in a closed system within which the laws of thermodynamics are operative.

Chambers refused to publish his observation because he considered it to be trivial. He believes, to this day, that the Form I arose spontaneously by the three steps alluded to above, "catalyzed" by water. He cannot accept the possibility that the Watson-Crick structure is wrong.

Since the data is unpublished, the reader may feel free to doubt it. Nevertheless, it is my duty to report to you that Robert Chambers' reputation for exacting scientific methodology was such that his colleagues gave him the nick-name "Bullet Bob". Neither Chambers nor I have any doubt that Form I appeared in his test tube of complementary single-stranded circular DNA in 1978, although, to the present day, he sticks to his own peculiar explanation for it.

I stick to mine.

You'll have to decide for yourself.


T.T. Wu's Experiment


T.T. Wu concluded, from a study of existing x-ray crystallography patterns of DNA, that DNA was more likely 4-stranded than 2-stranded. Apparently this idea was not original with him, but was a well-established interpretation which was rejected on largely genetic (vs. structural) grounds after the publication of the Watson-Crick double-helix.

For reasons which are not clear to me -- because I do not understand x-ray crystallography -- Wu concluded further that in circular DNA, the 4-stranded structure he was proposing was best constructed from a topologically non-linked model. Although perhaps an over-simplification, the Wu structure can be thought of as the TN structure proposed by Rodley1, after supertwisting.

To prove his structure, Wu isolated plasmid DNA from cells in "stationary phase", where there is ongoing RNA synthesis, but no DNA synthesis. Taking advantage of the fact that RNA:DNA hybrids are more stable than DNA:DNA hybrids on agarose gels (a peculiar fact, which is discussed below), Wu reasoned that the presence of bound m-RNA in the stationary phase imparted a significant physical difference between the two strands of plasmid DNA, and designed an ingenious experiment to separate the strands.

Rather than describe it myself, I'll present to you Wu's own description:



Richard Wu and Tai Te Wu,   A novel intact circular dsDNA supercoil, Bull Math Biol, 58, 1171-1185, 1996


      "Since the complementary DNA strands of most plasmid molecules are of about equal molecular weight and their charges are the same, it will be very difficult to separate them on agarose gel electrophoresis. However, one strand of the plasmid DNA is the sense strand and one the antisense strand. While RNA transcription is occurring, D-loops are formed, with mRNA paired with one strand of DNA. Since under agarose gel electrophoresis conditions, RNA:DNA bonds are tighter than DNA:DNA bonds (J. Casey and N. Davidson, Nucl. Acids Res. 4, 1539-1552, 1977), these RNA:DNA bonds will be maintained and promote the separation, on the gel, of the weaker DNA:DNA structure. The separated strands will then consist of a DNA strand and a DNA strand paired, over considerable lengths, with mRNA. Thus, due to the molecular weight difference or due to the different configuration in the presence of bound mRNA, the two DNA strands might separate. On the other hand, if the circular dsDNA plasmid molecules are linearized by a restriction enzyme, the two strands would be intertwined with each other so that they cannot be separated".

      "To find an ideal condition for such studies, we grew the E. coli cells which carry the plasmid molecules for one to five days before harvesting for plasmid preparation using the lysis method (J. Sambrook et al., Molecular Cloning, 2nd ed. Cold String Harbor, NY: Cold Spring Harbor Laboratory Press, 1989); those grown for three days gave the best results. Note that at three days of growth, the cells are in stationary phase. Then DNA replication-intermediate structures are not present to complicate the separation of the strands by intermediate structures of various sizes. At this stage of a culture, moreover, transcription should still be actively maintained, providing the D-loop structures within the plasmid to promote the separation of the DNA:DNA strands".

     "Most of the intact plasmid molecules can also appear as dimers, trimers, etc. Thus, they will give several bands on agarose gel electrophoresis. To avoid this difficulty, we screened various plasmids in our laboratory on 0.8% agarose (type II: medium EEO from Sigma) gel and found that pHTB4, a gift from Dr. Szaba of Cornell University Medical college, consisted of essentially monomers. It was thus used in our attempt to separate the two complementary DNA strands".

     "In order to promote separation, we increased the pH of the TAE buffer (Sambrook et al., 1989) from 8.0 to 8.5. Since the strand separation might take some time, agarose gel electrophoresis was carried out at 10 mA for 12, 24, 36 and 48 hr on a 0.7 X 11 X 14 cm agarose gel slab. The result is shown in Fig. 7."

(Fig. 7)

     "The right lanes contained the intact circular dsDNA plasmid pHTB4 molecules. At 12 hr they began to separate into two bands. The excess mRNA ran in front of these bands. At 24 hr, the separation was clear, and at 36 hr, these two bands began to diffuse. This phenomenon was characteristic of single stranded circular DNA molecules as in the case of fX174. By 48 hr, the bands were so diffuse that they were barely visible".

     "The middle lanes contained the linearized pHTB4 molecules by digestion with Pst I purchased from New England BioLabs. They remained as a single narrow band even after 48 hr".

     "The left lanes were the molecular markers of 6.0, 3.6 and 2.4 kb in sizes. Since they are linearized DNA molecules, they also remained as narrow bands and did not diffuse".

     "Thus, we encountered an interesting dilemma. Without cutting, the intact circular dsDNA plasmid molecules on agarose gel electrophoresis gave two bands. However, if these molecules were cut once, they gave only one band. Treatment with proteinase K (purchased from Sigma) did not change the pattern".


Wu then went on to describe a second experiment employing essentially the same plasmid, to which had been added a known marker sequence 17 base pairs in length. Radioactive probes were synthesized; one complementary to the sense strand of the marker sequence, and one complementary to the antisense strand. After electrophoresis, the two bands were isolated, and the Southern blotting technique was employed to demonstrate unequivocally that the bands were nearly-pure populations of the sense strand of pHTB4, and the antisense strand of pHTB4, respectively.

What does this experiment prove? It proves that circular DNA is not a helix. What other conclusion can we come to? Even if we presume that the DNA became nicked during electrophoresis, that will not begin to account for the separation of the sense and antisense strands.

This experiment was elegant in design, and precise in its execution. What was Wu's reward for doing this work? His "reward" was that his grant support was cut off, and he almost lost his job. He was saved only by the fact that he had attained tenure the year before.

Furthermore, as if an old English "Bill of Attainder" had been written, his son (coauthor of the 1996 paper) suffered also. When the younger Wu discussed the experiment with his college professors, he was treated with such contempt that he decided to never go into science as a profession.

In religious writings, the phrase often used to describe this evil is "causeless hatred", to which is attributed most of the trouble in the world.



Why are RNA:DNA bonds stronger than DNA:DNA bonds?


Wu's experiment was dependent upon the previously-demonstrated fact that on agarose gels, RNA:DNA bonds are stronger than DNA:DNA bonds. Does that strike you as odd? Why should it be so?

The answer to the question is intuitively obvious, if one simply allows ones mind to dwell upon the TN structure. The control DNA on the gels was all-right-handed "Watson-Crick" DNA, whereas the experimental plasmid was Form I, which means that it is TN. TN DNA is topologically 50% left-handed, which means that it is inherently unstable at physiological pH, relative to all-right-handed DNA.

Furthermore, the binding of m-RNA creates additional instability. In order for m-RNA to be synthesized, the parental DNA strands must be separated locally, and the m-RNA complexed with the "sense" strand. Now, when the DNA is isolated in the lab, and freed from whatever binding it had to enzymes and/or other proteins, the local separation of the DNA strands will be maintained, since m-RNA is now bound, preventing re-annealing of the DNA. The "sense" strand of DNA, no longer base-paired to the "anti-sense" strand, will function as single-stranded DNA, with many degrees of rotational freedom. Thus, it will form, with the m-RNA, an all-right-handed helix, because there's nothing to stop it from doing so.

Meanwhile, the parental Form I DNA, already destabilized by being 50% left-handed, is additionally destabilized by having one or more locally-denatured regions, wherever m-RNA is bound. Each of these constitutes an initiation point for further denaturation, lowering the activation energy for complete strand separation.



Tyler Sumner's Experiment


Tyler Sumner was a 9th grade high school student in Cartagena, Columbia, at the time he read this NotAHelix web site. He was looking for an idea for a science project, and he Emailed me. I suggested that he consider doing a "simple experiment" I had been proposing for many years, namely to attempt to split Form I DNA into its component single strands by boiling it in 1M NaCl at pH 9.95. The choice of these conditions is explained in the text which follows.

I never did the experiment myself, because I have never had a laboratory, or access to a laboratory, wherewith to do it. Furthermore, in the days when I first thought of it, the cost would have been prohibitive.

Tyler was able to complete this work for about $300-400, because of advances in technology (such as the ready availability of plasmid DNA, which, until relatively recently, had to be laboriously synthesized). However, the most extraordinary aspect of his achievement was that he was able to complete an experiment which would have been a challenge for a post-doctoral student, and that he did so without having had so much as a single high school chemistry class.

Although the experiment did not work out as planned, it produced interesting and potentially important data. Tyler, by the way, won First Prize in the high school science fair. However, when we submitted his results to Nature for publication, they were, quite predictably, rejected.

The following is the Nature article, edited only slightly for the Internet. It provides the third piece of experimental evidence that DNA is not a helix.




Electrophoretic evidence that pAMP Form I DNA can be created by reannealing of separated single-stranded circles without the breaking of any covalent bonds

Tyler Sumner and *Ken Biegeleisen

Student, Colegio Jorge Washington, Cartagena, Columbia
*Physician in private practice, 133 East 73rd Street, New York, N.Y. 10021, U.S.A.


Evidence is presented that Form I DNA may have appeared in an annealing mixture containing complementary single-stranded DNA of which approximately half was circular and half linear.

The discovery of circular DNA1 was quickly followed by the realization that there was a replication problem, since the two single strands of a Watson-Crick circular duplex ("Form I") are topologically linked, and cannot be separated without (1) the disruption of covalent bonds, (2) the total unwinding of all duplex turns and (3) the re-winding of the turns in the daughter chromosomes.

This problem was held to be solved by the presumption of a "swivel"2, a sort of enzymatic "ball joint" around which the duplex could twirl as it unwound.

The alternative, namely that DNA might have a topologically non-linked (TN) structure, has been rather poorly received in general, although the view has a number of proponents. The single most compelling reason for rejecting all TN structures out-of-hand is undoubtedly the observation that in an alkali titration of DNA3, the disruption of the ordered duplex structure at high pH does not lead to strand separation, as in linear DNA, but rather to a new structure ("Form IV") of great density, whose exact conformation, although unknown, is surely still duplex. This, of course, is entirely consistent with the traditional right-handed helical structure, where the strands are topologically linked, and would not be expected to separate under conditions promoting denaturation.

It is not widely realized that the entire alkali titration curve can be explained without invoking topological linkages. This can be done so readily (see the Supplementary Information ["SI"] file below) that we would propose that the right-handed helical structure for circular DNA remains unproven. There are two ways in which it can be tested, one "clean", and the other "crude":

The "clean" experiment consists of incubating complementary single-stranded circular DNA under renaturing conditions; not the randomly-selected conditions employed by Stettler and coworkers4 in their creation of so-called "Form V", but the narrow and precisely-defined conditions identified by Strider, Camien and Warner5,6 in their exhaustive investigation of the renaturation of Form IV, which, relative to Form I, may perhaps be viewed as being merely another form of complementary single-stranded circular DNA. Unfortunately, this "clean" experiment is a laborious undertaking, requiring a major commitment of time and effort.

The "crude" experiment consists of boiling Form I at a pH calculated to be one at which the component single strands of Form I might separate. This experiment is simple and inexpensive, but since boiling causes fragmentation of DNA, the results could conceivably be so complex as to defy interpretation.

The first author of this Letter, Mr. Tyler Sumner, is a 9th grade high school student in South America, who took it upon himself to perform the "crude" experiment mentioned above. His dedication and perseverance were remarkable. It took him three months to complete the work, and I report here his results. The materials and methods are given in the SI file.

The experiment was done with pAMP (Carolina Biological), a 4,500 base-pair Form I plasmid used throughout the world in high school electrophoresis labs. The first part of the experiment consisted of boiling pAMP in 1M NaCl, 0.1 M phosphate, 9 mM EDTA, at pH 9.95, for 1 minute. Based on Mr. Sumner's interpretation of the change in the electrophoretic mobility of the product (denatured DNA migrates more quickly in agarose gels than the parental Form I), he reported to me, by Email, that it had indeed split into its component single-stranded circles! It seemed too good to be true.

It was. When I received the photos of the gels, I saw that the boiling had indeed caused some of the DNA to migrate faster, but the band was broad, and most of it retained the electrophoretic mobility of Form I (Fig. 1, middle lane). Furthermore, there was also a clear line of demarcation between the faster and slower moving components of this band. Quite evidently, the boiling had merely nicked some of the strands of the DNA, and what the gel showed was a mixture of denatured DNA and intact Form I.


Fig. 1. Failed attempt to split Form I pAMP DNA into its component single strands by boiling for 1 minute in 1 M NaCl, 0.1 M phosphate, 3 mM EDTA, pH 9.95, as described in Materials & Methods (in Supplementary Information [SI] file). Left lane: Untreated pAMP DNA. Middle lane: The same DNA after the boiling. Note the clear line of demarcation between the majority of the DNA, which was evidently unaffected by boiling, and the minority of the DNA, which was denatured (presumably by the introduction of single-stranded nicks). Right lane: As a marker, an aliquot of pAMP DNA was subjected to alkali denaturation at pH 13 (see SI file). This converts Form I to Form IV, which is more compact, and hence migrates more rapidly.


By this time, however, Mr. Sumner had already proceeded with the second part of the experiment, which consisted of re-annealing the denatured DNA in 1M NaCl, 0.1 M phosphate, 9 mM EDTA, pH 10.75, for 20 minutes. The results, at first glance (Fig. 2, third lane to the right), were the sorts of results which can cause a scientist to throw up his hands in despair. There were no less than three bands, which, based on their electrophoretic mobilities, corresponded (top to bottom) to Form II (nicked circular duplex), Form III (linear duplex), and Form I.


Fig. 2. Reannealing of pAMP DNA which had been partially denatured by boiling. An aliquot of the same DNA shown in the middle lane of Fig. 1 was incubated for 30 minutes at 70 in 1 M NaCl, 0.1 M phosphate, 3 mM EDTA, pH 10.75, as described in Materials & Methods (see SI file). The reannealed DNA is shown in the third lane to the right. It migrated as three bands, corresponding in electrophoretic mobility (top to bottom) to Form II, Form III, and Form I. The top two bands represent two of the three possible outcomes of reannealing of a population of equal numbers of single-stranded linear and circular DNA molecules, as described in the text. Where is the third outcome, namely the product of the reannealing of a pair of intact single-stranded circles? Evidently, it is subsumed into the Form I band. Left and right lanes: These are both the same material, namely single-stranded DNA created by boiling pAMP Form I for a total of 3 minutes at pH 9.95. They are included here as a marker for denatured pAMP DNA, which is quite evidently not present in the third lane to the right. Second lane to the right: This is the same material as in the right and left lanes, after treatment with S1 nuclease (see SI file), which only degrades single-stranded DNA. This is included to establish the identity of the markers in the right and left lanes.


While in the process of preparing to discard this apparently useless data, I noticed something which I thought interesting, and which I [attempted to!] report to Nature readers.

If, as I perceive to be the case, the boiled material in the middle lane of Fig. 1 is about 1/3 denatured, then the number of nicks-per-chromosome induced by the 1 minute of boiling was clearly less than 1. Consequently, it may be deduced that very few chromosomes received more than one nick, and that therefore the ratio between the number of single-stranded linear molecules and single-stranded circular molecules in the reannealing experiment must have been almost exactly 1:1. This is illustrated in the following crude sketch:

This simply illustrates that, when conditions are such that few chromosomes receive more than a single nick, the denaturation of those nicked chromosomes gives rise to one linear single strand, and one single-stranded circle.

In other words, Mr. Sumner had inadvertently set up a mini-lab for analysis of the products of reannealing of equal numbers of linear and circular complementary DNA molecules.

This reannealing ought to have given rise to a mixture of double-stranded products at the same ratios as the products of a classical Mendelian heterozygous cross. If we call the single-stranded linear molecules "L" and the circles "C", then we should have seen the classic 1:2:1 ratio:

{LL, LC, CL and CC}, i.e. {1LL + 2LC + 1CC}

These species are illustrated in the following crude sketch:



"LL" is Form III, linear double-stranded DNA. "LC", which is electrophoretically identical to "CL", is Form II, i.e. nicked circular duplex DNA. Since the electrophoretic properties of Form II are independent of which strand is nicked, it may be stated, for our current purposes, that "LC"="CL".

This brings us to "CC". What is it? And where would it be on the gel?

The agarose gel clearly shows the 2 "LCs", i.e. the Form II, arising from the reannealing of a circle with a linear (please look back at Fig. 2, third lane to the right, uppermost band). It also shows a prominent band of "LLs", i.e. Form III linear duplex DNA, arising from the reannealing of a linear with a linear (please look back again at Fig. 2, third lane, middle band). The question is this: Where are the "CCs", i.e. the products of the reannealing of a circle with another circle?

If, as "classical" theory predicts, the circles cannot form a duplex structure without first being nicked and then re-sealed, then they should have formed a distinct band of denatured DNA, as is evident in the middle lane of Fig. 1. In Fig. 2, the right and left lanes are both denatured pAMP DNA, included as a marker. There is clearly no denatured DNA in the third lane to the right.

If, as Stettler and coworkers state4, complementary single-stranded circles have a propensity to form a non-Watson-Crick duplex, which they call Form V, we should perhaps see that as well, since Stettler describes Form V as being electrophoretically distinct from Form I. There is no obvious heterogeneity in the lower band of Mr. Sumner's re-annealed DNA.

Could it be, then, that the "CCs" reverted to native Form I, and were therefore subsumed into the Form I band? If not, then we are left with the question: Where is the product of the reannealing of complementary single-stranded circles in the third lane in Fig. 2? If it has blended into the Form I band because it is Form I, then native pAMP DNA does not have the Watson-Crick structure.

There is no point in repeating this experiment, because no one will ever be persuaded by data which arises from the boiling of DNA, with all the attendant fragmentation. The purpose of bringing forth data as inconclusive as these is to introduce an element of doubt into the minds of those who think that the Watson-Crick structure has been adequately questioned, because it has not been. It needs to be tested, by the "clean" experiment described above, namely the reannealing of complementary circles of single-stranded DNA under the conditions of Warner and co-workers5,6, not those of Stettler4.

If the current work was alleged to be a significant demonstration of topological non-helicity of DNA, I would concede that as such, it was as thin as a strand of DNA drawn out for an x-ray crystallography study. However, this is not the first such demonstration. It is the third, and by far the weakest. The other two (see SI file below, or this web site above) are, if the authors are to be taken at their word, positively thought-provoking.

Until the helicity question is taken seriously, I believe that the theory of non-helicity will persist, like a beggar stationed outside a rich man's front door, which rich man wonders to himself daily "What does that beggar want? Why doesn't he just go away?"



Note: If you arrived here by clicking on the "Experimental Results" link at the very top of the NotAHelix web site, you may wish to return there to read the complete description of the theory of TN DNA. However, that description contains much extraneous historical detail. The following "Supplementary Information" file, prepared for Nature (who did not want it), is considerably more concise. It begins immediately beneath the references below.



1. Cairns, J. The bacterial chromosome and its manner of replication as seen by autoradiography. J. Mol. Biol. 6, 208-213 (1963a).

2. Cairns, J. The chromosome of Escherichia coli. Cold Spring Harbor Symp. Quant. Biol. 28, 43-46 (1963b).

3. Rush, M.G. & Warner, R.C. Alkali denaturation of covalently closed circular duplex deoxyribonucleic acid. J. Biol. Chem. 245, 2704-2708 (1970).

4. Stettler, U.H., Weber, H, Koller, T. & Weissmann, C. Preparation and characterization of form V DNA, the duplex DNA resulting from association of complementary, circular single-stranded DNA. J. Mol. Biol. 131, 21-40 (1979).

5. Strider, W. Denatured replicative form and complex DNA of f X174: Isolation, renaturation, and sedimentation properties. Ph.D. Thesis, Department of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, N.Y. 10016, U.S.A. (1971).

6. Strider, W., Camien, M.N. & Warner, R.C. Renaturation of Denatured, Covalently Closed Circular DNA. J. Biol. Chem. 256, 7820-7829 (1981).

The authors gratefully acknowledge the assistance of Dr. Ana Verena Vargas and Jairo Pineda of Laboratorios Oceano, Cartegena, Columbia, without whose help with electrophoresis these studies would not have been possible.


Correspondence and requests for materials should be addressed to K.B.
(Click here for email).



Supplementary Information File


Electrophoretic evidence that pAMP Form I DNA can be created by reannealing of separated single-stranded circles without the breaking of any covalent bonds


By Ken Biegeleisen



Contents (click for immediate access)

Definitions and concepts
Theory of topologically non-linked (TN) DNA
Prior experimental evidence for the existence of TN DNA
Our proposed experiments to prove that DNA does have the TN structure
Materials and methods for Nature article



Definitions and concepts

Note: Every effort has been made to illustrate the topological statements made herein with drawings. Nevertheless, it is highly probable that some of the concepts presented will not be fully understood without the additional use of models made from string or rubber bands. The interested reader is strongly urged to make such models.

Form I: Duplex DNA closed into an intact circle, i.e. a circular structure with no nicks or gaps in either strand. "Traditional" theory holds this form of DNA, as found in nature, to be a "double helix". We hold otherwise, alleging it to be in the Rodley structure1, or a similar structure consisting of two parallel, "side-by-side" single-stranded circles which are topologically non-linked. See Fig. 1B.

Form II: Duplex DNA closed into a non-intact circle, with one or more nicks or gaps. Even a single nick in Form I destroys its unique properties, converting it into Form II.

Form III: Duplex linear DNA.

Form IV: The product of alkali denaturation of Form I. It is duplex, and extremely dense. Its structure is said to be unknown. It generally resists renaturation back to Form I, but renaturation can be achieved by scrupulous control of temperature, pH and ionic strength in the renaturation reaction mixture.

Superhelix: A tertiary winding in a circular DNA duplex chromosome. It is easier to draw than to describe. See Fig. 2.

Secondary helix: This is what is meant by the common household expression "the double helix". In other words, this refers to the helical winding of one single strand of DNA around its complementary strand.

Tertiary helix: This is synonymous with "superhelix". See above.

Swivel: A semi-hypothetical point in DNA where the DNA is enzymatically nicked, either in one or both strands. Like a ball-joint, the swivel is said to facilitate the rotation of the duplex about its own axis, allowing the unwinding/rewinding of secondary duplex turns during DNA replication.

TN: An abbreviation for "topologically non-linked", the structure for DNA proposed here. TN DNA may have short regions of right- and left-handed helicity, but the net number of secondary helical twists is zero, hence it can replicate without a swivel.

Rodley structure: The Rodley structure1 consists of short regions of right-handed helical DNA alternating regularly with left-handed helical DNA. The helical segments are less than 1 turn in length, so that when the chromosome is lying in a plane, neither strand ever crosses the other. See Fig. 1B. In our discussions of TN DNA, the Rodley structure is generally presumed to pertain.

R L transition: A shorthand term to indicate a rapid, cooperative unwinding of DNA and rewinding in the opposite sense. Such wholesale conversion will generally only be discussed with respect to nicked circular or linear duplex DNA (Forms II & III respectively), as it changes from the right-handed form to the left-handed form under conditions promoting denaturation.

Conversion of secondary winding into tertiary winding: For convenience, circular DNA topologists2 arbitrarily define a secondary helical turn as being a full 360, but a tertiary helical turn as only 180. The reason is that when the turns are defined thusly, there is a 1:1 numerical relationship between secondary and tertiary turns. This relationship is illustrated in Fig. 3, and explained below, as best as words can permit:

The winding of a right-handed superhelical (i.e. tertiary) turn causes either the simultaneous appearance of a right-handed helical turn in the secondary structure, or the disappearance of a left-handed helical turn from the secondary structure.

The winding of a left-handed superhelical (i.e. tertiary) turn causes either the simultaneous appearance of a left-handed helical turn in the secondary structure, or the disappearance of a right-handed helical turn from the secondary structure.

As best as is possible with drawings, this is illustrated in Fig. 3. Fig. 3A shows a "Watson-Crick" circular double helix with exactly two full turns. Remember that a secondary helical turn is that which results from wrapping one of the strands a full 360 around the other. If you look carefully at Fig. 3A, you will see that each of the strands wraps around the other exactly twice. You will also note that this drawing depicts a right-handed helix.

Now look at Fig. 3B. This is the very same chromosome depicted in Fig. 3A. It looks very different, does it not? What we have done is to add two left-handed supertwists to the structure. Keep in mind that the helix topologists define a "supertwist" as being not a 360 turn, but a turn of only 180.

What you will see in the figure, if you look carefully, is that the two left-handed tertiary supertwists have completely unwound the two right-handed secondary helical twists of the former Watson-Crick structure. Please go back now to Fig. 3A and follow it around again, and verify that either strand crosses the other two times as you go around. In Fig. 3B, however, neither strand ever crosses the other. The secondary helical twists are gone.

Topologically, the molecule is unchanged. But the Watson-Crick structure has most assuredly been unwound by the introduction of supertwists. If you cannot accept this principle from the evidence in the drawing, then please get some string, and spend 15 minutes making a string model, otherwise the arguments which follow will make no sense at all.


Theory of topologically non-linked (TN) DNA

The belief that circular DNA is a right-handed helix, and that it replicates by virtue of one or more "swivels" is based upon the presumption that certain key data, such as the alkali titration curve3 of Form I, cannot be explained otherwise.

This curve is shown in Fig. 4. Its usual explanation revolves about the presumption that native Form I DNA is a right-handed superhelix at physiological pH, a left-handed superhelix above pH 12, and a novel non-base-paired duplex (Form IV) above pH 13. Without going into unnecessary detail, let it simply be said that the starting point of the "traditional" explanation is that circular DNA is somehow "underwound" at the time of its creation, giving rise to right-handed supertwists in the native chromosome.

We agree with most of this basic formulation, disagreeing only with the explanation for the superhelicity. We shall therefore refrain from arguing against "traditional" theory, proceeding rather to a direct consideration of the alkali denaturation curve in terms of the TN theory.

DNA "prefers" the right-handed conformation at physiological pH

The major mistake made by opponents of the TN theory is the presumption that superhelicity "proves" the Watson-Crick structure. This is not true. The superhelicity of duplex circular DNA can be readily explained without the presumption of topological linkage of the strands. If DNA had the Rodley structure1(see Fig. 1B), with the Form I chromosome being topologically 50% right-handed and 50% left-handed, then it would be a right-handed superhelix. Why? Because at physiological pH's, the right-handed form of DNA is clearly preferred, yet the chromosome is constrained to be topologically 50% left-handed. It is therefore under strain. There is only one relief possible: The component of the chromosome which is right-handed can cooperatively force some of the left-handed turns to unwind into right-handed superhelical turns. This process will continue until these tertiary turns get too tight, which then introduces a new form of strain. At the equilibrium point, when the chromosome stops supertwisting, it will come to rest as a right-handed superhelix.

In Fig. 4 it can be seen that at physiological pH, Form I has a higher sedimentation coefficient than Form II, reflecting its superhelicity. Form II, which is a relaxed, open circle, sediments more slowly because it is less compact.

DNA "prefers" the left-handed conformation at higher pH

In 1970, Travers and his coworkers4 found that the optical rotatory dispersion spectrum of purified DNA inverted in aqueous methanol solutions. They proposed that this represented a transition from right-handed to left-handed helical DNA (an R L transition). Subsequent research on the circular dichroism spectrum of DNA at high salt concentration5, or following complexing6 with mitomycin C yielded similar data. In the case of certain synthetic polynucleotides, the significance of these spectral inversions, namely that they represented conversion to the left-handed helical form, were confirmed by x-ray crystallography7,8.

What do methanol, high salt and mitomycin C have in common? Chemically, they have nothing in common. But each, in its own different way, interferes with the forces which hold the right-handed double helix together. Might it therefore be that anything which significantly unwinds the right-handed form of DNA helix may bring about a transition to the left-handed form?

A rationale for this can readily be envisioned, based on the observations of Wang et al.8 on the pitch of the left-handed helical DNA fragment d(CpGpCpGpCpG), whose structure they referred to as "Z-DNA". This helix has a rise per residue of 3.7 angstroms, which is considerably larger than the 3.4 angstrom spacing between the bases of the "normal" right-handed DNA helix. With 12 residues per helical turn in Z-DNA (compared with 10 for right-handed DNA), it has a pitch of 45 angstroms (compared with 34 angstroms for right-handed DNA).

In other words, left-handed DNA appears to be a more loosely wound helix than right-handed DNA. If this is generally true of left-handed DNA, then it follows logically that any force which tends to unwind right-handed DNA might favor the formation of the left-handed helical structure.

Consequently, the TN theory states that at high pH, the left-handed form of DNA becomes favored. This is envisioned as follows: As the pH is increased above the physiological range, the DNA will eventually begin to unwind, and the bases will begin to move further apart, straining the right-handed component of the structure. When the inter-base spacing approaches the spacing found in left-handed DNA, the DNA will begin to prefer the left-handed helical conformation to the right.

Therefore, at high pH native circular duplex DNA should become superhelical in the left-handed sense, for the same reason that it was superhelical in the right-handed sense at physiological pH. In other words, at high pH, each left-handed superhelical turn removes one of the now-undesirable right-handed secondary turns.

In Fig. 4, it can be seen that in the pH range 11.5-12.0, there is a dip and then an increase in the sedimentation coefficient of Form I. We agree that this corresponds to an unwinding of the right-handed superhelical twists, followed by a re-winding of superhelical twists in the opposite, i.e. left-handed sense. We simply propose that the differences in superhelical winding result from differential stability of the right-handed vs. left-handed forms of DNA, rather than from the DNA being somehow "underwound" at the time of closure, as "traditional" Watson-Crick theory requires.

Why do the strands of Form II separate at about pH 12?

In Fig. 4, it may be seen that as the pH approaches 12, s begins to increase for both Forms I and II. For Form I this is readily explained. But why should this occur with Form II? The "classical" theory of DNA as a right-handed helix offers no explanation. But the theory of TN DNA, which incorporates the observation of the tendency of DNA to convert to the left-handed helical form under conditions tending toward denaturation, states simply that for both Forms, I and II, the DNA begins to express a "wish" to be left-handed, which translates into left-handed superhelix formation (since each left-handed superhelical turn unwinds a right-handed secondary helical turn).

At a pH just above 12, Form II denatures into single-stranded circular and single-stranded linear half-chromosomes (Fig. 4). Why? Form I, whether or not one presumes it to be a topologically-linked structure, is known to remain base-paired up to pH 12.310, therefore it cannot be said that Form II denatures because base-pairing is no longer possible at pH 12. Base-pairing, albeit weakened, clearly does exist at pH 12. Why, then, does Form II denature, and not Form I? Evidently, it must be because Form II undergoes an R L transition, whose rapid spinning and resultant centrifugal force drive the strands apart. Base-pairing, although still weakly present, is not sufficient to overcome this centrifugal force.

Why don't the strands of Form I separate at pH 12?

Form II denatures at about pH 12.0, but Form I does not irreversibly denature until pH 12.3. This has been painstakingly established R. Warner and his coworkers9,10. Can we account for these 0.3 pH units of incremental stability? Yes we can.

First of all, it is generally presumed that DNA denaturation, a cooperative process, begins at a free end. Form I has no free end. A crude estimate of the resultant protection against denaturation can be made by the simple "thought experiment" shown in Fig. 5, which reveals graphically that it will take about twice the disruptive force to initiate strand separation in a duplex with no free end. If we presume the disruptive force to be the hydroxide ion concentration, then a doubling thereof will correspond to a pH increase of about 0.3.

Why, at pH 12.3, do the strands of Form I not separate from the centrifugal force of an R L transition, as is evidently seen with Form II?

The answer lies in a consideration of the peculiar topological properties of a Form I TN duplex, as compared with Form II. In Form II the entire helix can at least attempt to undergo a rapid, cooperative R L transition, going from the all-right-handed to the all-left-handed conformation (and perhaps being disrupted in the process). This is possible because of the one or more nicks in Form II, each of which act as a "swivel", around which helical turns can be unwound/rewound. That is impossible with Form I. In a Form I TN duplex, the unwinding of a right-handed secondary turn inevitably must be accompanied by the unwinding of a left-handed secondary turn. This may be immediately appreciated by twisting a pair of rubber bands, wherein it may be seen that there is a mandatory relationship between the winding of right- and left-handed secondary turns, and of the unwinding of same.

We have surmised that at pH's above 12, DNA is "trying" to become left-handed. If the DNA has the TN structure, then it clearly cannot do that at all -- least of all quickly -- since in a Form I TN chromosome, every right-handed turn which is unwound must be accompanied by the unwinding of a left-handed turn! Conversion to the left-handed form, especially the rapid R L transition seen with Form II at pH 12, is impossible altogether in Form I.

But, Form I can become a left-handed superhelix, which it does become. Every left-handed superhelical turn which is wound, causes the unwinding of a left-handed secondary turn. Thus, the only conformational "relief" for Form I at pH's above 12 is to become an ever-more-tightly-wound left-handed superhelix.

By pH 12.3, the superhelix density of Form I has increased about 100% relative to Form I at physiological pH (Fig. 4). That is, the difference in sedimentation coefficient between Form I and Form II, which may be taken as a measure of superhelicity, is doubled at pH 12.3. This tight superhelical winding is a condition which is not seen in any other commonly-occurring form of DNA. If, therefore, this DNA behaves in ways which are not commonly seen, it perhaps ought not be surprising.

Form IV has the structure originally proposed by Linus Pauling

In 1953, Linus Pauling, considered by many to be the world's greatest chemist at that time, proposed a multi-stranded structure for DNA11. The structure was developed without regard to genetic considerations, because the principle of specific base-pairing was not yet known. Based only upon the principles of chemistry, Pauling concluded that the most likely structure for DNA was a multi-stranded helix with the phosphate groups pointing inward. He stated that this structure was most readily created with four strands, although he opted for a three-stranded structure in his publication, because he thought that it could be better made to conform to the existing x-ray diffraction data.

It might be noted, at this point, that DNA helices with the phosphate groups pointing inward have been discovered in nature12,13.

We note again that at pH 12.3, the superhelix density of Form I is twice as great as at physiological pH (Fig. 4). This is illustrated schematically in Fig. 6, which shows the superhelix getting tighter and tighter, which will cause water to be progressively squeezed out of the core. As the drawing shows, the final result of this process will be the unusually close juxtaposition of four DNA strands, all twisted together into a four-stranded helix. If, as Pauling & Corey proposed11, DNA has a predilection to form multi-strand helical structures with the phosphate groups pointing inward, interacting through salt bridges, then why wouldn't such structures form under these circumstances? Other than through the activity of some hypothetical enzyme, it would be difficult to imagine a process whereby four DNA strands could be effectively lined up in a four-stranded helical conformation more efficiently than the process which actually takes place in Form I at pH 12.3.

Therefore, it is hereby proposed that Form IV, a dense form of DNA which resists renaturation, is a Pauling four-stranded helix with the phosphate groups pointing inward. There is no reason for this structure not to form at pH 12.3, because (a) it is not at all dependent upon hydrogen bonding, and (b) the DNA -- if Pauling's chemistry is to be believed -- would simply be obeying the laws of chemistry by doing so.


Prior experimental evidence for the existence of TN DNA

1. Insufficiency of previous experimental evidence alleged to "prove" that Form I is an all-right-handed double helix


The single strongest challenge to the theory of TN DNA was the article published by Crick, Wang & Bauer in 197814, wherein it was alleged that the behavior of circular DNA in topoisomerase experiments "proved" that the Watson-Crick structure of DNA was correct.

In these sorts of experiments, DNA is treated with a nicking-closing enzyme called topoisomerase, after which a set of so-called "topoisomers" of DNA appears on agarose gels. These are bands of DNA populations which differ from each other by unity in the "linking number", or the net number of times one strand winds around the other.

It is (or ought to be) self-evident that once the topoisomerase has nicked native Form I DNA, the set of topoisomers which appears subsequently tells us nothing whatsoever about the original native structure. All we learn from these agarose gels is that the native Form I DNA moves faster than the topoisomer products, because it is more highly supertwisted, and hence more dense.

Therefore, all Crick et al have really demonstrated about the structure of native circular DNA is that it is a superhelix (which was already known before the topoisomerase experiment was ever done). But we explained above that superhelicity in Form I can be readily accounted for without resorting to the Watson-Crick structure, or to topological linkage of any sort whatsoever. Therefore Crick is not correct in alleging that the topoisomerase experiment "proves" the Watson-Crick structure and "rules out" the TN (e.g. Rodley) structure. In fact, upon reflection, it doesn't even address the question.

In the more distant past, it has been alleged that the electronic microscopic appearance of replicative intermediates of circular DNA15,16 "proves" the Watson-Crick structure. But again, these intermediates only display traits which demonstrate partial superhelicity in the daughter chromosomes, which is alleged to somehow "prove" that DNA is a helix. Since TN DNA is predicted to be superhelical, its replicative intermediates are also predicted to be partially superhelical.

Like the topoisomerase experiment, therefore, a consideration of the electron microscopic appearance of replicative intermediates of circular DNA doesn't even address the question of the topological status of Form I, much less prove anything.

There is, in fact, existing experimental evidence that circular Form I DNA does not have a topologically linked structure.


2. Previous experimental evidence that Form I is topologically non-linked

Observation #1


In 1978, Robert W. Chambers (personal communication) observed the spontaneous appearance of Form I DNA from complementary single-stranded circles of f X174 RF DNA. He was, at that time, the chairman of the Biochemistry Department at New York University School of Medicine, and he was "racing" Charles Weissmann to be the first to create "Form V", a duplex structure arising from the reannealing of separated complementary circles of single-stranded DNA. Weissmann won this "race"17, and Chambers put his complementary circles into storage in the refrigerator.

Three months later, he thought of another use for them, and subjected them to analytical cesium chloride density gradient centrifugation to ascertain whether or not they were still a relatively pure population of single-stranded circles. He found, quite to his surprise, that a significant amount of Form I had inexplicably appeared in his DNA.

Chambers communicated this observation to me, but he refused to publish it because he considered it to be trivial. He believed that the Form I arose from three spontaneous processes: (1) a single-stranded circle was nicked, (2) it wrapped itself around an intact circular complementary single strand, and (3) the resultant Form II chromosome was spontaneously re-sealed.

The "enzyme" catalyzing all this spontaneous activity, said Chambers, was water!

This explanation makes no thermodynamic sense. The system within which DNA becomes spontaneously nicked cannot also be a system within which the self-same DNA becomes spontaneously re-sealed. To put this differently, there are not, on this earth, any graduate students in the DNA sciences who have not experienced the nightmare of having their Form I spontaneously degrade into Form II upon storage. But how many graduate students have seen their Form II spontaneously "degrade" into Form I? It's never happened!

One must therefore conclude that either (1) Chambers was incorrect in reporting the appearance of Form I in his analytical ultracentrifugations, or, alternatively, that (2) Form I DNA is not a helix. I believe that the latter explanation is the correct one.

Robert Chambers, whose exacting scientific method led to his being nick-named "Bullet Bob" by his peers, stands by his 1978 observation to the present day, although he also sticks to his own explanation for it.

Observation #2

Tai Te Wu18 proposed, in 1969, that the x-ray diffraction pattern of DNA was, in some respects, more compatible with a 4-stranded structure than with a 2-stranded structure. In this 4-stranded structure, the 3.4 angstrom inter-base spacing was attained through stacking of bases from adjacent strands of DNA, each of which strands had an inter-base spacing twice that great. This concept was not original with Wu, but was a known possible interpretation of x-ray studies of DNA going back to the 1950's. Whereas most authors mentioned this interpretation as merely a possible structure of DNA, Wu considered it to be a probable structure.

In applying this concept to circular DNA, Wu found it expedient to propose that circular DNA is a topologically non-linked four-stranded helix. Although perhaps an over-simplification, it may be considered, as a first approximation, that the Wu structure is created by taking a circularized version of the double-stranded Rodley1 structure and supertwisting it.

To test this theory, Wu19 isolated plasmid DNA from confluent cultures of E. coli in stationary phase, where DNA replication had ceased but transcription to RNA was ongoing. Since duplex DNA has a "sense" strand which is transcribed, and an "antisense" strand which is not, it follows that in the stationary phase, there is a large physical difference between the two complementary strands of duplex DNA due to the presence/absence of bound m-RNA.

It is apparently known that, on agarose gels, the DNA:RNA bond for Form I is stronger than the DNA:DNA bond. Wu proposed to take advantage of these facts to separate the two strands of Form I plasmid DNA extracted from stationary phase E. coli cells.

The method he employed to effect the separation was prolonged electrophoresis. After 12 hours, the plasmid DNA began to split into two distinct bands. This split was complete at 36 hours. When the experiment was repeated with plasmids which had known genetic markers inserted, it was possible to show, by DNA hybridization, that the two bands at 36 hours were indeed the "sense" and "antisense" strands of the original duplex, respectively.

This demonstration would not be possible if the strands of plasmid DNA were topologically linked, as in the Watson-Crick structure. There, the separation of the strands would require breakage of one or both of them, something not expected to occur spontaneously during agarose gel electrophoresis.


Our two proposed experiments to prove that DNA does have the TN structure

Proposal #1: the definitive experiment


It is believed by many that Stettler et al17 ruled out the possibility of creating Form I from complementary single-stranded circular DNA. This is very far from the truth. Stettler et al -- whose conditions for incubating single-stranded DNA seem almost to have been arbitrarily and capriciously chosen -- should have considered the unique requirements for renaturation of Form IV before designing their experiment.

If Form I DNA has the TN structure, meaning that the single-stranded circles which comprise it are not bound together by topological linkages, then neither are the single stranded circles which comprise Form IV. Therefore, for some purposes it might prove expedient to view Form IV as being merely another form of separated circles of complementary single-stranded DNA. The requirements for renaturation of Form IV may therefore be a clue as to the requirements for the renaturation of separated circles of complementary single-stranded DNA floating freely in solution.

Form IV is difficult to renature into Form I, because the Form IV structure, once assumed by DNA, is stable under nearly all conditions of pH, temperature, and ionic strength (see, for example, the dashed line in Fig. 4). If Form IV were to turn out to have the TN structure, then it may also turn out that separated circles of single-stranded complementary DNA floating freely in solution also resist renaturation to Form I under most circumstances.

I would conceive of this resistance as being due to a predilection of separated single strands to form an anomalous structure, i.e. "Form V", which, like its Form IV "cousin", would form readily, but be converted to Form I only with great difficulty.

At the time it was first observed, Form IV DNA used to be referred to as "irreversibly denatured", but this was soon found by Pouwels and coworkers to not be the case20. The full elucidation of the remarkably narrow set of circumstances under which renaturation of Form IV occurs was studied in detail by Robert Warner and his coworkers9,10, who showed that only under very precisely defined and narrow conditions of pH, temperature, and ionic strength, could Form IV be made to rapidly reanneal into Form I. Some of their data is reproduced in Fig. 7, which shows the percent renaturation of Form IV at various temperatures and pHs. You will note that if the pH deviates from the optimum for any temperature by as little as 0.5 pH units, then the percent renaturation drops precipitously, from virtually 100% to about 20%. An additional change of 0.5 pH units would all but eliminate renaturation.

Fig. 8 shows Strider and Warner's pH optima plotted on a single graph. Please note the "+" symbol in the lower part of this figure. These were the conditions Stettler et al17 employed for the creation of Form V, which is distinctly different from Form I. But note that their pH is nearly 2.5 units from the Strider/Warner optimum for renaturation of Form IV at that temperature. Incubation of Form IV under Stettler's conditions would have yielded no Form I! Perhaps whatever prevents Form IV from renaturing to Form I under those conditions also prevents separated single-stranded complementary DNA in solution from renaturing to Form I.

We therefore propose that Form V, like Form IV, is an anomalous form of duplex DNA which is entirely non-base-paired. That this is the case is evidenced by Stettler's own data, which show that Form V, in spite of their claim that it is about 50% "Watson-Crick helix", has a thermal denaturation profile entirely devoid of cooperativity17. Furthermore, their experiment is essentially uncontrolled. There is no data showing that non-complementary DNA of the same species they used to create Form V, in the same solvent and under the same conditions of pH, temperature and ionic strength, wouldn't also have annealed anomalously to give Form V.

The definitive experiment we propose to test the TN theory is to incubate complementary single-stranded circles of DNA under any of the Strider/Warner9,10 conditions of optimal renaturation of Form IV (Fig. 7). We predict that this will result in the rapid and complete conversion of the single-stranded circles to duplex Form I, indistinguishable from native Form I DNA.

This experiment requires the preparation of complementary single-stranded circular DNA, which is a laborious undertaking. Yet, until this is done, and the work of Stettler repeated under the conditions of Strider/Warner, the question will remain unanswered.


Proposal #2: the crude experiment

A simple method of testing the TN theory would be to find conditions where Form I could be split into its component single strands. This might not be so easy, since efforts to denature it cause it to become a tightly wound superhelix, which then transforms itself into "irreversibly denatured" Form IV, a still-duplex structure which is stable under nearly all conditions of pH, temperature and ionic strength.

Some years back I deduced that the denaturation of Form I into single-stranded circles could be accomplished by extrapolating the data of Strider/Warner to the temperature of boiling water. The rationale was that under the conditions of the Strider/Warner optima, circular duplex DNA was in an intermediate state between Form I and Form IV. This intermediate state had to be one in which the complementary single-stranded circles which comprise Form IV could rotate freely with respect to one another, so that the complementary base pairs could once again "find" one another, allowing Form IV -- a non-base-paired structure -- to renature into Form I, a specifically base-paired structure.

I deduced that the single strands comprising duplex circular DNA, when in this intermediate state, not being locked together into a rigid conformation, might be induced to separate if the optima were extrapolated to the temperature of boiling water. This deduction, of course, is based upon the behavior of common sorts of linear duplex DNA, which denature when boiled. It is also based upon the presumption that the Strider/Warner data can be accurately extrapolated from the 5 optimum points they provided.

Based upon these 5 data points, from W. Strider's Ph.D. thesis9, I deduced that the pH at which to boil DNA, to effect a separation of its component single strands, would be about 9.95 (see Fig. 8).

As reported in this issue of Nature, this prediction was not borne out. Yet the hypothesis may not be entirely wrong. In the process of preparing the protocol for this experiment, I learned, from Dr. Warner, that about 10 years after the publication of W. Strider's Ph.D. thesis, there had been a subsequent publication on renaturation of Form IV10. This publication essentially confirmed all of Strider's findings, but added two additional data points to the plot of pH optima (Fig. 9). A consideration of these new points suggests that the plot of pH optima for renaturation of Form IV is not a straight line, as suggested by Strider's earlier data9. Thus, the pH at which Mr. Tyler Sumner, the first author of our present Nature manuscript, boiled his DNA, could have been off by an entire pH unit or more.

Nevertheless, we have reported these data because of the interesting finding that in the reannealing portion of Mr. Sumner's high school science project, there was no evidence of a final product resulting from the reannealing of complementary circular single-stranded DNA (see Nature article, Fig. 2). The reannealing of a circular single strand with a linear single strand gave a Form II band on gel electrophoresis, and the reannealing of two linear single strands gave a Form III band. This leaves the product of reannealing of a pair of circles unaccounted for. They definitely did not remain single-stranded, since a band of denatured DNA would have been evident. Could they have turned into "Form V"? It's possible, but Stettler et al17 state that Form V is electrophoretically distinct from Form I. There is no electrophoretically distinct Form V band in the gel.

If the reannealing of pairs of single-stranded circles gave rise to Form I, as Chambers observed to occur spontaneously on storage of single-stranded circles in the refrigerator, then that will explain why there is no distinct band resulting from the reannealing of pairs of single-stranded circles. They are mixed in with the Form I band because they are Form I.

If so, then pAMP DNA is not topologically helical, since the individual strands of duplex circular DNA -- with any linking number whatsoever, other than zero -- are topologically linked, and such linkage cannot occur unless one of the strands is nicked, then wrapped around the other, then re-sealed. Without the action of some sort of enzyme system, such a sequence of events is unimaginable.

The purpose of the present publication is to impress molecular biologists with the fact that the "double helix" is not proven for circular DNA. Since human DNA may prove to be circular, the problem is entirely non-trivial. Hopefully, someone, somewhere, will read this report and find a way to create complementary single-stranded circles of plasmid or viral DNA, and incubate them under the conditions prescribed by Warner and his co-workers for the reannealing of Form IV (see Figs. 7, 8 and 9). This is the definitive experiment which needs to be done.



Sodium chloride, sodium phosphate dibasic heptahydrate FW 268.07, pH 4 buffer solution, pH 10 buffer solution, sodium hydroxide and hydrochloric acid (for adjusting pH), and EDTA (disodium salt dihydrate, FW 372.24), were all "ACS" grade or better, from Fisher Scientific.

pAMP DNA, pipettes, micropipets, and Micro Reaction Tubes (0.5 cc, for storing DNA) were from Carolina Biological.

S1 Nuclease was from Promega. S1 is supplied at a concentration of 100 units/microliter. It is accompanied by a "10X reaction buffer" which consists of 500 mM sodium acetate pH 4.5, 2.8 M NaCl and 45 mM ZnSO4. This stabilizes the pH in a range which is optimal for S1, and provides zinc, which is necessary for the enzyme to function. In the experiment employing S1, 0.4 mg of (presumably) single-stranded pAMP DNA, in 10 ml of 1 M NaCl, 0.1 M phosphate, 3 mM EDTA, pH 9.95, was mixed with an equal volume of 0.1 M sodium phosphate, pH 1.35, to get the pH to about 4.5 (close to the optimum for S1 nuclease). Two ml of "10X reaction buffer", and 1 ml of S1 nuclease (100 units) were added. The mixture was incubated at 37 for 60 minutes.

All glassware was washed and rinsed thoroughly with distilled water, then heated to 160 C in a dry oven for 1 hour.

pH meters were calibrated with two buffers before every session. All pH determinations were made at room temperature. In experiments done at elevated temperatures, the pH's reported are those determined at room temperature before the experiment began.

All electrophoresis was done in 1.2 % agarose gels, at 65 volts. Gels were stained with ethidium bromide.


Forty mg of pAMP was diluted to 0.5 ml by addition of 1 M NaCl, 2mM EDTA, pH 7.4, and dialyzed overnight against the same buffer, for a final concentration of about 80 mg/ml.

Using a disposable micropipette tip, 200 ml of DNA was transferred to a clean plastic tube. To adjust the pH to 9.95, 200 ml of 1 M NaCl, 0.2 M phosphate, 4 mM EDTA, pH 9.95, was added (it was verified in advance that the pH of the final solution was indeed determined by the pH of the added buffer).

This DNA, now at pH 9.95, and in the solvent employed by Strider and Warner9,10 for all their studies (see Fig. 7), was boiled for one minute, then immediately cooled in an ice bath.

To further establish the Form I nature of the original pAMP DNA, and to provide an additional marker for gel electrophoresis, 200 ml of pAMP was transferred to another tube, and 200 ml of 1 M NaCl, 0.2 M phosphate, 4 mM EDTA, pH 13, was added. Alkali denaturation should convert it to Form IV, which should migrate more rapidly in agarose gels due to its great density.

Electrophoresis of 10 ml of DNA from each tube was performed. See the Nature article, Fig. 1. The left-hand lane was the untreated DNA (0.8 mg). The middle lane was DNA which had been boiled at pH 9.95 (0.4 mg). The right-hand lane was pAMP denatured by alkali.

From the pAMP DNA which had been boiled at pH 9.95, an aliquot of 300 ml was dialyzed overnight against 1 M NaCl, 0.1 M phosphate, 3 mM EDTA, pH 10.75. The next day, it was transferred to a covered plastic tube and heated in a dry oven at 70 C for 30 minutes. These conditions (i.e. 1 M NaCl, 0.1 M phosphate, 3 mM EDTA, pH 10.75, 70 C) represent one of Strider & Warner's9,10 optima for renaturation of Form IV (Fig. 7).

Electrophoresis was performed (Nature article, Fig. 2). The left and right lanes were markers for denatured DNA. Both contained the same material: pAMP DNA (0.4 mg), which had been boiled at pH 9.95 for a total of 3 minutes in three separate experiments (Fig. 1 of the Nature article shows the first such experiment, the other two are not reported here). The DNA was, at that point, essentially all single-stranded.

The second lane to the right was 0.2 mg of the same material after treatment with S1 nuclease, as described above. This was done to prove that the DNA markers in the right and left lanes were indeed single-stranded. The astute observer will note faint bands of Form II and Form III in this lane, which evidently formed during the 60 minutes of incubation at 37. An examination of these two bands reveals that the boiling had introduced very close to 2 nicks per chromosome, so that the great majority of DNA was now single-stranded linears, with very few intact single-stranded circles. Therefore, the product of reannealing of two single-stranded circles -- which one might reasonably inquire about since Forms II and III are present -- was probably too scarce to be detected on these gels.

The third lane to the right -- the experimental lane -- was 0.4 mg of the pAMP DNA which had been boiled at pH 9.95, then reannealed at pH 10.75, as described above. It shows strong bands corresponding, in electrophoretic mobility, to Forms II & III (top and middle band). The lower band is Form I, plus or minus any products of reannealing of complementary single-stranded circular DNA (which, if present, has had no evident effect on the homogeneity of the band).






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8. Wang, A.H.J. et al. Molecular structure of a left-handed double helical DNA fragment at atomic resolution. Nature 282, 680-686 (1979).

9. Strider, W. Denatured replicative form and complex DNA of f X174: Isolation, renaturation, and sedimentation properties. Ph.D. Thesis, Department of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, N.Y. 10016, U.S.A. (1971).

10. Strider, W., Camien, M.N. & Warner, R.C. Renaturation of Denatured, Covalently Closed Circular DNA. J. Biol. Chem. 256, 7820-7829 (1981).

11. Pauling L. & Corey, R.B. A proposed structure for the nucleic acids. Proc. Natl. Acad. Sci. USA. 39, 84-97 (1953).

12. Day, L.A., Wiseman, R.L. & Marzec, C.J. Structure models for DNA in filamentous viruses with phosphates near the center. Nuc Acids Res 7(6), 1393-1403 (1979).

13. Liu, D.J. & Day L.A. Pf1 virus structure: helical coat protein and DNA with paraxial phosphates. Science 265, 671-674 (1994).

14. Crick, F.H.C.; Wang, J.C. & Bauer W.R. Is DNA really a double helix? J. Mol. Biol. 129, 449-461 (1979).

15. Jaenisch, R., Mayer, A. & Levine, A. Replicating SV40 molecules containing closed circular template DNA strands. Nature New Biol. 233, 72-75 (1971).

16. Sebring, E.D., Kelly Jr., T.J., Thoren, M.M. & Salzman, N.P. Structure of replicating Simian Virus 40 deoxyribonucleic acid molecules. J. Virol. 8, 478-490 (1971).

17. Stettler, U.H., Weber, H, Koller, T. & Weissmann, C. Preparation and characterization of form V DNA, the duplex DNA resulting from association of complementary, circular single-stranded DNA. J. Mol. Biol. 131, 21-40 (1979).

18. Wu, T.T. Secondary structures of DNA. Proc. Natl. Acad. Sci. 63, 400-405 (1969).

19. Wu, R. & Wu, T.T. A novel intact circular dsDNA supercoil. Bull. Math. Biol. 58, 1171-1185 (1996).

20. Pouwels, P.H., Van Rotterdam, J. & Cohen, J.A. Structure of the replicative form of bacteriophage fX174. VII. Renaturation of denatured double-stranded f X DNA. J. Mol. Biol. 40, 379-390 (1969).



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Figure 1. Two models of topologically non-linked (TN) DNA.
A. In this rather unlikely model, there are 3 right-handed helical turns in the upper half of the chromosome, and 3 left-handed helical turns in the lower half. The net number of helical twists is therefore zero.
B. A more likely structure for TN DNA is the Rodley structure1, depicted schematically here. This structure consists of short alternating regions of right-handed and left-handed helicity, each such helical region being less than 1 helical turn in length. Therefore, when the chromosome is constrained to lie in a plane, neither strand ever crosses the other. During replication, the strands can separate without any disruption of covalent bonds.








Figure 2. Secondary and tertiary structure in circular DNA.
A. Standard "Watson-Crick" all-right-handed circular helix. It is depicted as lying flat in a plane, which would not be the case in real life.
B. This is the actual state of circular duplex DNA, which arises from the twisting in of a higher-order, or tertiary winding, often referred to as a superhelix.








Figure 3. Relationship between right-handed secondary helical turns and left-handed tertiary superhelical turns.
A. "Watson-Crick" Form I chromosome with exactly two secondary helical turns.
B. The same chromosome as shown in Fig. 3A, after the winding of two left-handed superhelical turns. Although the strands obviously cross each other in three-dimensional space, it may be readily appreciated that there are no crossings in the secondary structure, which now consists of two strands lying entirely in parallel.







Figure 4.  Alkali titration of Form I DNA. The y-axis is the sedimentation coefficient (s) of the DNA in cesium chloride velocity gradients. Data are from Rush & Warner3, who utilized fx174 RF. Similar data have been obtained with DNA of other species, and the characteristics of these curves appear to be generally applicable to all, or most Form I DNA. Form I exhibits a dip in s at about pH 11.8, then a marked increase characterized by a shoulder (c) at pH 12.3 and a plateau at pH 13. If the DNA at the plateau is neutralized, it is not converted back to Form I, but remains in the new dense structure (dashed line, "Form IV"). Form II, i.e. circular DNA relaxed by the introduction of at least one nick, shows a straight line up to pH 11.8, where it mysteriously begins to increase in density along with Form I. At pH 12, it separates into single-stranded circles and linears.

At physiological pHs, Form I has a higher s value than Form II, because Form I is superhelical, and hence is denser than Form II. At pH 12.3, which has been precisely determined10 to be the point of irreversible denaturation to Form IV, Form I has twice the s value, relative to Form II, that it does at physiological pH. It is therefore very highly superhelically twisted; much more so than any DNA in the native state. If, therefore, it were to assume a conformation not seen in nature, it should perhaps not be surprising.








Figure 5. "Thought experiment" to estimate the magnitude of protection against alkali denaturation of Form I due to the absence of a free end. As the drawing shows, if the force necessary to initiate denaturation on either side of a single-stranded nick is represented as being a weight of "100 lbs" (upper drawing), then clearly the force necessary to initiate strand separation in the absence of a single-stranded nick will be the total, or "200 lbs" (lower drawing).








Figure 6.   Steps in the formation of a Pauling 4-stranded helix with the phosphate groups pointing inward.
A. Form I at pH 11.8, in the relaxed, open circular conformation (see Fig. 4).
B. Form I at pH 12.0, beginning to show superhelicity in the left-handed sense.
C. Form I at pH 12.3, having become highly superhelical in the left-handed sense. D. Artist's representation of a point in the interior of a Pauling 4-stranded helix, with phosphate groups interacting through a salt bridge.







Figure 7.   Renaturation of "irreversibly" denatured Form I DNA.   Form I was denatured into Form IV by high pH treatment. Aliquots of denatured DNA in 1M NaCl, 0.1 M phosphate, 3 mM EDTA were incubated at the temperatures and pH's shown. At any given temperature, there was an optimum pH for renaturation. At these optimal conditions, renaturation was rapid and complete. Straying from these pH optima by as little as 0.5 pH units caused a drastic reduction in the renaturation rate.







Figure 8.   The pH optima from Fig. 7 plotted on a single graph. The data appear here to be linear (but see Fig. 9). If the presumption of linearity were correct, then the pH for optimum renaturation at 100 would be about 9.95.

The "+" at the bottom represents the seemingly randomly-selected conditions employed by Stettler et al17 in their creation of "Form V" through renaturation of complementary single-stranded circular DNA. Note how far they are from the pH-temperature optima for renaturation of Form IV.







Figure 9.   Additional renaturation optima for Form IV, from Warner's last publication on the subject10. The data are now seen to be non-linear, and the linear extrapolation shown in Fig. 8, to determine the pH of optimal renaturation of Form IV in boiling water, is seen to be off by as much as a pH unit or more.




















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