In order to apply the Wu structure to superhelical circular DNA duplex chromosomes, all four strands must be twisted into a right-handed helix, as shown to the right.
Tai Te Wu
Separation of the strands of intact duplex circular DNA
Tai Te Wu is a professor of Biochemistry, Molecular Biology, and Cellular Biology, and also of Biomedical Engineering, at Northwestern University. He studied medicine at the University of Hong Kong, mechanical engineering at the University of Illinois in Urbana, and applied sciences at Harvard University.
When the helical structure of purified, protein-free “crystals” of DNA was first deduced from x-ray crystallography, it was not clear whether it was a two-stranded or four-stranded structure. Wu noticed that the x-ray diffraction patterns obtained for DNA were different under two different conditions:
Employing a characteristic mathematical property of Bessel functions, Wu deduced that there were two different DNA secondary structures corresponding to the two different diffraction patterns observed. In 1969, Wu1 proposed that at 66% relative humidity DNA fibers were in the classical double helical configuration, while at 92% relative humidity they were four-stranded. The four-stranded structure was proposed to consist of two mutually intercalating stretched out double helices.
This was not considered a radical proposal in 1969, although in the process of time, and in the process of what some might call “progress”, it has apparently become so.
Wu’s four-stranded structure can be thought of as consisting of two loosely-wound Watson-Crick-type helices twisted together, with the base-pairs mutually intercalated. Prior to the discovery of circular DNA, the proposal met with academic interest only. After the discovery of circular DNA, however, the Wu model suddenly became a possible structure for DNA in the real world, since superhelically-wound circular DNA is, in effect, a four-stranded structure consisting of two Watson-Crick-type duplexes wound together.
To the right is a hypothetical picture of a chromosome bearing this structure, only with all the twists removed, to illustrate its basic principles. The picture can be difficult to understand at first. The elongated circles with the arrows represent the two circular, single-stranded anti-parallel sugar-phosphate backbones. The parallelograms are the hydrogen-bonded base-pairs. Those in "front" are arbitrarily colored white; those in the "back" are shaded to make the diagram easier to grasp (there are no physical differences between the base-pairs in the "front" and "back"). Note that the inter-base distance (after supertwisting -- not shown here) is 6.8 angstroms, twice that found in the traditional Watson-Crick model. After the base pairs in the “front” and “back” of the picture are mutually intercalated, however, the final inter-base-pair distance becomes 3.4 angstroms, as in the “traditional” model.
In order to apply the Wu structure to superhelical circular DNA duplex chromosomes, all four strands must be twisted into a right-handed helix, as shown to the right.
No detailed molecular model for the Wu structure has been published.
The Wu model, insofar as we have described it thus far, would decrease the magnitude of the "unwinding problem" during DNA replication within living cells at least ten-fold, but would not eliminate it entirely. Wu, however, goes farther, stating that the change he proposes for DNA structure, as the humidity increases, can be extrapolated from 66% (W-C structure), beyond 92% (Wu 4-stranded helix), all the way to 100% -- the "humidity" inside living cells, i.e., the state of being completely submerged in aqueous solution. At 100% humidity, Wu states, the structure of DNA will have no twists at all! In his words:
"The DNA molecule can thus be visualized as two base-paired strands in the form of a straight ladder held to another ladder by mutual intercalation."
He proposes the following distances between nearest neighboring base-pairs on the same strand:
Thus, insofar as the Wu structure can be applied to living systems, DNA can exist with no twists at all, and the unwinding problem can be dismissed entirely. Then the drawing above, depicting two straight, untwisted "ladders", becomes a fairly literal depiction of DNA structure, except that the 6.8 angstrom interbase distance in the drawing would become 7.1 angstroms.
Experimental Evidence
Wu and his son went beyond mere speculation, and designed an ingenious experiment2 to prove his theory. If circular DNA has no topological twists, then it ought to be possible to separate its single strands intact, without rupture of covalent bonds. The Wu's reasoned that this would be possible if the DNA was examined by gel electrophoresis under conditions where the strands could be induced to have different electrophoretic mobilities.
Toward this end, they grew E. coli cells to stationary phase, in which a monolayer of cells covers the entire growth surface, whereupon DNA synthesis, for the most part, ceases. Transcription of RNA, however, continues unabated. At any given time, a considerable amount of m-RNA will be bound to the “sense” strand of the chromosome, but none will be bound to the complementary “anti-sense” strand, since transcription occurs on one strand only. It is known that DNA:RNA bonds are stronger than DNA:DNA bonds during agarose gel electrophoresis, and the “sense” and “anti-sense” strands therefore do not have the same structure under these experimental conditions. One (the “sense” strand) has much bound m-RNA, and the other has none.
Wu isolated two different species of plasmid DNA, from cells grown to stationary phase. He subjected the DNA to electrophoresis at low voltage, so that it took about 2 days for the DNA to reach the end of the gel. After the first 24 hours, the DNA was clearly beginning to split into two bands. After 36 hours the separation was complete. Wu’s own words, and electrophoresis results, are shown below:
From:
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"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 repeated the experiment with plasmid DNA into which had been placed marker sequences, and the identities of the two bands was confirmed beyond a doubt. But could they have arisen by strand breakage during the electrophoresis?
Not likely. Wu ran a control; plasmid DNA which had been intentionally nicked to convert it to relaxed Form II. This DNA remained a sharp band, even after 48 hours. This indicates that the mere presence of bound m-RNA alone is not sufficient to give rise to strand separation during electrophoresis.
Wu does not attempt to explain this, and any explanation will be conjecture at this point. Therefore, I will offer the following conjecture: As I have shown previously (see ref. 3), in TN theory, Form I is inherently unstable due to the topological impediment of being 50% left-handed. When pure, however, its stability is enhanced by its superhelicity, which reduces the left-handedness by virtue of the Wu structure (or something like it). But in the presence of much bound m-RNA, the enhanced stability is again reduced; enough to predispose to strand separation during prolonged electrophoresis.
When nicked, however, the resulting Form II chromosome will immediately assume the all-right-handed Watson-Crick structure, or something like it. Since this is the structure spontaneously assumed by DNA when unconstrained by bound impurities (i.e., RNA or protein), we can surmise that it is inherently more stable than Form I, and that it therefore remains duplex during electrophoresis, the presence of bound m-RNA notwithstanding.
REFERENCES
Wu, T.T. Secondary structures of DNA. Proc. Natl. Acad. Sci. 63, 400-405 (1969).
Wu, R. & Wu, T.T. A novel intact circular dsDNA supercoil. Bull. Math. Biol. 58, 1171-1185 (1996).
Biegeleisen, K. Topologically non-linked circular duplex DNA. Bull. Math. Biol., 64, 589-609, (2002).
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