Finding of a zero linking number topoisomer

Biochimica et Biophysica Acta 1790 (2009) 126–133

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g e n

Finding of a zero linking number topoisomer You Cheng Xu University of Texas Southwestern Medical Center at Dallas, 1451 Huckleberry Cir. Issaquah, WA 98029-7651, USA

a r t i c l e

i n f o

Article history: Received 4 June 2008 Received in revised form 26 August 2008 Accepted 31 October 2008 Available online 20 November 2008 Keywords: pBR322 Gyrase Left-handed DNA DNA replication Linking number Figure 8 structure

a b s t r a c t It is generally assumed that native deoxyribonucleic acid (DNA) is a right-handed double helix. A reasonable deduction is that during replication, the two parental strands have to unwind very quickly. However, this surmised quick unwinding is problematic and has never been proven experimentally. It is hypothesized that the two strands of DNA are winding with each other ambidextrously rather than plectonemically. The successful assembling and disassembling of a zero linking number topoisomer supports this hypothesis. It was further proven by quick separation of singly nicked DNA. The new DNA model was also verified by the “figure 8” structure, which is the annealing product of two single-stranded circular DNA with a 2 kb complementary insert in opposite directions. These experimental results are hard to be explained by the traditional Watson–Crick model. The significance of this finding in the understanding of DNA replication is briefly discussed. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Shortly after the discovery of the double helix [1], it was noticed that there is a topological problem in deoxyribonucleic acid (DNA) replication [2]. Reportedly the Escherichia coli genome contains 4,639,211 base pairs [3]. According to the Watson–Crick model, 10.4 bp per turn [4,5], the linking number (L) of the circular chromosomal DNA of E. coli should be around 4 × 105. After each round of DNA replication, about 40 min in rich medium, this linking number must reduce to exactly zero so that the newly synthesized chromosomal DNA can be segregated into two daughter cells. It is well documented that the replication of the chromosomal DNA in E. coli is semi-conservative and bi-directional [6]. Each replication fork advances at the rate of 1 kb per second [7] which, according to the double helix model, means the unwinding rate should be 6000 rounds per minute (RPM). It is questionable that the delicate long DNA can unwind so rapidly in the viscous cytosol where friction and hindrance are expected to be high. Evidence gleaned in recent years has overturned the long-held belief that the topological problems, inherited from the double helix, can be solved by the magic power of enzymes. While there are many enzymes in E. coli, only various topoisomerases can change the linking

Abbreviations: EthBr, ethidium bromide; DNA I, native supercoiled DNA; DNA II, nicked DNA; DNA III, linear DNA; ssc-DNA, single-stranded circular DNA; ssl-DNA, single-stranded linear DNA; DNA V, annealing product of two complementary ssc-DNA; I′n, topoisomer produced in the presence of n μg EthBr/ml at room temperature; bp, base pair; L, linking number; T, total twist; W, writhing number; AGE, agarose gel electrophoresis E-mail address: [email protected]. 0304-4165/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2008.10.012

number of circular DNA. It was reported that type 1 topoisomerases (topoisomerase I and topoisomerase III) are not necessary in E. coli replication, since Δtop A mutants lacking topoisomerase I or Δtop B mutants lacking DNA topoisomerase III are both viable [8,9]. However, type II topoisomerases (gyrase and topoisomerase IV) are necessary for the viability of E. coli. Gyrase catalyzes an intermolecular strandpassing reaction, which reduces 2 linking numbers per reaction during the elongation of DNA replication. Whereas topoisomerase IV catalyzes an intra-molecular strand-passing reaction, which decatenates the interlocked two daughter molecules appeared at the end of replication. Evidently, gyrase is the main operator for unwinding the parent DNA duplex in replication. E. coli gyrase is composed of two A and B subunits. Although there are 1000 and 3000 molecules respectively of GyrA and GyrB per cell in a rapidly growing E. coli, mostly dispersed within the cytoplasm [10], they do not associate to form active gyrase (A2B2). Its reaction rate is 6 times per minute [11] and the linking number changed by one gyrase molecule is 12 per minute, or 480 per 40 min. There are only 50 gyrase molecules actively working in an E. coli [12]. Even if all the 50 gyrases work cooperatively, continuously and solely for DNA replication, it is still not sufficient to accomplish the duty of unwinding, i.e. ΔL = 50 × 480 = 2.4 × 104, an order of magnitude less than the 4 × 105 predicted by the current double helix model. In addition, viewed from a topological point, only the gyrases in front of the replication fork are effective in separating parent DNAs. Each gyrase needs to bind 150 base pairs at the toposite [13], and the available toposites get fewer as the replicating fork reaches the terminal region; both situations would make unwinding even more difficult. It is becoming increasingly clear that right-handed B-DNA model cannot explain the topological problem in E. coli. However, E. coli has

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Fig. 1. The AGE purified pBR322 topoisomers were denatured and examined by EM. Relaxed pBR322 DNA dimers were purified by AGE and checked again on AGE. The electrophoresis buffer containing 1 μg/ml chloroquine. Lane 1, 1 kb DNA marker; Lane 2, supercoiled dimer; Lane 3, relaxed dimer; Lane A, B, C, D, E, F, G, H, I, J, K, L, are purified fractions; Lane 16, supercoiled monomer. (Only the nicked monomer appears in this picture.) The fractions B, E, K were denatured by glyoxal and checked by EM. The bar represents 0.5 μm. The red numbers represent absolute topological number, |Lk|.

lived on the earth for millions of years and never has encountered any problem. It reminds us to be aware that it is uncertain whether the Watson–Crick model is a universal truth or just a theory. Based on our previous electron microscopic (EM) observation [14,15] and present experimental investigation, we hypothesize that in native DNA, a relatively high percentage of left-handed DNA coexists with the right-handed DNA. A zero linking number topoisomer was found by disassembling and assembling of covalently closed circular DNAs. It indicates that the two strands of DNA are not always winding plectonemically in one direction. Quick separation of singly nicked DNA further proved it. The formation of “figure 8” structure provided additional evidence to evaluate the validity of our proposed DNA model. All of these experiments indicate that this phenomenon of polymorphism in various native DNA (pBR322, pBluescript and λ Hind III fragment) is much more prevalent than previously thought. The biological meaning and the related implications of this finding are briefly discussed. 2. Materials and methods All plasmid DNAs of pBR322, head-to-tail dimer pBR322 and pBluescript, are harbored in HB101 respectively. Supercoiled DNA was obtained by published procedures [16]. DNA was relaxed by calf thymus topoisomerase I at room temperature [16]. Singly nicked

DNA was made according to the method of Greenfield et al. [17]. Linear DNA was generated by cutting DNA I with EcoR I. Purified topoisomers were recovered from a preparative agarose gel by squeeze and freeze method [18]. The collected samples were concentrated by isopropanol precipitation and resuspended in TE buffer. ssc-DNA was made by 15–30% sucrose gradient centrifugation in 0.25 N NaOH from singly nicked pBluescript DNA with the method of Stettler et al. [19]. ssc-M13mp9-λHindIII2kb-DNA+ and ssc-M13mp9-λHindIII2kb-DNA− were prepared according to Sambrook et al. [20]. The “figure 8” structure was annealed with equal amount of two ssc-DNAs in 50% formamide, 50 mM NaCl, 10 mM EDTA, 5 mM Tris–HCl pH 8.5 at 22 °C. Electrophoresis was carried out with a 20 cm long horizontal 1% agarose gel slab in TBE buffer (90 mM Tris–HCl, 90 mM Boric acid, 2.5 mM EDTA, pH 8.0) at 2 V/ cm for 20 h and the buffer was constantly circulated at room temperature. After the electrophoresis, the gel was stained with EthBr, washed and recorded as before [16]. The images were inverted for presentation. The second dimension AGE was done according to Lockshon and Morris [21]. The purified topoisomers were denatured in 6% glyoxal and 20% formamide solution at 22 °C for 1 h. Denatured samples were adjusted to 50% formamide, 2 mM EDTA, 20 mM Tris–HCl, pH 8.6, 50 μg/ml cytochrome C and spread onto hypophase of 20% formamide, 2 mM EDTA, 20 mM Tris–HCl, pH 8.6. The sample was picked up with parlodion coated grid after the spreading. The DNA samples on the grid were rotary shadowed

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with platinum for EM observation [14]. A Phillips EM-400 electron microscope was used for observation and photography. 3. Results 3.1. The disassembling of covalently closed circular DNA The two strands of each relaxed pBR322 dimer can be completely separated from each other. Fig. 1 shows the purity of topoisomer fractions obtained from a preparative agarose gel and the EM pictures of completely denatured topoisomers from several different fractions. Some important information could be obtained from these pictures: a) the prepared dimer pBR322 DNA sample is pure and clean. There is no detectable contamination of monomer pBR322 DNA, cantenated dimer pBR322 DNA or chromosomal DNA (lane 2 and lane 16). b) Due to operational difficulties, some of the purified fractions contain two adjacent topoisomers. It is very difficult to avoid the nicking of the long circular DNA; hence DNA II is present in almost every part of the purified circular DNA preparations. c) Each double stranded pBR322 dimer was denatured by glyoxal and turned out to be a pair of ssc-DNA loosely linked with each other. The glyoxal reacts with the NH2 group of guanine inside the DNA and forms an adduct, which prevents the formation of hydrogen bonds between the denatured ssc-DNAs. The contour length of each pair of ssc-DNA is in the range of 2.5 ± 0.5 μm, corresponding to the length of a dimer pBR322 DNA. The linking number of the totally denatured topoisomers can be counted easily on EM pictures by dividing the crossing number of each pair of the ssc-DNA with 2. d) The linking number determined by EM is reliable, since the probability of the two broken ends of a nicked DNA meeting at the same place on the sample grid is less than 1/1,000,000. e) The present EM photo does not provide the information of winding direction of two strands. The measured linking number can only be expressed in absolute integers. Fig. 1 shows that | L| of purified topoisomers from fraction B is always 5 or 6, from fraction E is always 3 or 4 and from fraction K is either 1 or 2. One might ask whether there are three consecutive over-crossings or under-crossings in these figures. If that is true, a topoisomer with m linking number would appear 3 possible linking numbers: L = m ± 2 or m (m is an integer). However such case has never been found in our experiment. Evidently, all these crossings are effective crossings, which are similar to the published EM pictures in determining the linking number of a specially designed DNA [22]. f) The linking number obtained from each fraction keeps a constant number. This phenomenon strongly suggests that the observed linking number is not an artifact of EM. Otherwise, any linking number may be found from each purified fraction. g) Comparing the linking numbers obtained from different fractions indicates that the linking number of adjacent bands differs by one. Actually, this is the topological property of closed circular DNA duplex as explained by Crick et al. [23]. h) Usually the difference of linking numbers between relaxed and native supercoiled DNA is 5% of the total twist number. It would be around 42 (0.05 × 4361 × 2 / 10.4) for pBR322 dimer. We found that the |L| of relaxed pBR322 dimer is smaller than that of supercoiled DNA. This is the understanding contrary to the traditional DNA topology. i) According to the rule of one topoisomer that keeps one linking number, a zero linking number topoisomer can be definitely located on the agarose gel (Fig. 1). pBR322 DNA contains 4361 base pairs; its dimer contains 8722 base pairs. According to the double helix model, 10.4 base pairs per turn, the linking number of a relaxed pBR322 dimer should be around 838. The measured low linking number from pBR322 dimer molecules greatly deviates from the expected. Similar results were also obtained from purified relaxed monomer pBR322 DNA. How to explain this is a big challenge to our wisdom. The experiment implied that the two strands of the pBR322 dimer are actually linked with each other with very low linking number. One

Fig. 2. The first and second dimension AGE of pBluescript DNA. First dimension AGE in TBE buffer containing 1 μg/ml chloroquine. Lane 1, 1 kb molecular marker; Lane 2, supercoiled DNA; Lane 3, singly nicked DNA; Lane 4, linear DNA; Lane 5, ssc-DNA; Lane 6, annealed ssc-DNA; Lane 7, ssl-DNA; Lane 8, annealed ssl-DNA; Lane 9, DNA relaxed in the presence of 3.8 μg/ml EthBr; Lane 10, DNA relaxed in the presence of 2.0 μg/ml EthBr. b) Second dimension AGE in the TBE buffer containing 5 μg/ml EthBr. Three slides of the sample in the first dimension were turned 90° for second dimension AGE. In the 3 square boxes, the 3 samples were electrophoresised in first dimension only and pasted in the way that keeps the nicked DNA lined with the corresponding nicked DNA in second dimension gel.

of the possible reasons is that lots of left-handed DNA coexists with almost the same amount of right-handed DNA in the relaxed pBR322 dimer. The cancellation of the opposite turns makes the apparent low linking number. Other scientists have reported similar phenomenon. Brack et al. [24] provided three EM pictures of the supercoiled pM2 DNA, a 10,078 bp circular DNA [25]. After denaturing, they appeared as two tangled ssc-DNAs with the linking number much less than that expected from the traditional double helix model. These pictures are not consistent with the claims of the authors of the paper, which is actually the call of nature implying that the two strands of DNA are not always winding plectonemically. We found it is possible to repeat their result and to detect the two ssc-DNAs of pBR322 DNA by EM [14]. Serendipitously, the discovery of the extremely low linking number was found in relaxed DNA [15], which led us to search for a zero linking number topoisomer. 3.2. The assembling of the zero linking number topoisomer To confirm that the zero linking number topoisomer is not a product of our imagination, a special zero linking number topoisomer was prepared and demonstrated by AGE. As shown in Fig. 2, two pBluescript ssc-DNAs were annealed in 75% formamide for 72 h at 4 °C, a new topoisomer was formed, which is different from DNA V [19], a partially annealed DNA. It is neither nicked DNA (DNA II) nor linear DNA (DNA III). On a two dimensional gel, it is located as one band of the relaxed topoisomer. This indicates that the new band is a topoisomer with a zero linking number, since it is formed from two ssc-DNAs without the involvement of any topoisomerase. The formation of this band is slow and the yield is low. Since it is the annealing product of two complementary ssc-DNAs, its formation must follow the topological rule of L = T + W = 0 at all times. This newly found topoisomer has never been formally reported yet, although it has been mentioned in literature [26].

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Fig. 3. AGE of pBR322 DNA II and DNA III denatured by NaOH. One second experiment was done in this way: a) add the buffer to the agarose gel to the margin of the gel (no buffer entered to the well) and turn on the power supply on at 2 V/cm; b) put 10 μl sample of DNA on the top of clean parafilm; c) pick 10 μl of 0.5 N NaOH into the tip with pipet; d) adjust the pipet volume to 20 μl; d) put the NaOH on the droplet of DNA and quickly pick the 20 μl mixture up and loaded into the dry well of agarose gel. The estimated time of this operation is one second. When all the samples entered the gel, more buffers were added to completely cover the surface of agarose gel. The AGE was conducted overnight at room temperature with constant circulation of the buffer. Lanes 1 and 7, 1 kb DNA marker; Lanes 2 and 8, supercoiled DNA; Lane 3, 0.5 μg of singly nicked DNA; Lanes 4, 5, 6, 0.5 μg singly nicked DNA denatured by equal volume of 0.5 N NaOH after 10, 1 min and 1 s; Lane 9, 0.5 μg linear DNA; Lanes 10, 11, 12, 0.5 μg linear DNA denatured by equal volume of 0.5 N NaOH after 10, 1 min and 1 s.

This experiment provides additional evidence indicating that the two strands of DNA are not always winding plectonemically in the right-handed direction. There is a relatively high percentage of lefthanded DNA in this zero linking number topoisomer. 3.3. Quick denaturation of singly nicked DNA There is an even simpler test that will give us insightful knowledge into the double helix. When the singly nicked pBR322 DNA II was mixed with equal volume of 0.5 N NaOH at room temperature, it quickly denatured and formed ssc-DNA and ssl-DNA in 10 min, 1 min and even 1 s. The denatured products can be separated by AGE as shown on the agarose gel (Fig. 3). Linear DNA behaved similarly and generated two ssl-DNA. The test is simple and easy. However, this result needs careful speculation. Due to both ends of a linear DNA are movable, the problems involved in the quick separation of two ssl-DNAs were frequently neglected. If we investigate the dissociation process more cautiously, a spacial (not a spelling mistake of special) problem emerges. Suppose the hydrogen bonds between the two strands were destroyed instantly after the addition of NaOH, according to the double helix model, the newly formed two ssl-DNAs are still tightly tangled with each other. (It should be noted that the linear DNA in solution is a 3 D thread, not always stretched out!) The physical separation of this pair of ssl-DNA needs the unwinding movement of both strands. Since the long slander ssl-DNA is highly hydrophilic carrying many water molecules at its vicinity, its movement should be very sluggish. Suppose the Newton's law is still applicable in this case, great force is needed to quickly separate the two tangled ssl-DNA strands. The singly nicked DNA would encounter the same problem when denatured by NaOH.

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The more difficult question is why is there no spacial hindrance for the quick separation of the ssc-DNA and ssl-DNA? In this singly nicked DNA, the two strands are supposedly winding 430 times; the complete separation of them within 1 s should not to be as easy as that of a pair of ssl-DNA. The reasons are: a) An ssc-DNA in solution is not always expanded wide enough waiting for both ends of its complementary ssl-DNA to pass through. b) The two ends of ssl-DNA unwinding around the ssc-DNA will have a head-to-head collision since they are all winding in the same manner inside the same ring of an ssc-DNA. The two ends of the ssl-DNA would inevitably tangle with each other, especially at the end of the process. c) Unwinding 430 turns per second (equal to 25,800 rpm) is a very fast rotation; great force is needed to push the ssl-DNA into rotation around the ssc-DNA in solution. Where does such a force come? d) The fast unwinding of the ssl-DNA in solution would inevitably cause the breakage of its backbone. One might argue that the two kinds of ss-DNA were not instantly separated while mixing the DNA II with NaOH, but those were probably separated during the long period of electrophoresis. However, this scenario is unlikely to be true. Once the denatured DNA sample enters the agarose gel, the immediate big pH change is not only unfavorable for further denaturizing of the DNA II, but provides conditions of low temperature and salt favorable for renaturation. Besides, the nick is located randomly on either strand of the plasmid and the AT rich region is distributed unevenly in the plasmid. The fringe effect would preferably help the separation of the two strands near the nick together with AT rich region. That would cause the unwinding not to be a synchronized process. The time of the dissociation of individual DNA II molecule should be different and that would lead to the formation of smear bands on the agarose gel, which is not observed. This test revealed that the two strands of pBR322 DNA are unlikely winding 430 times. This result is consistent with other findings. 3.4. A test for the “figure 8” structures In order to find a way to distinguish the two kinds of DNA structural model, a “figure 8” test is carried out. When two M13mp9 ssc-DNAs inserted with a 2 kb λ Hind III fragment in opposite directions were annealed, there would be two possibilities: a) The annealed 2 kb DNA fragment is in the Watson–Crick model. There would be 200 right-handed turns, the rest of the ssc-DNA have to be in 200 left-handed turns to keep L = T(right-handed) + T(left-handed) + W = 0 or in a highly supercoiled state. That would be a good evidence to confirm the Watson–Crick model. b) If the two strands of the 2 kb fragment do not all wind plectonemically in the Watson–Crick model, something different from the above expectation may occur. Fig. 4A shows that there are three kinds of annealing products formed from two ssc-DNAs at different times. The annealing is a relatively slow process since the topological constraints require the two ssc-DNAs to keep the L = T + W = 0 at all times during annealing. The final product F (Fig. 4A) was carefully recovered from the agarose gel (with no EB staining and no UV exposure) and examined by electron microscopy. A remarkable fact is that there are a lot of “figure 8” structures formed as shown in Fig. 4B. Some molecules are not perfect “figure 8” structure, which contain two broken singlestranded ends. They were probably generated during the spreading process while preparing the sample for electron microscopy. Fig. 4C shows three typical “figure 8” structures. Each “figure 8” structure contains two Y forks linking the single-stranded DNA and the doublestranded DNA. The length of the double-stranded part is, as expected, proportional to 2/9 of the total contour length of an ssc-DNA. A remarkable fact is that there is no annealed molecule in the form expected from the traditional double helix model. Since the ssc-M13mp9-λHindIII2kb-DNA preparation contains negligible ssl-M13mp9-λHindIII2kb-DNA, it is unlikely that the final

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Fig. 4. “Figure 8” structures obtained from two ssc-DNAs with a 2 kb fragment inserted in opposite orientation. A) AGE of annealing products at different times. Lane 1, 1 kb DNA marker; Lane 2, ssc-DNA+; Lane 3, ssc-DNA−; Lanes 4, 5, 6, 7, 8, 9, annealing of the two ssc-DNA after 1 min, 1, 2, 4, 8, 24 h. B) The EM of annealing product from fraction F. The bar represents 0.5 μm. C) The EM of three typical “figure 8” molecules. The bar represents 0.5 μm.

product of “figure 8” structure all contains a nick hiding in the 2 kb double-stranded region. Besides, the annealing of an ssc-DNA with an ssl-DNA should be much faster than the formation of a real “figure 8” structure, because there is less spacial constraint. They should be detected by AGE at the early stages of annealing. The annealing mixture contains no enzyme. Hence the linking number of the annealing product should be zero, i.e., L = T(right-handed) + T(left-handed) + W = 0. From these EM pictures, evidently W ≈ 0, so, T(right-handed) + T(left-handed) ≈ 0. That means, in the double-strand DNA region, the right-handed turns and left-handed turns cancel with each other. The net turns in the 2 kb duplex are close to zero. This 2 kb DNA duplex of the “figure 8” structure was further verified by digesting purified final product F by S1 nuclease with or without Nco I. There is a unique Nco I cutting site (23901) located in the 2 kb DNA λ Hind III fragment (from 23130 to 25157). Fig. 5 is the result of AGE for the analysis of the final annealing product on 10 cm long 0.7% agarose gel in TAE buffer. After the digestion of fraction F by S1 only, a 2 kb fragment is produced, whereas when both S1 and Nco I were used, two fragments with expected sizes (1.3 kb and 0.77 kb) were produced. It indicates that the annealing product of the 2 kb DNA is a normal double stranded DNA. This test strongly suggests that there are no 200 right handed turns in the 2 kb fragment. The result is consistent with above three results,

which cannot be explained by the Watson–Crick model. The 2 kb double-stranded DNA is probably composed of equal amounts of right-handed and left-handed DNA. The result of this test strongly suggests that the two strands of DNA are not winding plectonemically only in one direction. Similar EM study of the linking number was reported. Iwamoto and Hsu [22] constructed “figure 8” structure in a different way. Their 39 bp DNA duplex is apparently in the right-handed double helix. Their result indicates that the EM method is reliable. Their result rejects one kind of side-by-side DNA structural proposal [27]. It confirms Watson–Crick model, as well as our DNA model. 4. Discussion The discovery of circular DNA about 30 years ago emerges the new growth point of DNA topology. The unique topological properties of covalently closed circular DNA provide an interesting and important field to investigate the structure and function of DNA. The elucidation of the DNA double helix is definitely one of the greatest discoveries of the twentieth century. Nowadays, the prevalent idea is that after over half century of investigation, there is no more secret or mystery in the DNA double helix. Francois Jacob once stated, “The aim of modern biology is to interpret the properties of the

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Fig. 5. Identification of fraction F on 0.75% AGE. Lane 1, 1 kb ladder; Lane 2, ssc-DNA+; Lane 3, ssc-DNA−; Lane 4, fraction F; Lane 5, Fraction F digested with S1; Lane 6, Fraction F digested with S1 and Nco I.

organism by the structure of its constituent molecules.” One of the most important properties of an organism is reproduction, which distinguishes the living and non-living world. The discovery of the double helix paves the way for answering questions about life. Evidently, it cannot answer all the questions in “the secret of life”. The topological difficulties involved in E. coli chromosomal DNA replication are just one example of the unanswered questions. Today, every gene in E. coli is known. It is almost impossible to find a new enzyme other than topoisomerase IV and gyrase to reduce the linking number of parent DNA in E.coli. It should be mentioned that gyrase and topoisomerase IV are similar but different in structure and function. The unwinding reaction rate of topoisomerase IV is only 0.8 times per minute [28], slower than that of gyrase [11]. The independently measured slow reaction rates of gyrase and topoisomerase IV are reliable and reasonable. Since the catalytic strand passing reaction is rather complicated, its reaction rate cannot be as fast as those simple enzyme catalytic reactions [29]. From a topological view-point, based on the present knowledge of gyrase and all available information as explained in the introduction, it is very difficult to interpret the mechanism of DNA replication in E. coli with the canonical double helix model. We report here several experiments, which contain only a few simple chemicals and pure plasmid DNA. No unknown biological factor is involved. In this way, every component and all the experimental conditions are well defined and the result is reproducible. The interpretation of these experimental results will probably encounter many arguments. It is generally assumed that under the physiological conditions, DNA is a right-handed double helix with 10.4 bp per turn [4,5]. However, to precisely determine the winding direction inside the whole native DNA is very difficult. There is currently no protocol to detect it. Probably the strongest evidence comes from the X-ray crystallography of double stranded oligonucleotides. It has provided detailed structural data showing that the two strands of DNA are winding

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right-handedly. The only exception is the finding of the left-handed ZDNA [30]. After many years of investigation, Z-DNA is undoubtedly an experimental fact. Its presence in native DNA is believed to be very scarce, since it has a special sequence requirement. Up to now, all the X-ray crystallography data of DNA are obtained from oligonucleotides, mostly less than 20 base pairs. To extrapolate the results obtained from the short oligonucleotides to native plasmid DNA or chromosomal DNA leaves room for error. However, to obtain a crystal of native plasmid DNA with thousands of base pairs is probably very difficult. To determine the structure of a plasmid or chromosomal DNA is beyond the capacity of present day X-ray crystallography. What we did is exploiting the topological properties of covalently closed circular DNA to reveal the hidden secrets of the DNA duplex. It allows us a glimpse into the global structure of DNA macromolecules. Since a single nick turns supercoiled plasmid with thousands of base pairs into nicked DNA, and two nicks close enough on each strand can turn supercoiled DNA into linear DNA, the tremendous topological changes provide the basis for its detection. Although we did not directly see the winding direction of the two strands in native DNA duplex, the combination of EM, AGE and the rules of topology helps us to find out that the two strands of DNA are not coiled around each other plectonemically in only one direction. The assembling and disassembling of a zero linking number topoisomer provide evidences showing that the two strands of native DNA duplex cannot always wind in one direction. The quick separation of singly nicked DNA further proves it. The result of “figure 8” tests is unfavorable to the Watson–Crick model. All of these independent experimental results are consistent with each other and cannot be reasonably explained by the traditional double helix model. It should be noted that we did not give the answer to what the double helix really is. What we said is that, in native DNA, probably there is a lot of left-handed DNA coexisting with the right-handed DNA. Left-handed DNA may be in B-DNA form as proposed by Gupta et al. [31] or other unknown forms and Z-DNA is plausibly a member of the big left-handed family. An interesting finding of the co-existence of B-DNA with Z-DNA in oligonucleotides was found by X-ray crystallography at 2.6 Å resolution [32]. The left-handed DNA was also found in supercoiled pBR322 DNA [33]. It indicates that, in native DNA, the left-handed DNA can stably coexist with right-handed DNA. Crick et al. [22], claimed that the Watson–Crick model is suggestive rather than conclusive. It is true that if there is a DNA with zero linking number, its two strands should be able to be completely separated. According to their proposal, that kind of experiment can easily be done: to heat the circular DNA and run an AGE to detect the missing band with zero linking number. Until now, nobody has succeeded that kind of experiment, it might be deemed as the confirmation of the canonical double helix. That kind of experiment is by no means easy or simple. There are several conditions for the accomplishment of that kind of experiment: a) The denaturing treatment should be gentle enough to keep the backbone of the long circular DNA intact and strong enough to destroy all the hydrogen bonds. Simply heating the DNA solution can cause serious nicking and cannot be applied for this purpose (data not shown). b) The denaturing reaction should be reversible. Any reagent that permanently changes the DNA and causes loss of renaturation should not be considered. c) It is evident that there is only one zero linking number topoisomer in the whole set of topoisomers. The prerequisite of doing this experiment is to know where the zero linking number topoisomer could be found. Although our way of finding zero linking number topoisomer is indirect, we still hope our experience would help those who may try the experiment suggested by Crick et al. The complete separation of λ DNA, a long linear DNA with 48 k base pairs, by single strand DNA binding protein (gene32) was reported [34]. This result actually supports our suggested DNA model.

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The molecular weight of gene32 is 35,500. Each gene32 protein binds 10 nucleotides. Since the gene32 works in a cooperative way, it binds with ss-DNA preferentially to the vicinity of a gene32 that has already bound to the ss-DNA. This is the way that opens the double stranded λ DNA gradually. However, the long λ DNA has many AT rich regions, which are supposed to be occupied by gene32 first. The two ends of λ DNA are also supposed to be occupied by gene32 proteins preferentially. The Watson–Crick model requires the rotation of the rest of the strand since the AT rich region is only located at several different places of the strand. The huge mass of those parts already occupied by many gene32 molecules would prevent the rotation of the whole strand in solution. It is unlikely that the two strands of the long λ DNA can be completely separated by gene32. Whereas, if the two strands of λ DNA are not all winding in right-handed direction, their separation by gene32 would be much easier. Their EM picture shows that the two strands of the gene32 bound λ DNA are parallel as two side-by-side threads with no tangling of the two. Another interesting story is hidden in the application of point mutation by two polymerase chain reactions (PCR). Stratagene Co. has successfully developed the method and a kit is ready for any users. In this system, the only enzyme used is DNA polymerase, which starts DNA synthesis in opposite directions from two primers located at two strands of the same supercoiled plasmid site. It is inexplicable with the double helix model that two polymerases walking in a face-to-face way on the same plasmid molecule can produce the appropriate products with no topological hindrance. However, if the two strands are not wound plectonemically, it would be possible for the two DNA polymerase molecules working on two strands of the same circular DNA in reverse directions. All these reports published in literature and our findings reported here construct a chain of evidence implying that the two strands of DNA are probably winding ambidextrously rather than plectonemically. With our hypothesis, it would be easy to interpret the problems in DNA replication. It is our prediction that the linking number of circular E. coli chromosomal DNA is much less than 4 × 105. As mentioned before, it is plausible that the present double helix model causes the so-called “topological problem”. It is not a real problem for each E. coli to solve. It is likely that there is no high speed unwinding during DNA replication. This idea is significantly different from the present assumption on the mechanism of DNA replication. However, it is consistent with the recognition that parent DNA reels into the complicated processing factory of replisome and newly synthesized DNA is delivered out of the factory [35]. The replisome contains many enzymes including DNA polymerase holoenzyme, gyrase, helicase, and a lot of other elements. It is assumed that helicase opens the double helix quickly and the positively supercoiled stress generated in front of the replication fork is released by topoisomerase IV [36,37]. However, how the topological stress quickly transfers to the terminal is questionable. In a rapidly growing E.coli, the chromosomal DNA is composed of 6 or more replication forks, each occupied with a huge replisome. At the same time, it transcribes tRNA, rRNA and many mRNAs. That makes the chromosomal DNA very clumsy and it is hard to believe that the unwinding force can be easily transferred from each replication fork to the terminal site in a limited small space. According to our hypothesis, the helicase can do the unwinding work quickly and probably no topological stress is generated outside the replisome. The suggested DNA secondary structure may provide a different scenario for explaining RNA transcription. The question of how the newly transcribed RNA separates from its DNA template has never been clearly answered. Such a topological problem would not exist if the RNA–DNA duplex were not always winding right-handedly. Since Miescher, the long history of the discovery of DNA is full of twists. It had been halted several times by the prevalent ideas, which were abandoned by later experimental findings. It teaches us a good lesson showing that scientific knowledge is provisional and when new

facts are revealed, it is subject to re-examination. Our findings lead us to propose that in native DNA, the two strands are winding with each other ambidextrously rather than plectonemically. Such a subjective proposal about the double helix provides a different answer for interpreting DNA structure and its functions. It not only fits many experimental facts, but also fits the general frame of molecular biology. Crick et al. has stated: “DNA is such an important molecule that it is almost impossible to learn too much about it.” It seems crucial to collect all the essential information to make a theory of the double helix, which not only can explain all the facts, but also can predict the result of certain designed experiments. For this purpose, based on our hypothesis, the following predictions were made: 1. The linking number of E.coli is much less than 4 × 105. 2. A zero linking number topoisomer can be made from many different kinds of plasmid. 3. The absolute linking number of positively supercoiled or negatively supercoiled DNA is larger than that of their relaxed counterparts, i.e. |Lsupercoiled DNA| N |Lrelaxed DNA|. 4. In a plasmid with very long palindromic structure, the length of the two arms of the cruciform is proportional to the introduced supercoiling. All these conjectures or predictions could be verified if our DNA model is correct. Acknowledgements The author is grateful to Professors H. Bremer and L. Terada for their support; to D. Lang for his help in EM experiments; to J. C. Wang for the suggestion of “figure 8” test and his gift of calf thymus topoisomerase I. Thanks to Dr. L. Xu and Miss V. Poffenberger for their careful examination of the manuscript. References [1] J.D. Watson, F.H.C. Crick, A structure for deoxybibose nucleic acids, Nature 171 (1953) 737–738. [2] M. Delbrück, On the replication of desoxyribonucleic acid (DNA), Proc. Natl. Acad. Sci. U. S. A 40 (1953) 783–788. [3] BlattnerF.R. , The complete genome sequence of Escherichia coli K-12, Science 277 (1997) 1453–1462. [4] J.C. Wang, Helical repeat in solution, Proc. Natl. Acad. Sci. U. S. A 76 (1979) 200–203. [5] D. Rhodes, Klug, A. Helical periodicity of DNA determined by enzyme digestion, Nature 286 (1980) 573–578. [6] A., Kornberg and T.A. Baker (1992) DNA replication. Freeman and Company, N.Y [7] Z. Kelman, M. O'Donnell, DNA polymerase III holoenzyme: structure and function of a chromosomal replication machine, Ann. Rev. Biochem. 64 (1995) 171–200. [8] M.A. Schofield, R. Agbunag, M.L. Michaels, J.H. Miller, Cloning and sequencing of Escherichia coli mut R shows its identity to top B encoding topoisomerase III, J. Bacteriol. 174 (1992) 5168–5170. [9] V.A. Stupina, J.C. Wang, Viability of Escherichia coli top A mutants lacking DNA topoisomerase I, J. Biol. Chem. 280 (2005) 355–360. [10] M. Thornton, Immunogold localization of GyrA and GyrB protein in Escherichia coli, Microbiology 140 (1994) 2371–2382. [11] C. Ullsperger, N.R. Cozzarelli, Contrasting enzymatic activities of topoisomerases IV and gyrase from Escherichia coli, J. Biol. Chem. 271 (1996) 31549–31555. [12] M. Synder, K. Drlica, DNA gyrase on the bacterial chromosome: DNA cleavage induced by oxolinic acid, J. Mol. Biol. 131 (1979) 287–302. [13] G. Condemine, C.I. Smith, Transcription regulates oxolinic acid-induced DNA gyrase cleavage at specific site on the E.coli chromosome, Nucleic Acids Res. 18 (1990) 7389–7396. [14] Y.C. Xu, L. Qian, Z. Tao, A hypothesis of DNA structure, Scientia Sinica 25B (1982) 827–836. [15] Y.C. Xu, L. Qian, Determination of linking number of pBR322 DNA, Scientia Sinica 26B (1983) 602–613. [16] Y.C. Xu, H. Bremer, Winding of DNA helix by divalent metal ions, Nucleic Acids Res. 25 (1997) 4067–4071. [17] L. Greenfield, L. Simpson, D. Kaplan, Conversion of closed circular DNA molecules to single-nicked molecules by digestion with DNAse I in the presence of ethidium bromide, Biochem. Biophys. Acta 407 (1975) 365–375. [18] D. Tautz, M. Renz, An optimized freeze–squeeze method for the recovery of DNA fragments from agarose gel, Anal. Biochem. 131 (1983) 14–19. [19] U.H. Stettler, et al., Preparation and characterization of form V DNA, the duplex DNA resulting from association of complementary circular single-stranded DNA, J. Mol. Biol. 131 (1979) 21–40.

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