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Nucleic Acids Research, 2000, Vol. 28, No. 11 E51-e51
© 2000 Oxford University Press

Early melting of supercoiled DNA topoisomers observed by TGGE

Viktor Víglasky*, Marián Antalík1, Jozef Adamcík and Dusan Podhradsky

P.J. Safarik University, Faculty of Sciences, Department of Biochemistry, Moyzesova 11, 041 54 Koice, Slovakia and 1Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 043 53 Koice, Slovakia

Received January 21, 2000; Revised and Accepted March 31, 2000.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have used temperature gradient gel electrophoresis (TGGE) to measure the progress of local denaturation in closed circular topoisomer DNA as a function of temperature and superhelicity ({sigma}). We describe the versatility of this method as a tool for detecting various conformational modifications of plasmid DNAs. The early melting temperature of a structural transition for any topoisomer is dependent on the value of superhelicity. Supercoiled topo­isomers represent a system of molecules that is sensitive to changes in temperature. We show that the topoisomer with the highest absolute value of superhelicity melts earlier than topoisomers with lower values. Thermal sensitivity of highly supercoiled plasmids could play a biologically important role in regulation of replication and expression in cells under thermal stress. The estimated melting temperature for plasmids with {sigma} < –0.05 is very significant because these temperatures for early melting are below physiological temperatures.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The most common method for the study of DNA topological properties is agarose gel electrophoresis (1). Unlike linear or nicked circular DNA, whose mobility in agarose gels is primarily determined by the molecular weight, the mobility of closed circular DNA also varies with the linking number (Lk) (2). These species are topological isomers of the molecule and are generally called topoisomers.

The geometric distortion of a supercoiled molecule means that the average shape and frictional properties of the molecule are changed as a result of its topology. Electrophoresis separates DNA molecules on the basis of size and compactness; smaller and/or more compact molecules will migrate more rapidly through the matrix of the gel under the influence of the electric field. Within a certain range of the absolute value of specific linking difference, | {sigma} | < 0.05, the gel electrophoretic mobility of a topoisomer of linking number Lk increases with the magnitude of {Delta}Lk (the linking number relative to that for a relaxed form) (3,4). The resolving power of agarose gels for topoisomers is impressive, but unfortunately the range is limited, so that more highly supercoiled species co-migrate as a broad band. The major problem with one-dimensional gel electrophoresis is the relatively limited range over which migration is a function of topology. The traditional approaches to the study of linear DNA melting proved ineffective in this case. The resolution of topoisomers has been greatly improved with the recent use of two-dimensional gel electrophoresis (5).

Information about conformational changes of separated molecules is also offered by denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE). The ability of DGGE to detect changes in plasmid DNAs was used to study the effect of superhelical density on melting in different topoisomers (6). Meanwhile, very little is known about the formation of melted regions in supercoiled DNA (scDNA). TGGE is easy to perform and allows sensitive detection of the conformational stability of DNA under a wide variety of conditions. The sample of DNA in TGGE experiments moves through different denaturing conditions during electrophoresis (7). TGGE very sensitively separates covalently closed DNA of different structure. On one side of the gradient the biopolymers migrate as native molecules, on the other they are denatured, while in between the whole transition curve may be recorded. Aside from technical ease, TGGE has other attributes which can be exploited. The method allows one to fractionate plasmids according to size and shape and to monitor structural changes in the same experiment. Topoisomers in TGGE are electrophoresed through an agarose gel which contains a temperature gradient perpendicular to the direction of the electric field. We present a novel view on the conformational transitions of a system of topoisomers induced by temperature.

In the present investigation we examine the TGGE behavior of an extensive family of topoisomers of plasmids pBR322 (4361 bp) and pUC19 (2763 bp) over a temperature range of 25–75°C. We find that a series of mobility transitions occurs as linking number and electrophoresis temperature are systematically varied and reveal the fine structure of early melting in closed circular DNA. These transitions are manifested as cyclic variations in mobility with increasing temperature. The marked differences in electrophoretic mobility between supercoiled and relaxed DNA molecules made it possible to observe the denaturation of each DNA topoisomer leading to topological relaxation. Our results confirm others data (6) that such transitions occur before the melting of linear molecules; the transition width varies for different topoisomers. We have shown that thermal stability varies as a function of superhelical density for different topoisomers.

The degree of supercoiling and topological equilibrium in the cell is strongly controlled by specific enzymes (DNA topoisomerases and gyrases) that are capable of either adding or subtracting supercoiled twists in DNA. The distribution of topoisomers in the various supercoiled states and their biological function in the cell are not understood.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and enzymes
All reagents and chemicals used in the experiments were obtained from commercial sources. Plasmids pUC19 and pBR322 and agarose type II no. A-6877 was purchased from Sigma, calf thymus topoisomerase I was purchased from Amersham Pharmacia Biotech and LB medium from Fluka Biochemika. The plasmids were used to transform Escherichia coli HB101.

Culture of bacteria and plasmid isolation
Aliquots (250 ml) of exponentially growing E.coli cultured to an optical density of 0.3–0.4 at 600 nm in LB medium supplemented with ampicilin (75 µg/ml) at 37°C were further incubated in the presence of ampicilin (150 µg/ml) for 16 h. Plasmids were obtained from these cultures according to the method of Birnboim and Doly (8). The residual impurities were removed from cloning vector pUC19 by the following cleaning steps: equimolar phenol/chloroform extraction, chloroform and isoamyl acohol extraction (24:1) and precipitation with isopropyl alcohol and then with 2 vol of ethyl alcohol. The precipitate of DNA was dried and dissolved in TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0). scDNA must be prepared and handled with care because cleavage anywhere within the entire circular molecule completely changes the topology and releases the superhelical constraint.

Topoisomerase treatment
A method modified from those previously described (2,9,10) was used. An aliquot of 5 µg of plasmid DNA from a stock solution (0.5 mg/ml) was incubated for 2 h in 40 µl of TOPO buffer (5 mM dithiotreitol, 0.1 M Tris, 1 M KCl, 50 mM MgCl2, 1 mM ethidium bromide) and 1 µl of topoisomerase I from a stock solution (~5 U/µl) at 37°C. The reaction was stopped with 2% SDS. Topoisomerase and ethidium bromide were removed by the same procedure as was used for the removal of impurities from plasmid DNA.

Two-dimensional gel electrophoresis
Electrophoresis was performed as described previously (11). Briefly, the first dimension gel electrophoresis was conducted in tube gels of 1.0% agarose in 0.5x TBE buffer. The tube gel was prepared with a 40 x 0.4 cm (i.d.) glass tube, partially constricted at the bottom. Electrophoresis was performed for 60 h at 100 V (2.5 V/cm) in an electrophoresis apparatus maintained at a constant temperature (25°C) by a water incubator/circulator. The second dimension gel electrophoresis was conducted by placing the appropriately sliced tube from the first dimension into the upper slot of a slab gel, also of 1% agarose in the same buffer and containing chloroquine at a concentration of 1.0 µM. The second dimension electrophoresis was conducted for 23 h at 5 V/cm and 25°C.

Apparatus for TGGE
The equipment was basically similar to the device described by Riesner (7). A temperature gradient was formed in a gel perpendicular to the direction of the electric field. The gradient was established on a copper plate adjacent to the electrophoretic apparatus by cooling and heating of the plate at opposite sides with two independently circulating water baths. The gradient of temperature was linear along the plate, as was confirmed by measuring the temperature over the whole plate with a thermistor.

TGGE experiments
The agarose gel was in 0.5x TBE buffer (40 mM Tris, 2 mM EDTA, 90 mM boric acid, pH 8.3). Gel electrophoretic separation of DNA topoisomers was performed in 1% agarose in the same buffer as was used to prepare the gel. Before application of the DNA sample it is advisable to pre-electrophorese the gel for ~10–15 h. This step helps to remove reactive charged compounds and low molecular weight impurities from the gel that may cause artifacts. Electrophoresis was started without a temperature gradient. After 30 min the temperature gradient was established. The electric field was switched off during formation of the temperature gradient. The lower temperature part of the apparatus was set up at 20°C and the higher part at 80°C. After temperature gradient stabilization the DNA sample was applied to the starting slot of the gel. Before electro­phoresis 0.5 µg of DNA were dissolved in 40 µl of gel loading buffer (10 mM Tris, 1 mM EDTA, pH 8.0, 20% glycerol, 0.1% bromophenol blue). During electrophoresis the gels were submerged in the selected buffer, the sample was run at a constant 5.6 V/cm for 8 h and the electrophoretic buffer was recirculated. The nucleic acids were visualized by staining with the fluorescent intercalating dye ethidium bromide (1.5 µg/ml) and destained in distilled water.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Comparison of two-dimensional gel electrophoresis and TGGE
Two-dimensional electrophoresis was carried out to determine the distribution of pBR322 plasmids of different superhelical densities at room temperature (~25°C). The second dimension was performed in the presence of the intercalating agent chloroquine (1 µM), which causes a decrease in twist (Tw). This changes the twist of each DNA molecule by about four turns. More than 24 discrete topoisomers could be resolved in this gel, as shown in Figure 1. Clearly, the position of open circular DNA (ocDNA) is not identical to the linear, supercoiled or relaxed forms of DNA. The level of linear form was estimated to be <1% of total plasmid.



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  		Figure 1. Two-dimensional gel electrophoretic analysis of topoisomers of pBR322. The position of nicked (oc) and linear (L) DNA are designated. (Left) Gel photograph; (right) schematic representation of the gel photograph. The first dimension was performed in a 1% agarose gel at 2.5 V/cm in 0.5x TBE for 25 h. For the second dimension, the gel was turned through 90° and run for another 22 h at 5 V/cm in 0.5x TBE containing 1 µM chloroquine.

 
Figure 2 shows the corresponding TGGE records of pBR322 topoisomers. The chloroquine concentration used in TGGE experiments was 1 µM. Every band represents the mobility of one topoisomer at different temperatures. The most intensive band consists of ocDNA. The less intensive but well-separated bands correspond to scDNAs; their mobilities increase with the absolute value of writhe (Wr) at a temperature of ~35°C.



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  		Figure 2. Temperature gradient gel of topoisomers of pBR322. The direction of mobility of the sample is from anode (up) to cathode (down). The gels were electrophoresed with the indicated temperature gradient in 0.5x TBE buffer containing 1 µM chloroquine at 6 V/cm for 12 h.

 
If the shape and charge of a DNA molecule remain constant, mobility is a linearly increasing function of temperature. The structural transition of a topoisomer is seen as a continuous or discontinuous band which is retarded or accelerated in the temperature range of transition (12). It is possible to observe a decrease and an increase in mobility at certain temperatures characteristic for each topoisomer. We observe clear cut transitions, but the transition curve for some of the topoisomers intersect, making it difficult to identify unambiguously the transition curves for all topoisomers.

To get rid of the ambiguity, we performed analogous experi­ments with shorter scDNA preparations containing fewer topoisomers under the same conditions. TGGE of pUC19 is presented in Figure 3. Figure 3a presents the result of gel electrophoresis for a DNA preparation containing only eight topoisomers. In another electrophoretic experiment the DNA was electrophoresed without a temperature gradient for 14 h at 4 V/cm and only after this procedure was the temperature gradient applied and electrophoresis was continued for 9 h at 6 V/cm (Fig. 3b). Clearly, we get a much better separation of the less supercoiled topoisomers. The comparison of two-dimensional and TGGE under identical conditions enabled us to identify all transitions, which are numbered in the figure. The numbers denote the absolute value of the writhing number. The difference between the mobility of DNA without writhe (Wr ~ 0) and ocDNA is close to 0 and marked conformational changes cannot be seen with a temperature increase (5,13).



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  		Figure 3. Temperature gradient gel of topoisomers of pUC19. The electrophoresis conditions in (a) were the same as in Figure 2. The sample of DNA was run for 9 h. The experiment in (b) was also performed under the same conditions. However, the topoisomers were run at constant temperature (20°C) without a temperature gradient at 4 V/cm for 14 h, then the temperature gradient was switched on at 6 V/cm for 9 h. The numbers on both records denote absolute values of writhing number.

 
The transitions for topoisomers 1–8 are very clear. We can see that topoisomer 8 is the first to melt; followed by 7, then 6, etc. Generally, from both figures (Figs 2 and 3) it is obvious that the topoisomer with the highest absolute value of super­helicity melts earlier than topoisomers with lower values.

Analysis of melting curves
The curves are evidently not sigmoidal, as in experiments with a denaturing gradient (6). A sigmoid curve is characteristic for two-state processes. For data from TGGE it is not advisable to fit by means of a two-state mechanism (14,15). For a more than two-state system it is impossible to determine strictly the temperature of transition.

Therefore, to obtain more precise information on initiation of the melting process, we determined the derivative of mobility with respect to temperature separately for each topo­isomer (Fig. 4). The temperature Ti,m at the first local maximum of dµ/dT is an intrinsic property of the topoisomer with superhelicity {sigma}i. However, this temperature is not exactly the transition temperature. The peaks are clearly defined for most supercoiled topoisomers of pBR322 with an absolute value of Wr > 5. It is possible to estimate the transition width for various topo­isomers at Ti,m. It amounts to ~3–7°C. The accuracy of fit analysis for supercoiled pUC19 is higher then for pBR322 for the reason described above.



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  		Figure 4. The derivative of mobility with respect to temperature for each topoisomer. dµ/dT for topoisomers of (upper) pUC19 and (lower) pBR322. Temperature at the first local maximum of a topoisomer with {sigma}i is denoted Ti,m.

 
Figure 5 shows the plot of Ti,m versus corresponding superhelicity {sigma}i for pUC19 and pBR322. It reflects a topological unfolding (melting) of the DNA structure. From this plot it is evident that Ti,m decreases with increasing superhelicity.



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  		Figure 5. A plot of Ti,m versus superhelicity {sigma}i for pUC19 (closed circle) and pBR322 (open circle). It reflects a topological unfolding (melting) of the DNA structure. The error in Ti,m determination increases with more relaxed topoisomers.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We show in the present paper the effect of temperature on the early melting of superhelical DNAs with different superhelicities. The results were obtained by two-dimensional gel electrophoresis and TGGE. The results presented above demonstrate that TGGE makes it possible to observe the melting of scDNA caused by supercoiling. Since it is possible to observe this transition for individual topoisomers, one can conclusively prove that the previously observed early melting of scDNA leads to transient states of supercoiled molecules with lower values of negative writhe.

The so called ‘native’ negatively supercoiled plasmid DNAs isolated from bacteria consist of a distribution of topoisomers. One requirement for the investigation of DNA melting of different topoisomers by TGGE is a good distribution of supercoiled plasmids. This should cover the required range, i.e. topoisomers of | {sigma} | = 0–0.045, where the distribution of topoisomers can be described by an ideal Gaussian curve at room temperature (3). The electrophoretic resolution of DNA topoisomers is more sensitive for topoisomers with lower values of Wr. We obtained such preparations by treating the original plasmids pUC19 and pBR322 with topoisomerase I in the presence of ethidium bromide and the electrophoretic buffer and gel contained the intercalating agent chloroquine, which also affects twist (9,13). The average value of linking number obtained on relaxation depends on the solution conditions under which the relaxation took place. Specifically, the change in twist caused by topoisomerase I and/or altered conditions forces the writhe to change from its initial average value to a new value, and hence the DNA will run with a different mobility in the gel, as we can see in Figures 1–3. Structures such as cruciforms and Z-form DNA, which are unstable in relaxed or linear DNA, are formed cooperatively at a threshold level of supercoiling (16). We have attempted to select conditions where the values of superhelicity of topoisomers are under this threshold.

The mobility difference between adjacent topoisomer bands is a hydrodynamic property and is therefore determined as first order by the associated difference in Wr. Changes in Wr between successive topoisomer bands might not be accompanied by changes in Tw (2,17). The fact that any non-linear mobility behavior reflects a conformational change in the molecule allows the study of topological changes in scDNA. The decrease in mobility of an individual topoisomer (Figs 2 and 3) during the early melting process is based on induction of consecutive decoupling of weak interactions (hydrogen bonds and stacking interactions) in the structure of DNA. The change in temperature alters the helical repeat (h) of the DNA and hence changes the value of Tw0 (twist) and hence Wr. Next, the increase in mobility for all DNA topoisomers with total decoupled weak interactions is observed at temperatures above 65°C. In this case the covalently closed complementary interwound strands cannot be completely separated to a larger distance. This state is analogous to the denatured state of proteins without aggregation.

The nicked form of plasmid DNA (ocDNA) undergoes partial or/and total denaturation of base pairs (helix to random coil transition) similarly to linear DNA, resulting in a rapid decrease in mobility at higher temperatures. Denaturation is detected by electrophoresis as part of a band with a very low mobility value. Nicked circular molecules are not necessarily present in vivo, but may be formed by breakage of one strand of the closed circular molecules during ‘rough’ purification and by treatment with topoisomerase I. However, at temperatures >70°C separation of ocDNA and relaxed scDNA without writhe is possible by electrophoresis.

Under our conditions (see Materials and Methods) the melting of linear pUC19 and pBR322 molecules starts at temperatures >70°C. Consequently, the Ti,m values in Figure 4 for all topoisomers are below the melting temperature of linear and ocDNA. Hence, what we observe is early melting of scDNA. Our further experiments with others plasmids have shown that Ti,m is independent of the sequence and length of scDNA. It appears to be a function only of superhelicity. Local denaturation is closely coupled to supercoiling in closed DNA. However, a correlation based on existing models of melting of scDNA between a calculated denaturation profile for an individual topoisomer, the transition temperature and cooperativity were not executed (18,19).

It is worthwile to estimate the Ti,m of the ‘higher’ topo­isomers, which were not directly detected by TGGE, by linear extrapolation, because highly supercoiled DNA topoisomers are naturally occurring in cells (20–22). The estimated Ti,m for plasmids with {sigma} < –0.05 is very significant because these temperatures for early melting are below physiological temperatures.

Many recent experiments have shown the importance of DNA supercoiling and topoisomerase action in gene activation, replication and recombination (5,20–23). The topo­isomers with higher negative values of {sigma} (more condensed) are more sensitive to structural changes induced by temperature. The thermal sensitivity of supercoiled plasmids could play a biologically important role in regulation of replication and gene expression under thermal stress. The excess free energy associated with negative supercoiling of DNA may be utilized in many cellular mechanisms. In general, processes that require untwisting or writhing of DNA or which stabilize such deformations are facilitated with negatively supercoiled as compared to relaxed DNA. Examples of such processes include the replication and transcription of DNA, which require unwinding of the DNA helix, and the formation of nucleosomes and other protein complexes on DNA, which stabilize negative writhing of the helix (5,10,20–25).


   ACKNOWLEDGEMENT
 
This study was supported in part by grants nos 5053 and 6116 from the Slovak Grant Agency.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +421 95 62 235 82; Fax: +421 95 62 221 24; Email: viglasky@kosice.upjs.sk Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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