Distributions of Knots and Links in Circular DNA

Alexander V. VOLOGODSKII ABSTRACT

The distinctive feature of closed circular molecules is that they occupy particular topological states that cannot be altered by any conformational rearrangement short of breaking DNA strands. This topological constraint opens unique possibilities for experimental studies of the distributions of topological states created by different ways. Primarily, the equilibrium distributions of topological properties are considered in the review. We describe how such distributions can be obtained and measured experimentally and how they can be computed. Comparison of the calculated and measured equilibrium distributions of knots and links formed by circular molecules gave a lot of valuable information about properties of the double helix. Study of the steady state fraction of knots and links created by type II DNA topoisomerases exposed surprising property of the enzymes, their ability to reduce these fractions essentially below the equilibrium level.

1. Introduction A typical DNA molecule adopts many different conformations in solution, and therefore its properties have to be analyzed in probability terms. The probability distributions of DNA properties can be calculated with good accuracy now and can be measured experimentally in many cases. From the experimental point of view the distributions of topological states are especially attractive. The topology of a circular DNA is not altered as long as the strands of the double helix stay intact, and one can manipulate samples in different ways to determine the distributions. In particular, it is possible to resolve different topological forms by gel electrophoreses and obtain the distributions by measuring the intensity of different bands. On the other hand, there are methods which allow reliable computing topological properties of circular DNA. Due to these experimental and theoretical possibilities, distributions of topological states are not only very interesting objects, but also powerful instruments in biophysical studies. In this review we will consider distributions of knots and links between pairs of circular DNA molecules and show how they can be used to study DNA conformational properties. In the last Section of the review we will show how these concepts and methods can be used to study enzymes which change DNA topology, DNA topoisomerases.

There are three levels of topological variables which are needed to describe a state of double-stranded circular DNA. For the first and second levels we need to consider DNA molecule as a simple curve which coincides with DNA axis. The fist level describes topology of isolated closed curve which topology can correspond to unknotted circle or to a knot of a particular type. If we have many DNA molecules, part of them can form topological links with others, and the second level of description specifies types of these links. In this review we will consider only links between two chains, also gigantic network of topologically linked DNA circles are known in biology [1]. The third level of the topological variables specify the links formed by complementary strands of the double helix. The links exist only in socalled closed circular DNA where both complementary DNA strands are closed. The linking of the DNA complementary strands, which is very important for DNA conformational and biological properties (see reviews [2,3], for example), will not be considered here. Our main emphasis will be the equilibrium distributions of topological states. There is one distinctive feature of such distributions. Normally, initial distribution of conformational properties relaxes, as a result of thermal motion, to its equilibrium form. This is true for all properties of linear polymer chains. After some time the distribution of the end-to-end distance of a polymer chain, for example, or the distribution of loops randomly formed by the molecules will correspond to the thermodynamic equilibrium. This is not the case for the equilibrium distributions of topological states, because exchange between different topological states is impossible for circular molecules. The system of circular polymer chains does not relax to the equilibrium distribution of topological states. The concept of the equilibrium distribution, however, is very useful in this case. The definition of the equilibrium distribution of topological states of circular molecules does not differ from the corresponding definitions for other properties. This is the most probable distribution which has to be reached by random exchange between different states in solution. Thus, the distribution minimizes the free energy of the polymer molecules. We would get the equilibrium distribution of topological states if our circular molecules had phantom backbones, so that their segments could pass one through another during the thermal motion. Thus, to calculate the distribution we should forget that the exchange between different topological states is forbidden and use usual rules of statistical mechanics. It means that, regardless of the topology, the probability of conformation i , Pi , is specified by the Boltzmann distribution:

Pi exp( Ei / kT ) ,

(1)

where Ei is the energy of conformation i and kT is the Boltzmann temperature factor. To calculate the probability of a particular topological state we have to take a sum of Pi over all conformations which correspond to this state. In the Section 3 we consider computer simulation of such equilibrium conformational distributions as well as the analysis of the simulated conformations. Experimentally we can obtain thermodynamic equilibrium over topological states by slow cyclization of linear molecules. In the case of double-stranded DNA, the cyclization can be provided by joining complementary single-stranded ends (cohesive ends) [4]. If the cohesive ends are long enough, about dozen of nucleotides or longer, a circular form is stable at room temperature. Such circular molecules have single-stranded nicks, so only topology specified by the double helix axis is defined there. Comparison of the experimentally measured distribution with computer simulations provides a lot of valuable information about DNA properties. Such studies will be described in Sections 4-6. Distributions of topological states created by type II DNA topoisomerases will be described in the last section of the review. 2. Description of topological states of circular DNA 2.1. Knots A knot is understood to be any closed curve, and in particular the topological equivalent of a circle (in this case it is called a trivial knot). There is infinite number of topologically distinguished knots. For the classification, knots have to be deformed to obtain the standard form of their projection on a plane. A standard form of a knot projection is such an image thereof when the minimum number

Figure 1. Table of simple knots with less than seven intersections in the standard form.

of intersections on the projection is achieved. One must be always careful, though, that there should be no self-intersection of chains in

the course of such reduction. Deformations of this kind are known in topology as isotopic deformations. Two knots which can be transformed into each other by way of isotopic deformation belong to the same isotopic type. Knots can be simple or composite. A knot is composite if there is an unlimited surface crossed by the knot at two points, which thus divides it into two nontrivial knots. The simplest knot has three intersections in the standard form and is called a trefoil. Fig. 1 shows the initial part of the table of simplest knots - namely, all knots with less than seven intersections in the standard form. A knot and its mirror image are considered to belong to the same type of knot, although they may belong to the same or different isotopic types. In particular, the trefoil and its mirror image cannot be transformed into each other by way of continuous deformation without selfintersection, and therefore belong to different isotopic types. The figure eight knot (41) and its mirror image belong to the same isotopic type. The table of knots is, in fact, a table of knot types, for it features only one representative of mirror pairs. With the growth of the number of intersections the number of types of simple knots grows very fast. As it turns out, there are 49 types of simple knots with nine intersections, 165 with ten and about 552 types of knots with eleven intersections [5,6]. The table of simple knots with less than 17 crossings in the standard form was completed recently [7], there are 1,701,936 of such knots. 2.2. Links In chemistry and biology links of two or more circles are usually called catenanes. The tables of two-contour links are based on the same principle as the tables of knots [5]. The initial part of the table of link types is shown in Fig. 2. Although three links in this table, 21, 41 and 61, belong to the torus class [8] which corresponds to

Figure 2. Table of links with less than seven intersections in the standard form.

the links of the complementary strands of the double helix in closed circular unknotted DNA, the fraction of torus links among more complex links is very small. 2.3. Identification of topological states Suppose we want to determine the type of knot formed by highly coiled closed cord. The fact that persistent attempts to untangle the cord have produced no result cannot be taken as a proof that we are dealing with a nontrivial knot. What is needed for resolving this problem is an algorithm of verification of the topological identity of the configuration in question. Topologists developed many of such algorithms based on invariants of the topological states, characteristics thereof which remain unchanged with any isotopic deformations of the chains, which are possible without the disruption of their integrity [8]. The simplest invariant of topological state is the Gauss integral which defines the linking number of two chains (see below). Of course, for classifying the state of chains with a topological invariant, the latter must assume different values for different topological states. Not a single topological invariant meets this requirement in full measure, but there are very powerful ones among them which help identify many elementary types of knots (links) and distinguish them from more complex ones. The Gauss integral is a fairly weak topological invariant and is of no use for distinguishing many linked states of chains from unlinked states. Still the integral is very useful for analysis of DNA supercoiling, since it identifies all links of the torus class. Another important invariant is the Alexander polynomial which is very convenient in computer simulation (reviewed in [9]). It is a polynomial of one variable in the case of knots and of two variables in the case of links of two closed contours. The invariant was used in all studies dealing with the computation of the equilibrium distribution of topological states. The great majority of the simplest knots have different Alexander polynomials, (t) , and thus the invariant allows to distinguish all simplest knots one from another and from unknotted chains [9]. For unknotted chain (t ) = 1 . (t ) for all knots with less then 11 crossings in standard form can be found in Ref. [5]. An invariant in the form of an Alexander polynomial also exists for links, but it is a function of two variables rather then one. The algorithm of calculating the Alexander polynomial for two chains, ( s, t ) , is described in ref. [10].

3. Computer simulations of equilibrium conformational distributions of circular DNA 3.1. DNA model and its relation to the actual molecule If we want to compare computed results with measured properties of an actual polymer we have to establish the correspondence between a model chain and actual polymer molecule. This is done over the concept of Kuhn statistical segment, b. The idea of this correspondence can be described as following. It can be shown that for any model of sufficiently long polymer chain the average square of the end-to-end distance, contour length, L:

R 2 , is proportional to the chain R 2 = bL .

(2)

Eq. (2) corresponds to random walk with step length b. It does not account for excluded volume effect, related with the chain thickness, but the effect does not change Eq. (2) for DNA molecules a few thousand base pairs in length. The value of R
2

can be determined

experimentally and, if we know L, Eq. (2) allows us to determine b. The value of b is directly related to the chain persistence length, a = b/2, and characterizes the bending rigidity of a polymer chain [11]. For DNA molecule b is close to 100 nm or 300 base pairs. Now we can express the molecule length in the number of Kuhn segments, L/b. The concept of Kuhn statistical segment is equally applied to any theoretical model of the polymer chain. Modeling conformational properties of a polymer chain we should use a model chain of the same number of Kuhn segments. DNA double helix of n Kuhn statistical segments can be modeled, for our purposes, as a closed chain of kn rigid cylinders of equal length and diameter (d). The energy of the chain, E , is specified by angular displacements i of each segment i relative to segment i + 1 :

g kn 1 2 E = i . 2 i =1

(3)

Eq. (3) can be naturally transformed for the case of circular chain. All conformational properties considered here do not change, within a good accuracy, if k 10 , and for majority of these properties we can obtain accurate results even for k = 1 ( g = 0 ). In the polymer

statistical physics the chain with k = 1 is called the freely-jointed chain, since all its conformations have the same energy. The freelyjointed chain is the simplest model of a polymer chain, often used in general analysis of polymer properties. If we set a value of k, we can calculate g [12]. DNA effective diameter, d, specifies the electrostatic interaction between DNA segments: d is the diameter of an uncharged polymer chain which mimics the conformational properties of actual electrically charged DNA. Its value depends strongly on ionic conditions. The dependence of d on [Na ] and [Mg ] is known with accuracy [13-16]. The value of d can be several times greater than the geometric diameter of the double helix. For the purposes of computer simulation it is convenient to express d in the units of Kuhn length, so the topological properties of two chains should be similar if they have the same number Kuhn segments and the same diameter. For physiological ionic conditions DNA effective diameter is close to 4 nm, or 0.04 in Kuhn length units. Thus, the DNA model has two parameters, the chain length measured in the number of Kuhn statistical lengths, n, and the effective diameter, d. 3.2. Computation of the conformational properties Despite the simplicity of the model described above, few of its statistical properties can be evaluated analytically. There are, however, well developed computational methods which allow estimating many conformational properties, including topological properties of circular chains. The methods are based on the random sampling of the equilibrium conformational distribution followed by direct analysis of the constructed conformations. Metropolis Monte Carlo procedure is the most general way to perform the sampling which satisfies condition (1) [17]. At each step of the procedure, a trial conformation of the chain is generated by displacement from the previous conformation. The starting conformation is chosen arbitrarily. The probability of accepting the trial conformation is obtained by applying the energy test. If the energy of the trial conformation, E new , is lower than that of the previous conformation, E old , then the trial conformation is accepted. If the energy of the trial conformation is greater than the energy of the previous conformation, then the probability of acceptance of the trial conformation is equal to exp[( Eold E new ) / kT ] . It is assumed that a conformation has infinite energy if the distance between two non-adjacent segments is smaller than the d value. Therefore, the minimum distance between
+

2+

all pairs of non-adjacent segments of a trial conformation is calculated. If any distance is less than d, the trial conformation is rejected. This procedure is repeated numerous times to obtain an ensemble of conformations that represents the equilibrium distribution. If for any reason a trial conformation is rejected, the current conformation is taken as the next one in the constructed set of conformations. Displacement of the chain is usually performed by a crankshaft rotation of a randomly chosen subchain [18]. A subchain containing an arbitrary number of adjacent segments is rotated by a randomly chosen angle, , around the straight line connecting the vertices bounding the subchain. The value of is uniformly distributed over an interval ( 0 , 0 ) chosen so that about half of the moves are accepted. Segments of the chain are allowed to cross each other during the displacements. With such a phantom, or incorporeal, chain, topology of the chain is changing and we obtain a sampling of the equilibrium distribution over topological states. To analyze the distribution we need to determine knot type of each constructed conformation. It can be done by calculating (t ) for t = -1 and t = -2 for all conformations of the set [9,19,20].

Figure 3. Typical simulated conformations of circular DNA 10,000 base pairs in length. The illustration was obtained by computer simulation of the equilibrium conformational set.

Sometimes we need to evaluate equilibrium properties of the chains with a particular topology. In this case we have to start simulation from a conformation with the desired topology and keep the topology

during the displacements by rejecting all trial conformations which topology is different from the topology of the starting conformation. A typical DNA conformation from the equilibrium set is shown in Fig. 3. The size of this DNA corresponds to DNA of bacteriophage P4. Similar approaches are used to compute equilibrium properties of two closed DNA molecules which can be unlinked or form links of different types [9,10,21,22]. 5. Equilibrium distribution of knots Let us consider a diluted solution of nicked circular DNAs at thermodynamic equilibrium, so the probability of links between two molecules is negligible. In this case the equilibrium distribution of topological states is reduced to the probabilities of unknotted chains and various knots. Let us first consider theoretical aspect of this problem. Starting from 1974 [19,20], about a dozen of studies have been devoted to computations of the equilibrium probability of knots (reviewed in [9,23]; see also [24] and refs. therein). The results show that, for DNA molecules thousands base pairs in length, the equilibrium fractions of nontrivial knots are rather small although increases with DNA length (Fig. 4).

4 Fraction of knots, %

3
31

41 52

51

0 10 20 30 DNA length, thousands of base pairs

Figure 4. Equilibrium probability of the simplest knots in circular DNA. Results of computer simulations [15] are shown by solid lines, symbols correspond to the experimental data for trefoils [15,42]. The data correspond to the physiological ionic conditions (0.2 M of NaCl).

The computations also showed, that the probabilities of knots reduce dramatically when the thickness of a polymer chain increases [25,26]. The probability that closed chain of n Kuhn segments ( n < 50 , d / b < 0.2 ) will form nontrivial knot, P ( n, d ) , can be approximated by the following empirical equation [26]:

P(n, d ) = P (n,0) exp(23d / b) .

(4)

This dramatic dependence of the knot distribution on the chain thickness clearly shows why lattice models of a polymer chain are not suitable to model DNA topological properties. For a self-avoiding lattice model the value of d / b is close to 1, when for DNA molecule it equals 0.04 at physiological ionic conditions [16]. As a result, the probability to form nontrivial knot for a chain of 200 Kuhn segments is 0.23 for the proper DNA model and 0.0004 for the face-centered cubic lattice of 200 segments [27]. The difference is nearly 3 orders of the magnitude, and it is even larger for shorter chains. This striking difference clearly shows that topological statistical properties of lattice models have very little in common with the corresponding properties of DNA molecules. However, in a some sense the lattice models are simpler, and it stimulates their continuing use in theoretical studies of DNA topological properties (see [28,29], for example). Knots and links of different types formed by circular DNA molecules have different electrophoretic mobility in gels, which makes it possible to separate them by the electrophoresis (Fig. 5). The method requires special calibration, for it is impossible to say in advance what position a particular topological structure must occupy relative to the unknotted circular DNA form. A sufficiently large body of experimental results on the mobility of various topological structures has been accumulated by now [30,31]. To separate knotted and linked DNA molecules by gel electrophoresis they must have singlestrand breaks, for otherwise mobility will also depend on the linking number of the complementary strands. Comparison between measured equilibrium fractions of trefoils and the corresponding calculated values for different concentration of sodium ions is shown in Fig. 6. The agreement is really remarkable,

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Figure 5. Electrophoretic separation of knotted (right lane) and linked (left lane) DNA molecules 4363 bp length. Each band corresponds to a knot or link with a specific number of intersections in the standard form. These numbers are shown next to each band. All links belong to the torus family. OC dimer is open circular DNA molecule of double length. (Illustration provided by E. M. Shekhtman and D. E. Adams.)

taking into account that there were no adjustable parameters in the DNA model used in the computations. One of two model parameters, DNA effective diameter, increases greatly when the ion concentration reduces, and this causes the decline of the trefoil fraction shown in the figure. Comparison between measured and calculated fractions of trefoils allowed determination of DNA effective diameter for other ionic conditions [16]. 6. Equilibrium probabilities of links If the concentration of DNA molecules during their random cyclization in solution is not too small, a fraction of the molecules forms catenanes at thermodynamic equilibrium. Let us suggest that the concentration is still small enough so that only small fraction of the molecules is linked with others at the equilibrium; in this case formation of links from 3 or more molecules can be neglected. Thus we can consider formation of catenanes as bimolecular reaction which is characterized by its equilibrium constant, B . Equilibrium concentration of linked DNA molecules, ccat , can be found from the equation

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5
Equilibrium fraction of trefoils, %

4 3 2 1 0

0.01

0.1 [Na+], M

Figure 6. Comparison of the measured and simulated equilibrium fractions of trefoils for different concentrations of sodium ions. P4 DNA, 10 kb in length, was cyclized in solution of different NaCl concentrations via joining the cohesive ends [15]. Each point on the graph (filled circles) is the mean of 6 to 20 determinations. Computational results (open circles) account for the dependence of DNA effective diameter on NaCl concentration [13,14].

ccat = B(c 2ccat ) 2 Bc 2 ,

(5)

where c is the total concentration of circular molecules. The value of B depends on DNA length and ionic conditions in solutions. Dependence of B on DNA length for physiological ionic conditions is shown in Fig. 7. We can also consider topological equilibrium for a system which includes two types of circular DNAs (A and B). Such equilibrium catenation was studied first by Wang and Schwarz more than 30 years ago [32]. We recently used the approach to probe conformations of supercoiled molecules [22]. The system consisted of supercoiled molecules of one length (molecules A) and cyclizing molecules of another length (molecules B). The system was prepared by such a way that there was only partial topological equilibrium: there were no AA catenanes although the concentration of molecules A was rather high but there was the equilibrium regarding AB and BB links. Since the goal was to measure the equilibrium constant for AB links, such partial equilibrium simplified the analysis of various

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products in this system. Comparison between simulation and measured data obtained in this study was used to test simulated conformational properties of supercoiled DNA [22].

Figure 7. Equilibrium constant of catenation of two identical nicked circular DNAs. The data obtained by computer simulation for the physiological ionic conditions (see ref. [22] for details of the computation).

7. Unknotting and unlinking circular DNA by type II topoisomerases Type II topoisomerases are important enzymes that pass one DNA through another and thereby change DNA topology. They make a transient double-stranded break in one DNA segment that allows passage of another segment of the same or another molecule and then reseal the break (reviewed in [33,34]). Thus, these enzymes might convert real DNA molecules into phantom chains that freely pass through themselves. There was a widely held belief that, with the obvious exception of DNA gyrase, which introduce supercoiling in circular DNA [35], topological equilibrium is also achieved in the reactions catalyzed by type II DNA topoisomerases. They can unlink knotted [36,37] or catenated DNA [38], and catalyze catenation if the DNA concentration is high enough [39]. In all these reactions the systems approach equilibrium. However, the question of whether the enzymes create an equilibrium distribution of catenated or knotted molecules had not been carefully examined till the study by Rybenkov et al. [40]. It was unexpectedly found that the fractions of knotted and catenated DNAs produced by type II topoisomerases are up to two orders of

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magnitude lower than at equilibrium (Fig. 8). Thermodynamically, there is no contradiction in this finding because the enzymes utilize the energy of ATP hydrolysis. This property of topoisomerases has an important biological consequence. It helps to explain how they can remove all DNA links under the crowded cellular conditions which favor the opposite outcome. Whereas ATP hydrolysis allows type II topoisomerases to remove topological links from DNA below their equilibrium values, it was not clear from the beginning how this topology simplification is achieved. Free strand passage during the thermal motion of circular DNA molecules should establish an equilibrium distribution of topological forms. This distribution would not change even if only a fraction of the segment collisions results in strand passage, as long as the probability of passage is independent of DNA topology. However, a single topoisomerase, given its small size compared to DNA, cannot recognize the topological state of DNA by binding the whole molecule. Topology is a global property of circular DNA molecules,

Figure 8. Type II topoisomerase, topo IV from E. coli, removes topological links from DNA to level below equilibrium. The reaction reached its steady state at substoichiometric values of enzyme/DNA ratio. Equilibrium value of the knot fraction for the given conditions and DNA length, 7 kb, is shown as a reference level by dashed line. Also shown, as a control, the fraction of knots found for topo III, type I topoisomerase from E. coli, which does not consume the energy during the catalysis and thus must shift the fraction of trefoils to the equilibrium level [40].

and it is impossible to design a machine which could determine topology by using only a local interaction with these long molecules. Enzymes are very small compared with DNA molecules, so they definitely belong to the category of such locally acting machines,

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although they can interact simultaneously with two DNA segments separated along the molecular contour. We found, however, that these enzymes do so by using internal statistical properties of DNA molecules rather than by determining their overall topology [41]. Type II topoisomerases create a sharp bend upon binding with a DNA segment, forming a hairpin (Fig. 9).

Figure 9. Model of type II topoisomerase action. The enzyme (black pacman) bends the bound DNA segment into a hairpin. The entrance gate for another segment of DNA is inside the hairpin. Thus, the second segment can pass through the first segment only from inside to outside the hairpin.

It turns that the probability to find another segment of the circular DNA inside the hairpin is a few times higher for knotted DNA than for unknotted one. Therefore, if the strand passing goes only from inside the hairpin, which is provided by specific orientation of the entrance gate of the enzyme relative to the hairpin, the strand passing should reduce the steady state fraction of knots. It is important to note that the hairpin itself does not notably affect the equilibrium fraction of knots. The steady-state fraction of knots is reduced dramatically in this model only because the enzyme catalyzes the strand passage reaction in one direction, from inside to outside the hairpin. The quantitative analysis of the model prediction as well as supporting it experimental data can be found in ref. [41]. This work was supported by NIH grant GM54215 to the author.

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[32] Wang JC, Schwartz H. Noncomplementarity in base sequences between the cohesive ends of coliphages 186 and lambda and the formation of interlocked rings between the two DNA's. Biopolymers 1967;5:953-66. [33] Wang JC. DNA topoisomerases. Ann. Rev. Biochem. 1996;65:635-695. [34] Wang JC. Moving one DNA double helix through another by a type II DNA topoisomerase: the story of a simple molecular machine. Q. Rev. Biophys. 1998;31:107-144. [35] Reece RJ, Maxwell A. DNA gyrase: structure and function. Critical Rev. in Bioch. and Mol. Biol. 1991;26 (3/4):335-375. [36] Liu LF, Liu C-C, Alberts BM. Type II DNA topoisomerases: enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break. Cell 1980;19:697-707. [37] Kato J-i, Suzuki H, Ikeda H. Purification and characterization of DNA topoisomerase IV in Escherichia coli. J. Biol. Chem. 1992;267:25676-84. [38] Peng H, Marians KJ. Decatenation activity of topoisomerase IV during oriC and pBR322 DNA replication in vitro. Proc. Nat. Acad. Sci. USA 1993;90:8571-8575. [39] Krasnow MA, Cozzarelli NR. Catenation of DNA rings by topoisomerases: mechanism of control by spermidine. J. Biol. Chem. 1982;257:2687-2693. [40] Rybenkov VV, Ullsperger C, Vologodskii AV, Cozzarelli NR. Simplification of DNA topology below equilibrium values by type II topoisomerases. Science 1997;277:690-693. [41] Vologodskii AV, Zhang W, Rybenkov VV, Podtelezhnikov AA, Subramanian D, Griffith JD, Cozzarelli NR. Mechanism of topology simplification by type II DNA topoisomerases. Proc. Natl. Acad. Sci. USA 2001;98:3045-3049. [42] Shaw SY, Wang JC. Knotting of a DNA chain during ring closure. Science 1993;260:533-536.

New York University, Department of Chemistry, 31 Washington Place, New York 10003, USA

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