Statistical

mechanical

theory for the plectonemic

DNA supercoil

John E. Hearst
Department of Chemistry, University of California, Division of Chemical Biodynamics, Lawrence Berkeley Laboratory, Berkeley, California.94720

Nathaniel G. Hunt
Department of Physics, University of California, Division of Chemical Biodynamics, Lawrence Berkeley Laboratory, Berkeley, California 94720

(Received 5 August 199 1; accepted 6 September 199 1) The eigenfunctions for circular boundary conditions of the differential equation fust used by Harris and Hearst in 1966 to represent the dynamic properties of the wormlike coil have now been applied to the closed circular coils of high writhe. In order to avoid problems of knotting and excluded volume, the discussion here has been restricted to three-dimensional eigenfunctions with near plectonemic symmetry, i.e., eigenfunctions which cross each constant z plane only twice. It is concluded that at the natural levels of superhelical density that are found for DNA in vim, the DNA free of protein must be in a highly reduced configurational entropy state. The impact of this conclusion on issues of entanglement, chromosome replication and segregation, and chromosome organization are discussed.

I. INTRODUCTION DNA supercoiling and packaging are essential features of cellular architecture and must be understood before gene regulation and the dynamics of cell division can be properly addressed. Many points of view have been presented in efforts to provide enlightenment on these problems. First, the DNA alone condensation has been studied both experimentally1~2 and theoretically.3,4 Chromatin and the nucleosome problems, both with respect to structure5 and with respect 6 to the dynamics of this structure in relation to gene regulation and expression, have been studied. DNA supercoil-lo ing has received extensive study since its discovery, tist experimentally as a static property,-I6 and then dynamically, primarily as linked to transcription., * Mathematical rules for calculating linking number (Lk) and writhe ( Wr) have developed slo~ly~~-~~ and efforts at the determination of bending2 and torsiona128-31 force constants have led to js2 some computer modeling of DNA molecules with known writhe densities.32 In all of these studies what has been lacking has been a tractable mathematics which allows for the representation (albeit approximate) for the various quarternary folding structures which DNA is hypothesized to form. Furthermore, while the importance of writhe to the issue of folding and packaging has been obvious to most, and the mathematics for the calculation of writhe for any space curve has been reasonably well described in the literature, there have been no applications to continuous space-curve representations of DNA. We were first attracted to this problem by a suspicion that the oxygen regulation of the photosynthetic gene cluster of Rhodobacter capsulatus (a 46 kilobase-pair region of this bacterial genome committed to photosynthesis) would be related to DNA torsional tension.33 After several rounds of careful experimentation, there is no definitive evidence that significant changes in torsional tension occur during this cel9322 J. Chem. Phys. 95 (12), 15 December 1991

lular process,34 suggesting that this may be a conserved property in vivo. In an effort to understand the significance of linking number deficit or excess to the polymer configurations available to DNA in phase space, we have used the dynamic theory of Harris and Hearst3 for the worm-like coil. We have concluded that at the high-excess linking-number densities (pALk) found in nature ( - 0.05 turns per turns DNA duplex), a DNA molecule is effective in a single conformational state with very low polymer configurational entropy, and we believe that this low-entropy state is likely to be the evolutionary reason for DNA supercoiling. Such a state may avoid problems of entanglement during DNA replication and during chromosome segregation. c

II. THE NORMAL COORDINATES The Harris-Hearst theory for stiff-chain polymers- (the wormlike coil) is a dynamic representation of a polymer in terms of four parameters: p, the mass per unit length; J; the friction factor/unit length; ohend,a bending force constant or flexibility parameter; and L, the contour length of the chain. The principal assumptions are that the potential energy of the chain be given by the continuous integral,

B v=?
/2.

(1)

and s is a contour position coordinate having a range

- L /2<s<L

The equation of motion was then obtained by requiring that the variation of the action be zero.36 The constraint that the chain has average length (L ), is introduced by use of a Lagrange multiplier leading to a linear differential equay, tion for the motion of the wormlike coil:
@I 1991 American Institute of Physics

0021-9606/91/249322-07$03.00

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J. E. Hearst and N. G. Hunt: The plectonemic DNA supercoil

9323

-31 8 d 3 $P .P w
(3)
FIG. 1. A DNA plectonemic superhelix. The approximate properties of this positive superhelical structure are the following: superhelical pitch = 52; Wr = 3.95; Tw = 47.05; Lk = 51; Two = 46.10; Lk,, = 46.10; ATw,, = 0.95; ALk, = 4.90; c = + 0.106 turns/turn DNA helix; d = 90 A.

(4) This procedure symmetrizes the bending motions in three dimensions, leading to a longitudinal degree of freedom as well as the two transverse degrees of freedom associated with bending. In addition, a fluctuation in contour length is artificially introd.uced into the motion of the polymer. This approach converts an otherwise nonlinear problem into one requiring the solution of a simple linear differential equation. The theolry has been demonstrated to be highly successful in predici.ing quadratic moments of the polymer motion such as the pair-correlation function between contour positions on the chain, both in the limit of the rigid rod where the positions are very close to each other in contour and in the limit of the random coil where the positions are many statistical lengths away from each other on the contour. The time-independent eigenfunctions for circular boundary conditions are simple sines and cosines satisfying the circular condition. These functions represent a complete orthonormal set, capable of describing all configurations accessible to circular stiff-chain molecules. In particular, they are capable of describing configurations with writhe: Y, = (2/L)1 sin[ (2ms/L) 2 + 51, (5) where S is an arbitrary phase angle between 0 and 2a. Assessment of what is required for functions of definite writhe leads to some intcdtive simplifications which limit phase space to a certain class of molecular shapes, perhaps ignoring other classes which also have writhe. The logic for making these simplifications is that otherwise phase space would include highly knotted configurations, and configurations in which the polymer, as a line, touches itself (coincidence). Both such classes of structures must be eliminated for physical reality. We therefore initially limit ourselves to configurations of symmetry similar to those of plectonemic supercoils, an example provided in Fig. 1. The lowest-energy configuration for space curves with stiffness and writhe will typically have a plectonemic structure.21 An eigenfunction with definite writhe must be three dimensional. Thus, the constraint of writhe requires that the eigenfunctions for the x, y, and z directions are no longer independent. We arbitrarily assign the long axis (the screw axis) of the plectonemic helix to the z direction. In order to avoid knotting and coincidence, the sine function in the z direction experiences only one period, n, = 1, over the contour length L of the closed circle. The values of n, and nY are now allowed to take on any values, but the three-dimensional wave function is made up of only one simple sine or

cosine function along each direction. While this procedure is not the only one possible, more complex functions can be generated by taking linear combinations of these simplified three-dimensional functions. We have selected orthonormal eigenfunctions which retain the terminology of Harris and Hearst.26 In representing the approximate plectonemic helix, there is a necessary phase relationship between the trigonometric functions in the three directions. The only positions where both x and y equal zero simultaneously are at the ends of the helical structure where z is at one of its two extrema which occur at s = - L /2,O,L /2. This selection for the phasing of s on the continuous circular molecule is one of many possibilities, but this choice is sufficiently general. The vector eigenfunction X has three vector components X = xi + yj + zk, where the components are z== E,(2/L) /2COS(2rrS/L),. x=ex(2/L) 1 sin(2m,s/L), 2 (6) /2. y=~,(2/L) sin(25-+7/L), and s, the contour parameter, has a range - L /2<s(;L Ill. WRITHE Writhe is a property of any continuous space curve in three dimensions. It can be calculated by the Gauss integral:20*21 pL /2 , yL /2 dszf(s, ~2 1, (7) 4a -L/2 -L/2 .(; dX ) -(p&213r1, f(s,rSz 1 = J&2 (8) ds ~2 ds S, where

Wr=I J

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9324

J. E. Hearst and N. G. Hunt: The plectonemic DNA supercoil

(9) In the context of closed circular DNA molecules as shown in Fig. 1, the two phosphodiester backbones of the single DNA strands may be thought to be the edges of a ribbon. The linking number of this closed ribbon is established by wrapping the ribbon about a cylinder so that its center axis lies in a plane perpendicular to the cylindrical axis. If one starts with the ribbon flush with the cylindrical surface (untwisted), the linking number of a closed ribbon is the number of 360Q rotations the ribbon must undergo at a single cut relative to the other end of the cut in order to achieve the final state of the ribbon. Because the DNA strands have different polarities, 180rotations, leading to moebius strips, do not occur in the DNA. The linking number, Lk, is always, therefore, a positive or negative integer depending on the direction of rotation. Once the center axis of the ribbon is no longer constrained to a single plane, the linking number can be accommodated by writhe Wr as well as twist Tw. Whitem has proven that (10) In the DNA world the DNA duplex is assumed to have a definite pitch or helical repeat, h, of 10.4 basepairs per turn or 35.4 A/turn of right-handed helix and a 3.4 8, spacing per basepair along the helix axis. By convention, the linking number is replaced by a linking number difference, ALk, between the hypothetical linking number it would have if unconstrained, Lk,, and its actual value. Lk, equals the total number of basepairs N divided by 10.4. Therefore, ALk = Lk - Lk, = Lk - N/10.4. Because N/10.4 need not be an integer, ALk need not be an integer either. Changes in ALk still occur in integral steps, however. Twist, Tw for DNA is also measured as a difference from its expected unconstrained value, N/10.4: ATw = Tw - Tw, = Tw - N/10.4. Therefore, (11) For any definite circular DNA molecule, ALk is fixed, and the linking number deficit or excess in the DNA will distribute itselfby modifying the twist of the DNA duplex or by configurational modifications of the axis of the duplex to give its space-curve writhe. The relative importance of changes in twist, ATw, and writhe, Wr, in this process has been determined experimentally,16 and has been predicted by Monte Carlo simulations,32 however, no prediction is yet available from statistical mechanics. The following theory presented here achieves that goal for a restricted class of polymer configurations, those approximating the plectonemic helix and, therefore, having cylindrical symmetry about an axis in space. The dependencies of configurational potential energy on twist and writhe arc presumed to be uncoupled except by virtue of the constraint that ALk remains constant. Numerical evaluation of writhe by Eq. (7) by computer analysis for any space curve having a parametric representation in a contour parameter s is straightforward as long as care is taken with the limits of integration. The numerator and denominator of the argument of integration,f(s, ,s, ), in ALk = ATw + Wr. Lk=Tw+Wr.

x,, = X(s,1- X(s,1.

Eq. (81, both approach zero as s1 approaches s2. The argument, however, remains finite. IV. IMPORTANT ElGENSTATES AT THERMAL EQUILIBFWM Upon calculating the writhe for each of our three-dimensional eigenfunctions, a striking fact is observed. Taking some arbitrary ratio of e, to l X = ev which is far greater than 1, say 10, leads to the conclusion that the highest writhe is always found on the + 1 or - 1 off diagonals of a matrix in the variables n, and izg. This conclusion becomes even more convincing as one goes to large n. We conclude, using this set of eigenfunctions, the highest writhe for a given energy must be obtained from those functions for which n, = nY + 1 or np - 1. We are, however, required to determine the correct value for Q E,, the ratio of the major to minor axis of the plectonemic helix. It is here that Harris-Hearst theory proves useful. In terms of the eigenfunction expansion coefficients and the eigennumbers, the average length (L ) of the circular DNA molecule can be written as (L ) = j-;s (9. $) ds, (12)

m7m2(n3~2,) (13) The time-average potential energy ( V) of the molecule is (V> = Wbend/2) Gwl5~W(~)

(L>-

+ n;cq + (2)).

+ $!(6) + (6)>.
(14) )n],

The eigenvalues for the X, y, and z directions are (15) where A = y&-d. From the discussion which will follow, for molecules of large numbers of statistical lengths, the rz2 term dominates for low values of ri and the n4 term dominates at large values of 12.Small values of IZ relate to the random-walk characteristics of the distribution of elements; large values of n relate to the local bending of the stiff rod. As in all normal mode analyses, the distribution function for the expansion coefficients is just a statement of equipartitioning of thermal energy to each normal mode. From the distribution function of Harris and Hearst [Eq. (22) of Ref. 35 1 the mean-square average of each expansion coefficient is (16) 65 hE = (kT%,, I( 1/P, 1, where the subscript TE refers to a thermal equilibrium average according to Harris and Hearst. This equation is valid for a space curve with no cross-sectional area. The bending force constant, fibend,is related to the statistical length of the DNA. Again, retaining old terminology,26 the Kuhn statistical length is 1/;1 and the persistence length is l/u. The relationship between the force constant and the parameter il
is27,37

/JCL, (2%-/Jc)[n + ( l/2%q2(hL =

B bend= kT/W. (171 For the z expansion coefficient and large AL since , n, = 1, (<)TE = (1/2a)2(4;1L/3)(L3/hL ). (18)

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J. E. Hearst and N. G. Hunt: The plectonemic DNA supercoil

9325

The parameter AL 2 is discussed in detail by Harris and Hearst.35 It may be approximated by the expression AL2=(2U)2/(1-{[1-exp( -WL)]/WL)). (19) For the limit of a very large :number of statistical lengths, the only case considered in this work, AL = 4(n~C)~, or four times the square of the nurnber of statistical lengths in the circular DNA. In the random coil limit, where one ignores the n4 term in Eq. (15), ( (<).)TE/(<)rE) = n2. Table I is.a matrix of writhe values for this limiting case. These have been calculated by numerical integration using IQ. (7)) setting ez = 1, eX = l/n,, and eu = l/n,,. A circular contour of constant potential energy can be superimposed onto the matrix through any particular writhe values, for example, f 8. Only writhe values with absolute values smaller than the near diagonal values occur ,within the quarter circle indicating that there are no lower energy or equal energy states having equal or greater absolute values of writhe. This vali-

dates the conclusion that, in the random coil limit, the lowest energy state ofmaximum writhe is always the state for which n, 5 ny f 1. The constant energy contour on this matrix from Eq. ( 14) for the more general cases discussed later will occur in a field between this quadratic (circular) random coil limit and the quartic excluded volume limit which is even more restrictive in the area of accessible values of ti, and n, .

V. EXPANSION COEFFKYENTS AT HIGH WRITHE WHEN PREDICTED EQUILIBRIUM STATES ARE NOT ACCESSIBLE The more appropriate limiting case we wish to consider is one in which AL > 5 and the absolute value of the writhe densityC > 0.03 turns per turn helix. For the x and y expansion coefficients, thermal equilibrium requires that, in the limit of large n which is the useful limit for the high linking number density found for DNA in vivo,

TABLE I. Writhe matrix for the random coil.

nx
..I

1
2 3 4 5 6 7 8 9 b 10 I1 12 13 14 15 16 77 18 19 20

3 4 ::j:i::~:~$.~ .:.:.:.:.m... .

5 - 2

9 -1.

10

11

12

73

74

15

16

17

18

79

,,_.~. ~~~~~.:,:,:.:, .:.:!!.::?. :;qg$y - g; is$kJ$

1
_ ,

1
33

-1
-2 -

1
2

-1

1 2

. . . ::s:j:::.:r<< . ... ~*..~~~~.~: . . :..m~s ;;:3-~;.:p,:::,\ $$$$m 2

2 -5 -2 4

-1
1 -6 -3 -2 -3

-3 1 5 -7

-1

-1

1 1 -1

-2 -1

-1 2

-1

-1

-4

1 -1

1
2--

3 5 -2 -2 1 -4

if
2

-1

1
2 3 6 2 3 -5

:$i$@ ::::::::::-.p$ ::+:.:.:.;:.:; .%a% ...zQ+..<,r 1 .A......... g....q.q. .:p:.$ ;.py :g** J $g$jg 1 G&~~~ti .:.:.:.:.:.: _ .,...,.,, ,~, .~, .:.:.:.:.>:..:::.:
ggg$ :F;; ?z;a$$:! gj :s;p>>.,:.:r _ $&

:F$>?T;pz; :.:.:.:::~:~:t:~:~: :awa?

1 0 -G

2 -2 .l

-1

1 1 -2 4

!$z& :.:.:::::::. :.~~:::::i:::::: !+k?: $j@;$.~:; p& .::a:$: . p:

1
-,--

$Xggj. -.....:.:i :$.c

-1
2

3 -1

g$g g#$ .......,:.:,:.:. :::::::: fg y..>:.. .>..> . _ .:.:,:.!.:.:. 4jfii .......^

-2 .l g$g

7.

-1

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9326

J. E. Hearst and N. G. Hunt: The plectonemic DNA supercoil

(20) A quick calculation of the root-mean-square value of x, (x2) 1 for the conditions in which DNA is usually found, 2, leads to a value ~20 A. Since the diameter of the DNA cylinder is approximately 20 A, this result is unrealistic, indicating that the configuration of the plectonemic helix at high writhe is sterically determined and that the z axis is maximized to generate a helix with the tightest radius steritally allowed. Thermal equilibrium in the sense of Harris and Hearst can never be achieved for DNA molecules of high writhe density. Within this approximation, thermal fluctuations have only small fractional impact on the dimensions of the plectonemic helix and the helix may ,be represented by the eigenstates coming closest to the required writhe. There is thus a reduction of configurational phase space from 19,~~) the total number of degrees of freedom of the stiff chain circle which is of the order of magnitude of 3 times the number of basepairs, to a very small number of order 1. Since the thermally predicted values of the expansion coefficients are not accessible, it is necessary to relate the expansion coefficients, Ed and eu, to a distance of closest approach between DNA duplex regions. In the xy plane a typical plot of the distance between the centers of the DNA duplex in a selected eigenstate is shown in Fig. 2 as a function of s. The mean-square distance for a given eigenfunction is (21) (d2) = (4/L)(< + 6). It can further be shown that this value is a maximum if ex = 6,. We have thus established the rationale for setting

(E,&

==( 1/2;,4(4/lL/3)L

3( l/n,)4.

these two expansion coefficients equal to each other. These expansion coefficients are now considered to be constants in time. Using Eq. (2 1)) the x and y axis expansion coefficients are determined by a choice of the root-mean-square distance d of closest approach of the centers of the two helices..Because of the negative charge density on the DNA backbone, this number is not likely to get smaller than 50 A, although an experimental determination of its value is necessary and feasible. Thus, ex = ey = d( L , 1 8) 2. (22) From the constraint on L [ Eq. ( 13 ) 1, setting (L ) = L, and from the high writhe requirement that n = n, = n, * 1, the square of the z expansion coefficient becomes < = [L~/(~T)~] - [ni(n i-

1)21(L~8)(d2).

(23) For the very small values of d relative to L which we are considering, the first term is dominant even at large n. Nevertheless, this is the expression for the reduction in the z axis of the helix as the number of turns increases and its pitch decreases. Table II shows data calculated for DNA where the following parameters were used: L is in number of basepairs (bp) and, therefore, equals N, d is 15 bp, l/ii = 300 bp, and writhe = writhe densityx (3.4)N. The parameter IpWrl is the absolute value of the writhe density in units of writhe per unit length and the parameter 35.4 lpWr/ is the absolute value of the writhe density in turns writhe per turn DNA helix. VI. POTENTIAL ENERGY PER UNlT LENGTH INCLUDING BOTH WRlTHE AND TWIST For the conditions in Table II and writhe density per turn helix values greater than 0.03 and less than 0.05, it is possible to derive the following equation for the potential energy in A, pE,,, , of the plectonemic helix. The experimental approximation, that the force constants&,,, for bending and fltwist for torsion are equal, is often applied:

[2r4d2@Wr)41 +Ptwist [~~-YPATw)~I, P&t =Pbend

(24)

TABLE II. Representations for DNA plectonemic helices at physiological writhe densities. Notice that for DNA at these writhe densities, the integer index n and total writhe are nearly identical. FIG. 2. The distance between the center of the DNA duplex in constant x planes. The abscissaxis the contour parameters with range from - L /2 to 0 left to right and/or from 0 to L 12 right to left. The ordinatey is a relative distance with range from 0 to approximately 1.4d. The v) in this figure equals d The particular normal coordinate this plot applies to n, = 9, and . n, = 10. It should be noted that while v) is large enough to satisfy the steric constraints of a cylindrical structure like DNA of finite diameter d, this normal coordinate has~regions which clearly violate the steric constraint. To properly satisfy the steric contraint uniformly along the contour, linear combinations of the normal coordinates will have to be used. This issue will be addressed in detail in a future paper. Total writhe and 35.41pWr( = 0.03 in) 5.8(7) 17( 18) 29(30) 143( 145) 286(289) (integer index) 35.4)pWrl = 0.05 (n) 9.6( 10) 29(30) 47(49) 235(241) 472(482)

Ubp)

%/C

2000 6000 10 000 50 000 100000

60 180 300 1500 3ooo

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J. E. Hearst and N. G. Hunt: The plectonemic DNA supercoil

9327

where writhe density pWr and twist difference density pATw are defined as the turns of writhe and twist per unit length of DNA. The equation shows deviations at higher writhe densities because n does not predict the value of writhe at very high writhe densities where terms of order higher than the fourth power in writhe density become important. The high writhe density domain, equivalent to lower pitch helices, is discussed in some detail in the following paper.38 At lower writhe densities, thermal equilibrium begins to play a role and the number of configurational states that must be considered for the DNA grows rapidly. Equation (24)) for energy density, plus Eq. ( 11)) which provides a relationship between writhe density and twist density, allows one to predict the energy of a coil of constant linking number density but varying writhe and twist. The lowest-energy state is also predictable. Since Eq. (24) is essentially identical to a more general equation, derived in Ref. 38 by a different method, conclusions involving numerical predictions are presented there.

because the structural backbone of the DNA is thicker and stiffer, making the threading of one section of DNA into the loop of another section more unlikely. It may also provide the structural distinction by which a knot is distinguish from an ordinary supercoil. In the low-entropy limit, a supercoil has very uniform characteristics, making it readily distinguishable from a knot. It is being proposed here that the living cell is obliged to maintain a state of least DNA entanglement, and this principle of least entanglement requires that the DNA sustain a high writhe density in vivo.

ACKNOWLEDGMENTS We wish to extend our thanks to David Cook, Robert Harris, Vaughan Jones, and Paul Selvin for discussions and suggestions during the development of this theory. This work was supported in part by the National Institutes of Health Grant No. GM 41911 and by the Office of Energy Research, Office of Health and Environmental Research of the Department of Energy, under Contract No. DE AC0376SFOOO98.

VII. CONCLUSION By application of the principles of statistical mechanics in the form of Harris-Hearst theory to supercoiled closed circular wormlike coils, we have been able to predict some important properties of such coils in their plectonemic forms. ( 1) Above a minimum writhe density, the number of polymer configurational states available to the coil drops to a number near 1. This statement relates only to the configurational states within the context of the continuous model presented here. The internal vibrational and electronic states associated with the atomic structure of DNA have not been considered. (2) The writhe density that DNA has in vivo is larger than this minimum, indicating that in cells DNA has very little configurational entropy. (3) In these low-entropy states, the potential energy of the coil depends on writhe density to the fourth power and higher-order terms. It is of interest to speculate on the evolutionary advantage of low-entropy states for DNA in vivo, for the cell must expend considerable energy in order to sustain these lowentropy states. First, since DNA is compacted in volume by a factor greater than 10in living cells relative to the volume it occupies when free in solution, its entropic freedom must be greatly reduced in the cell anyway. Second, writhe density provides a possible systematic mechanism for the compaction, especially since DNA in viva is known to exist in topologically constrained domams. Finally, knotting of highly compacted DNA could present major difficulties to the processes of replication, transcription, and chromosome segregation. While enzymes exist which can unknot DNA in very dilute solutions where entropy drives knotted objects apart, the unknotting of highly compacted DNA is likely to prove far more difficult unless a local structural distinction can differentiate a knot from two DNA duplexes which happen to be close to each other. High writhe density can help solve this dilemma in two ways. It can make knotting very unlikely to start with

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