Roles of supercoiled DNA structure in DNA transactions

Roles of supercoiled DNA structure in DNA transactions Roland Kanaar and Nicholas R. Cozzarelli U n i v e r s i t y of C a l i f o r n i a , Berkeley, C a l i f o r n i a , USA The structure of supercoiled DNA has recently been ascertained using experimental and computational methods. Negative supercoiling restricts the ensemble of DNA conformations and facilitates the opening of the DNA double helix. These attributes of supercoiling are exploited in multiple ways during DNA transactions such as site-specific recombination, transposition, transcription, and the initiation of DNA synthesis. Current Opinion in Structural Biology 1992, 2:369-379

Introduction DNA supercoiling, the coiling of the double-helix axis, is ubiquitous in living cells. Supercoiling results from physical constraints such as the winding of DNA around proteins, or topological constraints, such as a linking number deficit. These constraints often occur simultaneously, because a linking number deficit greatly aids the winding of DNA around proteins. In this review, we focus on recent advances in the understanding of the structure of supercoiled DNA and on the ways in which supercoiling assists and regulates DNA transactions, particularly in the Gin DNA inversion system of bacteriophage Mu. Effects of supercoiling on transposition, transcription, and replication are more briefly discussed. We will not discuss the mechanism by which DNA supercoiling is generated in vivo [1], the supercoiling of DNA around nucleosomes [2], DNA topoisomerases [3], and the promotion of alternative DNA structures by supercoiling [4]. "1"

Definitions of topological terms We will give a brief introduction to DNA topology. A comprehensive primer that treats the topology and geometry of DNA supercoiling in a non-technical fashion is available [5]. Topological properties can only be rigorously defined for intact closed circular DNA. Linear chromosomal DNA is also supercoiled in vivo, however, because it is divided into topologically constrained do mains. The most basic topological property of DNA is its linking number (Lk). In any plane projection, the two strands of the DNA double helix will cross each other. These crossings or nodes are given a sign according to the convention shown in Fig. 1. Lk is defined as one half of the sum of these nodes. Remarkably, Lk is unchanged by all deformations that do not involve breakage of the DNA backbone. Lk, an integral topological property, is the sum of two continuously varying geometric properties, writhe (Wr) and twist (Tw), that describe the shape of DNA:

Fig. 1. Sign convention for DNA nodes. The diagram shows crossings in projection, or nodes, of the two strands of DNA double helices. By definition, both strands of the double helix have the same orientation, indicated by the direction of the arrows. The sign convention for nodes is given in terms of the rotation needed to make the arrow on top congruent with the underlying arrow. For angles < 180°, anti-clockwise and clockwise rotations define positive (+) and negative ( - ) nodes, respectively. The nodes of the double helix of B DNA are (+). Nodes also result when a DNA molecule has writhe, and their sign is defined in the same way. In a ( - ) supercoiled DNA, these nodes are ( - ) i n sign.

Lk = Wr + Tw.

(1)

Wr is a measure of the coiling of the axis of the double helix and Tw reflects the helical winding of the DNA strands around each other. The Lk of DNA in its lowest energy, or relaxed state, is designated Lk0. For relaxed DNA, Wr 0 = 0 and Tw o equals the number of base pairs divided by the helical repeat of ,-, 10.5 bp per turn. The Lk of DNA isolated from cells is less than Lk0. In order

Lk--linking number; o'--supercoiling density; T ~ t w i s t ; Wr--writhe. (~) Current Biology ttd ISSN 0959-440X

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Nucleicacids to minimize the free energy change associated with this Lk deficit, DNA adopts a supercoiled shape, thereby introducing ( - ) Wr and reducing Tw. Thus: ALk = Wr + ATw,

(2)

where ALk = Lk - Lk0 and ATw = Tw - Tw 0. Supercoiling density (c~) is often used to describe DNA supercoil ing because it is independent of DNA length: c~ = zXtk/ik0.

(3)

DNA isolated from virtually all organisms has a ~ value of - 0.06. In summary, ( - ) supercoiled DNA has a ( - ) ALk, ~, and Wr and the value of Tw is less than that of relaxed DNA.

Structure of supercoiled DNA Many reactions are strongly influenced by DNA supercoiling. Despite this, until recently, little was known about the structure of ( - ) supercoiled DNA in solution. Most standard physical methods are of limited usefulness in studying DNA supercoiling. Significant supercoiling requires a circular DNA molecule that is at least I kb in length, which is too large for high-resolution techniques such as X-ray crystallography. Measurements of hydrodynamic properties give information only about the average dimensions of DNA and show little variation with o. The structure of ( - ) supercoiled DNA has now been greatly clarified by the use of a combination of techniques, particularly electron microscopy, recombinase probing, and computer simulation.The results of these three methods agree on a number of features of ( - ) supercoiled DNA. Because the assumptions and limitations of each technique are different, this congruency gives confidence in the conclusions reached. We begin with a summary of the structural features and then give the supporting data. The results were obtained for DNA molecules at least 2.5 kb in length with cr between - 0.03 and - 0.07 in solutions of at least moder ate ionic strength ( > 0.05 M NaC1). The first five features were demonstrated by more than one technique. Deftnitions of structural parameters of DNA supercoiling are given in Fig. 2. (a)

(b)

(c) (d) (e)

Supercoiled DNA conformations are plectonemic (interwound) and frequently branched. Extensive solenoidal supercoiling is absent. Supercoiling diminishes both Wr and Tw in proportion to ]o~ so that Wr/ATw remains constant at ~ 3. The superhelix axis length is independent of (~ and is ,--40 % of the DNA contour length. The superhelix diameter varies inversely with (~. For (~ = 0.06, it is ~ 1 0 n m . The number of supercoils is directly proportional to Iol and equals ~0.9ALk.

The following are results of a computer simulation of ( - ) supercoiled DNA that have not yet been verified experimentally [6o.].

Fig. 2. Model of supercoiled DNA. A DNA double helix, represented by a line, is shown in a plectonemic (interwound), ( - ) supercoiled conformation. The superhelix is idealized so that its structure is completely regular. The molecule has one branch point and three superhelix ends. The superhelix diameter (D), the superhelix axis (dashed line), and a single superhelical turn (bracket) are indicated.

(f)

(g)

Supercoiling increases the local concentration of two DNA sites in a DNA molecule by two orders of magnitude. Branching of the superhelix is enthalpically unfavorable but is driven by entropy.

A thorough experimental study of DNA supercoiling by Boles et al. [7] examined 3.5 kb and 7.0kb plasmids whose cr varied from 0 to -0.12. The length of the superhelix axis and the number of supercoil nodes were determined by electron microscopy. Using another method, supercoiled nodes were converted by recombination by the phage ~. Int system to catenane or knot nodes. The number of catenane and knot nodes could be measured accurately and used to calculate the number of plectonemic supercoils in the substrate. Assuming a regular superhelix geometry such as that in Fig. 2, these data were used to obtain conclusions ( a ) - ( e ) above. In preparation for conventional electron microscopy, DNA is dried, flattened, stained and shadowed. It has been shown, nonetheless, that this makes no significant change in the Tw and Wr of relaxed DNA [7]. Thus, mi croscopy is a valid measure of some properties of supercoiled DNA, particularly if the c~ of the samples is varied systematically. A promising but technically demanding alternative to conventional microscopy is cryo-electron microscopy of vitrified samples. The key advantage is that DNA is viewed without staining, shadowing or flattening onto a grid. DNA supercoils viewed by this method are also plectonemic [8]. The diameter of the superhelix decreased approximately threefold when 10mM MgC12 was added to a 10mM Tris buffer. The conclusion that the conformation of supercoiled DNA is sensitive to ionic conditions

Roles of supercoiled DNA structure in DNA transactions Kanaar and Cozzarelli

agrees with other results cited below. Extensive measuremerits of supercoiling parameters were not made, but the results are qualitatively similar to those obtained by Boles et al.

[7].

A limitation of these experimental approaches is that an idealized regular geometry for the superhelix must be assumed in order to calculate quantities that cannot be measured directly. The theoretical modelling of supercoiling overcomes this limitation and, in principle, provides precise information about conformational and energetic equilibrium distributions. Three complementary computer simulations of DNA supercoiling have been performed recently. Of the three studies, Tan and Harvey [9] used the most detailed model of the double helix. Each base pair was represented by three particles in a plane, and double helical parameters such as Tw, roll and rise were explicitly considered. A particular advantage of this approach is that the results are sensitive to the effects of local DNA sequence variations, such as intrinsically bent regions. Because the model involved a large set of structural parameters, the calculations were restricted to small DNA circles. Hao and Olson [10] described the first Monte Carlo treatment of highly supercoiled DNA that involved an elastic model for the double helix. Subsequently, Schlick and Olson [11"] developed techniques for investigating both the dynamics and the energetics of supercoiling. In these simulations, DNA adopted continuously bending structures. The computational approaches described so far sought the minimum elastic energy conformation and found an interwound superhelix. Determination of the average properties of supercoiled DNA, however, can be obtained only by generating an ensemble of DNA conformations at thermal equilibrium. This was achieved by Metropolis/Monte Carlo investigations that modeled DNA as a wormlike chain, whose conformation is determined by DNA bending, torsional rigidity, and the effective double-helix diameter [6o.,12]. Branched plectone mically supercoiled conformations predominated. There was a close agreement with the experimentally determined values of Wr, superhelix axis length and number of superhelical turns as a function of cs. The simulations indicated that two opposing factors determine branching: the bending energy needed to form the branch, and the expanded set of conformations that branching affords. Branching varied sharply with cs, DNA length, and effective double-helix diameter. The free energy of supercoiling was shown to vary quadratically with cs only under certain conditions. Moreover, at low Id, the entropic contribution to superhelix free energy was negligible, whereas at high Io~, the entropic and enthalpic contributions were nearly equal. Supercoiling significantly changed the spatial distribution of DNA sites. The proba bility of juxtaposition of any pair of DNA sites, separated along the chain length by at least 150 bp, was two orders of magnitude greater in ( - ) supercoiled DNA than in relaxed DNA. The local site concentration in ( - ) supercoiled DNA was not strongly dependent on the contour separation of the sites. A critical parameter in the modeling of DNA is the effective diameter of the double helix. This excluded volume

parameter can be larger than the geometric diameter of 2 nm because of electrostatic repulsion caused by the negatively charged backbone of DNA. Monte Carlo calculations have revealed that the knotting probability during cyclization of linear DNA is highly sensitive to the effective double-helix diameter [13]. By comparing the computed knotting probabilities with the experimentally determined values, the variation of effective double-helix diameter with NaC1 concentration was determined. Independent measurements ( W Rybenkov, NR Cozzarelli and AV Vologodskii, unpublished data; S Shaw and J Wang, personal communication) agree very well with theoretical predictions [14], and with a previous measurement based on the osmotic pressure of highly concentrated DNA solutions [15]. The effective double-helix diameter was strongly influenced by electrolyte concentration, increasing from 3 nm in 1 M NaCl to 15 nm in 0.01 M NaC1. Hearst and Hunt [16.,17] have sought to rdate the conformations of supercoiled DNA to fundamental physical principles. A statistical mechanical treatment led to the conclusion that highly supercoiled DNA has very little configurational entropy [16.]. In another study, DNA was treated as an elastic rod, and supercoiling was modeled as an infinitely long, unbranched superhelix of constant diameter [17]. The authors calculated that the plectonemic form of supercoiling was energetically favored over the solenoidal form, and that there was a strong dependence of supercoil properties on ionic conditions. The calculated values for Wr were somewhat higher than in the other studies, perhaps as a result of the simplifications in the modeling.

Roles of DNA supercoiling in site-specific recombination Site-specific recombination, the reciprocal exchange between specific DNA sequences, exploits the properties of DNA supercoiling to achieve high efficiency and selectivity. Site-specific recombinations can be divided into two stages: synapsis and strand exchange. In synapsis, two distant recombination sites are brought together in a highly organized nucleoprotein complex. During strand exchange, both DNA sites are cut, crossed over, and religated. Depending on the connectivity of the recombination sites, inversion, deletion or intermolecular fusion results (Fig. 3). These DNA rearrangements have a wide variety of biological functions that include differential gene expression, resolution of transposition intermediates, plasmid amplification, and viral integration into and excision from host chromosomes [18-22]. We will discuss the multiple roles of DNA supercoiling in site-specific recombination, drawing examples primarily from the Gin DNA inversion system of phage Mu and also including the Hin and Tn3/75 resolvase site-specific recombination systems, and the phage Mu transposition system. Recombination by the Gin system is restricted to inversion and requires three specific sites on a ( - ) supercoiled DNA molecule and two proteins [22]. The phage-encoded Gin protein binds to the two inversely repeated recombination sites, called gix, and the host-

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~~"~,j~ Inversion~ Deletion Fusion

Fig. 3. Connectivity of recombination sites and DNA rearrangements. The diagram shows plasmid DNAs, represented by circles, containing recombination sites (split arrows] and markers (a, b, c and d). The directionality of the recombination sites is indicated by the arrowheads. Inversion of the sequence between the sites results from recombination within a plasmid containing inversely oriented sites. Deletion of the sequence between the sites, frequently called resolution, results flora recombination within a plasmid carrying directly oriented sites. The reverse reaction results in the intermolecular fusion of two DNA molecules.

encoded Fis protein binds to the recombinational en hancer. A synaptic complex containing all these elements is shown in Fig. 4 and further details of the system are

given in the figure legend. The Hin system is homologous to the Gin system and has similar requirements [22]. In contrast to these DNA inversion systems, the Tn3/78 resolvase system only mediates deletion. The requirements for this system are the resolvase protein and two directly repeated r e s sites in a ( - ) supercoiled DNA molecule [19,23]. The initial steps in Mu transposition require MuA transposase, the host encoded HU protein, ( - ) supercoiled DNA, two inversely oriented sites ~t the ends of the phage genome, and an internal enhancer element [24-27]. Negative supercoiling in recombination is postulated to: lower the free energy of binding of recombination proteins; provide proper recombination site alignment and interwinding; raise the effective concentration of the par ticipating DNA sites; provide energy for double helix un winding; and, promote directional strand exchange. We will examine each of these effects in turn. Free energy of binding of recombination proteins Negative supercoiling has long been known to reduce the free energy of binding of proteins that reduce Tw or introduce ( - ) Wr [28]. This is because the ( - ) supercoils cancel out the positive ( + ) supercoils that compensate for the DNA distortion resulting from protein binding. In the phage g, Int system, the formation of the nucleoprotein complex at the phage recombi nation site is promoted by ( - ) supercoiling and the DNA is thought to be ( - ) supercoiled around the proteins [20,21,29], Topological experiments proved that two ( - ) supercoils were entrapped by the Gin synaptic complex [30--32] and three by the resolvase complex [33-35]; thus, ( - ) supercoiling is expected to greatly assist synapsis. The binding of proteins that bend DNA

[ Fig. 4. The Gin synaptic complex. (a) A branched ( - ) supercoiled substrate DNA is shown with the split arrows representing the gix sites bound by Gin, and the grey box indicating the enhancer bound by Fis. The gix sites and the enhancer interwind at a branch point such that two ( - ) supercoils are entrapped by the synaptic complex [30-32]. The 21 kD Gin protein contains the active center that cleaves and religates the 34 bp gix sites [38]. The 11 kD Fis protein binds to two 12 bp sites located within the 60 bp enhancer [22]. (b) Electron micrograph of a Gin synaptic complex, which appears as an electron-dense mass at a branch point in ( - ) supercoiled DNA [60,61]. Bar = 1 lam.

Roles of supercoiled DNA structure in DNA transactions Kanaar and Cozzarelli

without altering Wr and Tw may also be favored by su percoiling, because DNA bends can reduce the enthalpic cost of a superhelix end [6°%28]. This can be important because many recombination proteins bend DNA [20]. A synergistic effect of DNA bending and DNA supercoiling was observed in phage Mu transposition [27]. At low ]¢~, the reaction became dependent on the DNAbending protein-integration host factor, a DNA-bending protein that binds to the enhancer.

more complex than gix. The arrangement of these binding sites renders the inverted-sites complex unproductive because the crossover regions are misaligned [34,42}.

Site alignment and interwinding, and local concentration of DNA sites

[36].

For the site-specific recombination systems whose action is restricted to either inversion or deletion, ( - ) supercoiling plays a key role in this selectivity. As illustrated in Fig. 5, DNA supercoiling is thought to favor energetically and/or kinetically the particular configuration of the sites required for the formation of a productive wnaptic complex [19,36,37]. Negative supercoiling of the substrate DNA favors parallel alignment only if the number of supercoils entrapped by the synaptic complex is even with inverted sites and odd with direct sites (Fig. 5a). The same orienting effect of supercoiling favors an antiparallel alignment if the number of supercoils entrapped by the synaptic complex is odd with inverted sites and even with direct sites (Fig. 5a). In order to change the alignment of the sites in the synaptic complexes shown, an additional supercoil has to be introduced, which is energetically unfavorable. In order to obtain a nucleotide sequence rearrangement, the recombination sites in the synaptic complex must be aligned in parallel. Figure 5(b) shows how antiparallel alignment of ggx sites precludes the rearrangement of the nucleotide sequence. Gin makes staggered breaks in the gix sites [38], and strand exchange can be described by a 180° rotation of the cut gix sites before ligation [30]. The antiparallel alignment of the gix sites demands an even number of strand exchanges before complementarity of the bases in the c ~ossover region is achieved and ligation can occur. An even number of strand exchanges will always rejoin the broken strands to their original partners and thus no cearrangement in nucleotide sequence occurs. Recent experiments provide strong support for this postulated relationship between the number of supercoils entrapped by the sTnaptic complex and the resulting DNA rearrangement. Gin synaptic complexes could form on ( - ) supercoiled DNA containing directly repeated gbc sites [32]. This complex entrapped two ( - ) supercoils, just as the complex with inverted sites. Remarkably, not only did the complex form, but strand exchange occurred at the same rate as with the inverted site synaptic complex. No nucleotide sequence rearrangement resuited, but the reaction was detected by the formation of DNA knots with a characteristic topology. Similar observations have been made for Hin [39,40] and resolvase [41] reactions catalyzed between recombination sites differing in the central dinucleotide of the crossover region. Tn3 resolvase only recombines directly repeated sites, but forms a synaptic complex containing three ( - ) supercoils with both direct and inverted res site substrates [34]. The multiple resolvase binding sites in res make it

The first in-depth study of the kinetics of resolvase recombination suggested that DNA supercoiling provides a kinetic assist to productive site synapsis [43°*}. Synapsis was shown to take place at a rate greater than that calculated for the random association of protein-bound sites. It was suggested that the sites synapse by an ordered motion directed by supercoiling, such as slithering The results of simulations of supercoiled DNA conformation help rationalize this assist, if effective site concentration is rate-determining. Supercoiling increases by two orders of magnitude the local concentration of two DNA sites in cis [6"]. Sites in these simulations were treated as points. If the directional effect of supercoiling on the alignment of the sites is also considered, the increase in effective concentration would be even greater. For the Gin system, ( - ) supercoiling should have an even larger effect on the effective site concentration because not two but three sites interact simultaneously. Recent elegant experiments on the initial steps of Mu transposition illustrate the importance of local site concentration [44"]. The usual cis-requirement for the enhancer was bypassed by a 40-fold molar excess of enhancer DNA. Under these conditions, the reaction rate was reduced 25-fold. The interpretation now is that synapsis is dependent on the three-dimensional diffusion of the enhancer DNA in trans rather than the one-dimensional diffusion in cLs. Dissection of multiple roles of DNA supercoiling in recombination was achieved by the use of catenated DNA substrates [31,37,45]. Gin and resolvase will not fuse two DNA circles containing a single recombination site each. In this trans configuration, the effective site concentration is several orders of magnitude lower than in the cis configuration on ( - ) supercoiled DNA under typical reaction conditions [6*']. Cater'~tion of two DNA rings containing single recombination sites will greatly increase local site concentration. As illustrated in Fig. 6, multiply interlinked catenanes can provide the oriented interwound DNA structure of supercoiled DNA in addition to high local site concentration. The increase in site concentration afforded by singly linked catenanes was sufficient for the function of some transcriptional enhancers [46,47]. For Gin and resolvase recombination, multiply linked catenated substrates were fully active, whereas singly linked catenanes weze completely inactive [31,45]. This suggests that, in addition to concentrating the recombination sites, ( - ) supercoiling is important in providing the correct interwinding of the sites in the synaptic complex. We conclude this section by considering in more detail how the requirement for specifically interwound sites in a ( - ) supercoiled substrate provides an elegant way to limit Gin recombination to inversions. We hypothesize that the Gin-bound gix sites synapse first, but that strand cleavage requires a transaction with Fis and the enhancer. We show in Fig. 7, hypothetical complexes on

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Site alignment Antiparallel

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Site connectivity

Inverted

Parallel sites 5' h

~5'

I--A ,A-T d-& G - C ~'

T A

A T

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Cut

Align

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Rotate 180 ° ligate

Antiparallel sites

T A

Align

Cut

C G

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AC

Rotate 180 °

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T-A

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H; Ro~

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ligal~

an inverted-site substrate that entrap up to tour ( - ) supercoils between the gix sites. The only complex that allows the enhancer bound by Fis to readily interwind with the Gin-bound gix sites is the one that entraps two ( - ) supercoils. Only in this complex does the branching provided by ( - ) supercoiling allow the additional slithering motion that brings the enhancer into the complex. Alternative complexes with a ( - ) supercoiled substrate DNA containing inverted, direct, or intermolecular sites cannot readily include Fis and the enhancer and are thus inactive. The only direct-site complex that can be activated in this

Fig. 5. Relationship between recombination site alignment, recombination site connectivity, and ( - ) supercoils trapped by the synaptic complex. (a) The diagram shows ( - ) supercoiled plasmid DNAs containing two recombination sites (split arrows). The shaded region represents the synaptic complex. For DNA substrates with directly repeated sites, trapping of an odd number of supercoils in the synaptic complex causes a parallel alignment of the recombination sites. Entrapment of an even number of supercoils causes antiparallel alignment. For inverted sites the opposite holds true. Examples with one and two trapped ( - ) supercoils are shown. The number of supercoils trapped by the synaptic complex is operationally defined as the number of supercoil nodes that would remain after nicking the substrate without dissociating the synapsed sites. (b) An illustration of the consequences of parallel and antiparallel site alignment for strand exchange in the Gin system. DNA is represented by bold and thin ladders in which the rungs represent base pairs and the circles the 5' termini. A gix site consists of two inversely oriented half-sites separated by a 2bp crossover region. Across this region a 2 bp staggered break is made during recombination [38]. The only asymmetric feature in gix is the nucleotide sequence in the crossover region, which gives a direction to gix (arrow). (i) Parallel aligned sites (Align). After strand cleavage (Cut), strand exchange takes place by a 180° rotation of the broken strands around each other, and a recombinant is obtained by ligation (Rotate 180°, Ligate). (ii) Antiparallel aligned sites (Align). Strand cleavage occurs as above (Cut). After a 180° rotation of the strands, however, ligation cannot occur because the bases in the crossover region are non-complementary (Rotate 180°). After an additional "180° rotation, ligation can take place (Rotate 360°, ligate). Note that the product is identical in primary sequence to the substrate.

model is one that entraps two ( - ) supercoils and thus has antiparallel sites (Fig. 5).As discussed above, Gin and Fis do indeed activate this complex but no sequence rearrangement can result. Consistent with this model for recombination selectivity is the phenotype of mutants of Gin that have lost the dependence on Fis, the enhancer, and ( - ) supercoiling: they no longer display site-orientation selectivity [48]. The explanation is that these mutants form multiple productive complexes that differ in the number of entrapped

Roles of supercoiled DNA structure in DNA transactions Kanaar and Cozzarelli

(a~

(b)

Fig. 6. Comparison of singly and multiply linked catenanes. Singly linked (a) and multiply linked (b) catenanes differ fundamentally in that there is an intrinsic relative orientation to the two rings only for the multiply linked catenane. Both forms are shown with a parallel orientation, indicated by the arrows, but the singly linked catenane can be converted to the antiparallel orientation by 180° rotation of one of the rings about a horizontal axis through the centers of the rings. In addition, the single interlock does not cause writhe in each ring, whereas multiple interlocks do [51]. In both these properties, multiply linked catenanes mimic the oriented interwound structure of ( - ) supercoiled DNA.

supercoils (NJ Crisona, A Klippel, R Kanaar, R Kahmann and NR Cozzarelli, unpublished data). Similarly, Mu transposition lost site-orientation selectivity under conditions that bypass the requirement for ( - ) supercoiling [26]. Site-specific recombination enzymes such as phage )v Int that mediate inversion, deletion and intermolecular fusion do not entrap a fixed number of plectonemic supercoils between their DNA sites [7,36]. Every synaptic complex formed by collision of the sites is productive. This property has made Int an excellent probe of supercoiled DNA structure in vivo and in vitro [7]. Double-helix unwinding The use of catenated DNA substrates has revealed yet another requirement for DNA supercoiling in Gin and resolvase recombination. The DNA rings of the multiply linked catenated substrates still needed to be supercoiled to support recombination [31,45]. Multiply linked DNA catenanes dissociate the two key attributes of ( - ) su percoiling. They provide an oriented interwound DNA conformation but little or no energy for double-helix unwinding, because Tw and catenane-induced Wr are not directly interconvertible, as is the case with supercoiled DNA. Thus, an additional required role of supercoiling, not shared by catenation, could be to assist the unwinding of recombination sites. Recent experiments suggest that localized unwinding of gix sites does occur and that this may be important for recombination (A Klippel, R Kanaar, R Kahmann and NR Cozzarelli, unpublished data). Mutants of Gin that carry out recombination in the absence of ( - ) supercoiling, Fis and the enhancer [48] unwound each gix site by approximately one half turn upon binding. Wild-type Gin did not unwind the gix site. In this case, the unwinding may depend on the inclusion of Fis and the enhancer in the synaptic complex. Localized unwinding could stim ulate recombination by disrupting base pairing in the crossover region and aiding juxtaposition of strands in

the ligation step. The crystal structure of the catalytic domain of 78 resolvase, which has been solved to 2.7A resolution, placed the two active-site serine residues too far apart to make a 2 bp staggered break in DNA [49,50]. Bending of r e , site DNA combined with a partial unwinding of the site may help position the serines more favorably (P Rice and T Steitz, personal communication).

Directionality of strand exchange The topological changes accompanying recombination have shown that, in a ( - ) supercoiled substrate DN& the direction of strand exchange for both Gin and resolvase is always right-handed [30,32,33,35]. A right-handed ro ration is defined as one that results in right-handed turns in the DNA (Fig. 8). Several experiments have shown that ( - ) supercoiling, and not the recombination proreins, impose this directionality. Mutants of Gin that no longer require ( - ) supercoiling mediated both rightand left-handed strand rotations with relaxed DNA, but only right-handed rotations with ( - ) supercoiled DNA (A Klippel et al., unpublished data). Resolvase performed left-handed rotations under conditions that bypassed the requirement for ( - ) supercoiling [35]. The rather subtle mechanism of the directional promotion of strand exchange was revealed by the study of processive recombination [32]. During this process, multiple strand exchanges occur without breakdown of the synapsed recombination sites (Fig. 8). In order to evaluate the driving force that DNA structure provides for processive recombination, we shall consider the changes in DNA coiling while the gix sites are still synapsed. The synaptic complex divides the substrate DNA into three domains, labeled A, B and C (Fig. 8). In each round of fight-handed strand exchange,-domains A and B interwind once to form a ( - ) node while the DNA double helix in domains A and B overtwist by one half turn, creating two + 1/2 nodes. A left-handed rotation causes a ( + ) node between domains A and B and two - 1 / 2 nodes in each domain. Processive recombination occurs efficiently and unidirectionally because the nodes introduced by strand exchange make different contributions to the free energy of DNA. The nodes introduced by each round of strand exchange that are between domains A and B are catenane-like nodes as long as the synaptic complex is intact. These nodes should thus be energetically similar for strand rotations in either direction. The nodes put into the double helix of domains A and B are Lk nodes. In ( - ) supercoiled DNA, these nodes are energetically favored if ( + ) , as in right-handed rotations, and disfavored ff ( - ) , as in left-handed rotations. This explains why right-handed rotations would be greatly favored by ( - ) supercoiling over left-handed rotations. If the catenane-like nodes between domains A and B and the supercoil nodes cancelled out by strand exchange are energetically equivalent, however, the free energy change for recombination would be approximately zero. The deformation caused by catenation is proportional to the number of interlocks minus one [51]. If this is also true for the catenane-like nodes from recombination, any number of rounds of right-handed processive recombination could, in effect, relax one supercoil.

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-1

-2

-3

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Fig. 7. A model for Gin recombination selectivity. The diagram shows a single ( - ) supercoiled DNA substrate for Gin with several different configurations of the gix sites. The symbols are the same as in Fig. 4 and the shaded region represents the synaptic complex. Two different movements of the DNA can change the positions of the Gin-bound gix sites [36]. One is slithering, the sliding of DNA segments past each other, which can also cause extrusion and resorption of branches. Slithering will generate an ensemble of DNA conformations, examples of which are shown on the left. The second motion, bending of the superhelix axis, allows synapsis of the Gin-bound gix sites, shown in the middle. The varying number of supercoils trapped in the depicted complexes is indicated on the far riglqt. Only the complex entrapping two ( - ) supercoils can be readily activated by inclusion of Fis and the enhancer. In this complex, the enhancer can interwind with the gix sites at a branch point, as shown on the right. The other DNA conformations shown need not be unbranched, but are depicted that way for simplicity.

DNA supercoiling and other DNA transactions Analogous to site-specific recombination and transposition, transcription and the initiation of DNA replication are mediated by highly organized protein-DNA complexes [29]. These complexes also capitalize on the conformation of ( - ) supercoiled DNA and its promotion of unwinding of the double helix to initiate their transactions with DNA. Recent results have demonstrated the importance of the ends of the interwound DNA superhelix for transcription. Escherichia coli RNA polymerase and certain transcrip tion factors tend to localize in this region. Because the su perhelix end is already folded back upon itself, it readily accommodates bent DNA structures and protein-DNA complexes that bend DNA. The first clear demonstration that intrinsically bent DNA sequences localize at the ends of the DNA superhelix was provided by eleo

tron microscopy [52]. Direct visualization of an E. coli RNA polymerase--promoter complex by cryo-electron microscopy showed that it too was located at a su perhelix end [53"]. Interestingly, this location of RNA polymerase was maintained during transcription. Studies on transcription initiation at the lac promoter suggested that localization of the highly bent catabolite actb vator protein-RNA polymerase-promoter complex at an end of the superhelix may facilitate the unwinding of the promoter to form an open transcription initiation complex [54"*]. Catabolite activator protein and its binding sites could, to some extent, be replaced by appropriately phased intrinsically bent DNA, but only if the DNA was ( - ) supercoiled [55"]. The semi-conservative replication of double stranded DNA requires the complete unwinding of the double he lix [33]. DNA replication, like transcription, is controlled by initiating the unwinding at a specific site, the origin

Roles of supercoiled DNA structure in DNA transactions Kanaar and Cozzarelli

/

),

"I !

!

Fig. 8. DNA coiling during processive recombination by Gin. Double helical DNA is schematically represented by a ribbon in which the edges (black and grey lines) are the complementary strands. One side of the ribbon is white, the other grey. All supercoils except the two stabilized by synapsis of the gix sites have been removed for clarity. The gix sites are represented by the split arrows and the enhancer by the grey box. The three domains emanating from the synaptic complex are labeled A, B and C. Each round of recombination overtwists the two strands of the double helix in domains A and B (+ 1/2) and creates a ( - ) node by crossing these two domains ( - 1). Two rounds of recombination involving successive 180° right-handed rotations of the strands are indicated.

of replication. The initiation of DNA replication in many organisms begins with the localization of the origin by site-specific DNA-binding proteins [29,56]. In E. coli, the DnaA protein initially assembles at oriC a structure consisting of DNA wrapped around multiple copies of the protein [57]. This DNA wrapping raises the local superhelix density and favors the subsequent unwinding of the DNA double helix in a region adjacent to the DnaA binding sites [58]. Unwinding initiates in this region because the energy required to unwind is so low that it could be provided by high ( - ) supercoiling alone [59]. The DNA unwinding creates an entry site for a DNA helicase whose action allows the elongation stage of DNA replication to begin.

Conclusions The study of the conformation of supercoiled DNA by electron microscopy and topological techniques has recently been effectively complemented by computer simu lations. The resulting detailed picture of the structure of supercoiled DNA illuminates the multiple roles of DNA

supercoiling in site-specific recombination, transposition, transcription, and the initiation of DNA replication.

Acknowledgements We thank R Kahmann, A Klippel, P Rice, T Steitz and J Wang for sharing unpublished data. We thank C Boles for the electron mi crograph shown in Fig. 4b. This work was supported by National Institutes of Health grants to NR Cozzarelli. R Kanaar is a fellow of The Jane Coffin Childs Memorial Fund for Medical Research. This investigation has been aided by a grant from The Jane Coffin Childs Memorial Fund for Medical Research.

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R Kanaar and NR Cozzarelli, Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California at Berkeley, 401 Barker Hall, Berkeley, California 94720, USA.

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