Proc. Nati. Acad. Sci. USA Vol. 82, pp.

1079-1083, February 1985 Biochemistry

Determination of the stereostructure of the product of Tn3 resolvase by a general method

(site-specific recombination/partial denaturation mapping/catenanes/RecA protein/strand exchange)

STEVEN A. WASSERMAN AND NICHOLAS R. COZZARELLI

Department of Molecular Biology, University of California, Berkeley, CA 94720

Communicated by Gunther S. Stent, October 22, 1984

ABSTRACT A method has been developed for determination of the absolute structure of DNA catenanes. The catenated DNA is partially denatured before being thickened with a coating of RecA protein and spread for electron microscopy. This treatment allows visualization of the orientation of each ring as well as identification of the overlying and underlying DNAs at crossing points. These determinations define the topology of a catenane, providing a powerful means for testing mechanisms of catenane-producing enzymes in DNA recombination and replication. The technique was used to show that the single interlock of the catenated products of site-specific recombination mediated by Tn3 resolvase is exclusively of negative sign. The unique topology of the products indicates that resolvase fixes the sum of the number of supercoils between recombination sites at synapsis and the number of such supercoils lost or gained during strand exchange. The data strongly suggest that there are in fact three negative supercoils between synapsed sites; one supercoil is dissolved in the cross-over mechanism, whereas the other two are metamorphosed into the unique catenane interlock.

FIG. 1. The two types of singly interlinked catenanes. Molecules shown in plane projection with the orientation at each node (crossing of 2 DNA duplexes) represented by an arrow. The sign of each node is assigned by convention: If the overlying arrow can be aligned with the underlying one by a clockwise rotation of less than 1800, the node is assigned a value of -1. If a counterclockwise rotation can produce alignment, the node is given a value of +1. Note that the two catenane types are identical except for the relative orientation of the rings.
are

Interlocked rings of DNA, or catenanes, arise frequently in both prokaryotic and eukaryotic organisms. Catenanes are formed in vitro and in vivo in the course of replication, recombination, and topoisomerase action (1-3). The structure of catenated DNA reflects the mechanism of its creation. Analysis of catenane topology has the potential, therefore, to help delineate the pathways of cellular DNA organization and rearrangement (4-7). Realization of this potential has been limited, however, by the lack of an analytical method capable of distinguishing each of the myriad possible types of catenanes. In reporting the development of such a method, we focus here on the catenated products of intramolecular recombination mediated by Tn3 resolvase. In an in vitro reaction equivalent to the cointegrate resolution step of transposition, resolvase recombines directly repeated resolution (res) sites in a supercoiled plasmid (8). The predominant product is a catenane of two singly interlocked rings (9) for which the stereostructure, the configuration of the interlock, is unknown. As shown in Fig. 1, the two possible types of singly interlocked catenanes are indistinguishable unless one knows both the overlap and orientation at the nodes (crossings of DNA segments) that comprise the interlock. The sign of a node is fixed by the convention shown in Fig. 1; a singly interlocked catenane has two nodes of the same sign. In theory, the single interlock of the catenated resolvase product could be exclusively of either sign. Alternatively, the product could be a mixture of the two catenane types. To distinguish among these possibilities, we developed a means of orienting DNA rings in an electron micrograph in which
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could also observe the path and overlap of the DNA. We considered decoration with nucleic acids or sequencespecific binding proteins to reveal sequence orientation but chose instead partial denaturation of the catenane DNA as an established and easily generalized method (10). Denaturation bubbles can be mapped to particular adeninethymine (A-T)-rich regions of a DNA, allowing orientation of the molecule. For visualizing DNA path and overlap, we used the recently developed RecA protein-coating technique (11). Coating of DNA with RecA heightens micrograph contrast, making it possible to distinguish the overlying and underlying strands at a node. By wedding partial denaturation mapping to RecA-coating, we have developed a general method for determining the structure of catenanes. We show here that the singly linked catenanes produced by Tn3 resolvase are of one sign and that their topology delimits steps in the mechanism of DNA strand recombination.
we

MATERIALS AND METHODS Preparation of Partially Denaturated DNAs. The substrate, pA2, is a head-to-tail dimer of the res site-containing plasmid pA (12). pA2 DNA was either digested with Nde I to yield monomer-length [3200-base-pair (bp)] linear molecules or reacted with Tn3 resolvase as described elsewhere (12). The catenated resolvase products were nicked once per recombinant ring with either DNase I (13) or Bgl I (14) in the presof ethidium bromide. Partial denaturation followed the procedure of Johnson (15): DNA (2 jig) was reacted at 57C for 20 min in 30 ,u of 10 mM potassium phosphate buffer (pH 7.6) containing 1 mM EDTA, 50% (vol/vol) ethanol (Gold Shield, Hayward, CA), and 0.1 M deionized glyoxal. The DNA then was passed over a 300-,ul Sepharose CL-4B (Pharmacia) column in 10 mM Tris Cl, pH 7.6/1 mM EDTA. Coating of DNA with RecA Protein. Partially denatured
ence

Abbreviation: bp, base pair(s).

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DNA was reacted with Escherichia coli RecA protein (gift of K. McEntee) by a slight variation of the method used by Koller et al. (16) for coating single- or double-stranded DNA. Successful reaction required premixing DNA (2 ,ug/ml) and RecA (65 ,&g/ml) for 5 min at room temperature in 25 mM triethanolamine chloride (pH 7.2). Magnesium acetate (2.5 mM) and adenosine 5'-[y-thio]triphosphate (0.5 mM) then were added, and the 25-/.l mixture was incubated at 370C. After 40 min the reaction was fixed by addition of one-ninth volume of 2% glutaraldehyde (Polyscience, Warrington, PA, EM grade) in 75 mM Hepes (pH 7.5). After an additional 15 min at 370C, the RecA-coated DNA was passed over a 300-/Al Sepharose CL-4B column in 5 mM magnesium acetate. Electron Microscopy. Preparation of shadowed grids for electron microscopy followed the general method of Williams (17). Samples (5 Al) of RecA-coated, partially denatured DNA were applied directly to carbon-coated specimen grids made hydrophilic by glow discharge. Grids then were stained with uranyl acetate and shadowed in two stages first rotary and then unidirectional-at a 70 angle and a source-to-sample distance of 9.6 cm. Micrographs were obtained using a Japan Electronic Optics Laboratory 100-B electron microscope with 25,000-fold primary magnification. The assignment of sign to catenane nodes depends on the shadowed image in the electron micrograph being a noninverted replica. This was ensured in two ways. First, by placing the grid in the microscope with the shadowed surface facing up (toward the electron source) and the negative in the enlarger with the emulsion side up (toward the light source), the stereostructure of the catenane is correctly preserved. Second, the visible right-handed helicity of shadowed DNARecA complexes (18) was used to verify the fidelity of all micrographs. Denaturation bubbles sometimes spanned the intersection of catenated DNA rings. Such intersections, which were treated as single nodes, provided an internal control for assignment of DNA overlap. Denaturation Maps. An experimental denaturation map was constructed from measurements obtained with a Numonics graphics calculator. The length distribution of partially denatured DNA was sharp; on average the two rings of a catenane differed in length by only 4.1%. All molecules therefore were normalized to equal length. For comparison, a denaturation map was generated from the nucleotide sequence of pSlA (refs. 19 and 20; R. Reed,

Funnell and Inman (21). This method is based on the correlation between regional A-T content and susceptibility to denaturation. A plot of the sliding average of A'T content over 360 bp (mean observed denaturation-bubble length) forms the predicted denaturation profile.

personal communication) by the computational method of

RESULTS Partial Denaturation Map of res-Containing Plasmid. Orientation of a circular molecule in a micrograph requires at least two distinguishable or three indistinguishable landmarks arranged asymmetrically on the DNA. We therefore first established a monomer denaturation map with the requisite features and then used it to orient each of the identical rings in a catenane produced from a dimeric substrate. The substrate, pA2, was cleaved with Nde I into monomer-length linear molecules and then thermally denatured in the presence of glyoxal. Glyoxal reacts with single-stranded DNA and thus prevents reassociation of the duplex (15) but fortunately did not block subsequent RecA protein-coating of the treated DNA. After denaturation for 20 min, a large proportion of the complexes of RecA protein and DNA had the appearance of the molecule shown in Fig. 2. Regions of denaturation are clearly identifiable as bubbles (pairs of bifurcations) in the linear complexes. The location of the denatured regions in 32 such molecules is plotted in Fig. 3; single- and double-stranded DNA are of identical unit length under the conditions used for coating with RecA protein (16). The three single-stranded sections of each molecule were asymmetrically distributed and could each be identified by their position relative to the three nondenatured sections of the molecule. In terms of a circular map, the double-stranded regions occupied 20, 6, and 40% of the monomer length (Table 1). In all cases, the size of a double-stranded region fell outside the ranges observed for the other double-stranded regions. Thus, a single denaturation pattern was seen for all 32 specimens. Fig. 3 also confirms that the location of denatured regions can be predicted accurately from the DNA sequence. The two closely spaced denatured regions (b2 and b3) sometimes coalesced into one large denatured region (b2-3)
40
32

64

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0)

640

1280

1920

2560

3200

bp

FIG. 2. Partially denatured res-containing monomer. pA2 was digested with Nde I to generate monomer-length linear molecules partially denatured, and coated with RecA protein before electron microscopy. Denaturation bubbles (bl, b2, and b3), the intervening duplex regions (d12 and d23), and the terminal duplex regions (d3la and d3lb) are indicated. In terms of the circular map, d3l d3la + d3lb. (x87,000.)
=

FIG. 3. Experimental and predicted denaturation profiles. The abscissa marks distance (in bp) from the unique Nde I site in pA in the clockwise direction on the standard circular pBR322 map. The unique Bgl I site in pA is at position 1190. For the experimental data (shaded profile), the left ordinate indicates the number of molecules (out of 32 measured) that are denatured at each position along Nde Igenerated linear DNA. For the computer-generated data (solid line) the right ordinate represents the average A-T content for a 360-bp region centered around each position along the circular substrate. A T-rich regions of <50 bp are not expected to form visible denaturation bubbles and are omitted from the predicted profile.

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Proc. Natl. Acad. Sci. USA 82 (1985)

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Table 1. Partial-denaturation map of res-containing plasmid Length, % of monomer Circular molecules Linear molecules Range Mean + SD Range Feature Mean + SD 6.9-12.9 9.9 1.8 5.7-14.4 9.9 2.3 b, 11.2-19.3 15.0 2.0 6.7-18.0 13.5 2.7 b2 5.3-15.4 8.3 2.3 4.8-21.1 10.3 3.4 b3 12.2-22.2 17.5 + 2.9 14.5-26.9 20.4 3.2 d12 3.0-10.4 6.1 1.8 2.5-11.8 6.0 2.3 d23 37.4-48.2 43.1 2.9 29.3-46.4 39.8 4.2 d3l 20.4-31.3 25.7 3.2 23.0-33.2 29.9 2.8 b2-3 SD and range of observed lengths for seven features of Mean the partial denaturation map for pA equivalents are given as percent of monomer length (see legend to Fig. 2 for nomenclature). Lengths of the three denaturation bubbles (bl, b2, and b3) and the distance separating them (d12, d23, and d3j) are shown for a set of 32 linear and 31 circular molecules. Length of the coalesced bubble (b2-3) for a second set of 11 linear and 9 circular molecules is also given. Linear monomers are pA2 digested with Nde I; circular monomers are catenane rings from recombined pA2.

(Fig. 4). However, formation of this large (20-33% monomer length) bubble did not make the orientation of a monomer ring ambiguous since b2-3 and the other bubble (b1) were asymmetrically situated and of measurably different length (Table 1, compare d12 with d31 and b1 with b2-3). Thus, we could reliably identify the sequence orientation of rings with either three denatured regions or one small and one large denatured region. Determination of Catenane Linkage. Molecules of pA2 were reacted with resolvase and then nicked once per recombinant ring to remove supercoils. This nicked DNA was partially denatured, complexed with RecA, and examined by electron microscopy. Micrographs were taken of catenanes in which all nodes and denaturation bubbles could be discerned clearly. When these molecules were measured, the distribution of single-stranded and double-stranded regions in both rings fit the established map very well (compare linear and circular molecules in Table 1), allowing unambiguous orientation of every catenated circle. All of the products of recombination examined were singly interlocked. Each ring of these catenanes was oriented, the overlap at each crossing was determined, and the sign of each node was evaluated. The catenane in Fig. 5 thus was seen to be held together by an interlock consisting of 2 negative nodes. Its linkage value, the sum of the node terms, is therefore -2 (5). Interpretation of some catenanes randomly nicked with DNase I was difficult, owing to denaturation-bubble scission and consequent mapping ambiguity. We therefore turned to a nicking method specific for-a site in a nondenatured portion of each recombinant ring. We used the restriction enzyme Bgl I in the presence of ethidium bromide (14) to nick at its recognition sequence in a guanine . cytosine (G C)-rich region of the DNA (see legend to Fig. 3). As seen in Fig. 6,

FIG. 4. Partially denatured dimeric substrate. (A) Electron micrograph of the dimeric substrate pA2 after nicking with DNase I, partial denaturation, and coating with RecA protein. (x78,000.) (B) Tracing of molecule. Single-stranded regions are drawn as thin lines, and double-stranded regions as thick lines. Nomenclature is described in legend to Fig. 2, except that the coalesced region formed from b2 and b3 is denoted b2-3. Primed designations distinguish one of the monomer lengths of pA2.

short tails of single-stranded DNA were frequently found at the position of the Bgl I site, providing an additional landmark. In addition to the catenanes shown in Figs. 5 and 6, 18 other catenanes charted in Table 1 were also analyzed. This set of molecules included both randomly and site-specifically nicked catenanes and 9 molecules in which one ring had the coalesced b2-3 structure. In all 20 cases the linkage value was -2.

croscopy. If the initial arrangement of A T-rich sequences in a substrate does not result in a denaturation pattern of unambiguous orientation, appropriate pieces of DNA can be in-

DISCUSSION We present a general technique for determining the structure of catenanes. The DNA is fixed in a partially denatured form, coated with RecA protein, and viewed by electron mi-

FIG. 5. Partially denatured resolvasegenerated catenane. (A) Micrograph of catenated product of resolvase reaction with pA2 after DNase I-nicking, partial denaturation, and coating with RecA. (x63,000.) (B) Tracing of molecule. The catenane is represented by black and gray rings. Nomenclature is that of Figs. 2 and 4. (C) Idealized drawing of molecule. Arrows point in the direction of the path from b, to b2 and b3. Sign of each node was assigned according to the convention illustrated in Fig. 1.

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b3-,,;

Ib

2/

bb

FIG. 6. Partially denatured recombinant catenane with site-specific nicks. Resolvase-generated catenane is shown after BgI I-nicking, partial denaturation, and RecA-coating. Arrows indicate singlestranded tails initiated at the BgI I recognition site. (x96,000.)

serted or removed by recombinant DNA techniques. The resultant denaturation profile can be predicted readily from the distribution of adenosine and thymidine residues in the DNA sequence (see Fig. 3). Treatment with glyoxal and RecA is comparable to other techniques for partial denaturation mapping in terms of accuracy and reproducibility. An advantage is that RecA-DNA complexes may be applied directly to grids for electron mi-

croscopy, using less material and giving a cleaner micrograph background than the cytochrome c method. Unlike other denaturation methods that rely on DNA-binding proteins, such as bacteriophage T4 gene 32 protein (22), this technique yields single- and double-stranded DNA of identical unit length and morphology. We have shown that the interlock of the catenanes produced by Tn3 resolvase is exclusively negative, the same sign as that of the supercoils in the substrate DNA. That the catenanes are always singly interlocked, irrespective of substrate superhelicity (12), indicates, however, that negative supercoils are not included randomly in the synaptic complex. Thus resolvase-mediated recombination contrasts sharply with that mediated by the phage X int gene product, in which a substantial and variable number of substrate supercoils are converted into catenane interwindings (7). The origin of the nodes that interlock resolvase catenanes can be stated precisely. The recombination sites divide the substrate into two domains that are converted by recombination into the two linked product rings. A crossing of a segment from each of these substrate domains defines an interdomainal supercoil. The number of such supercoils at site alignment plus the net loss or gain consequent to the strandexchange mechanism equals the catenane linkage value (5). Since the linkage value was a constant, -2, resolvase presumably fixes each of these two terms, controlling both the number and fate of synaptic interdomainal supercoils. We have devised a model, diagrammed in Fig. 7, that provides values for these linkage terms. This scheme accounts for the appearance of the singly interlocked catenanes as well as the rare, more complex products of the resolvase reactions. Such complex products each make up about 1% of the total product and include topological forms whose stereostructure is independent of DNA orientation. It therefore was possible to determine by the RecA-coating method alone that the simplest of these forms were a 4-noded knot and a figure-8 catenane, whose structures are shown in Fig.

Substrate

Singly linked catenane

4-noded knot

Figure-8 catenane

FIG. 7. Model of resolvase recombination. The two directly repeated res sites (thickened arrows) divide the supercoiled substrate into two domains of DNA, indicated by black and gray lines. For simplicity, only the three interdomainal supercoils are shown. Molecules in the upper row are imagined bound to resolvase, with three successive rounds of recombination drawn from left to right. The bottom row of structures represent resolvase-free substrate and products drawn in standard form. Each round of recombination is divided into two stages: the direct product of crossing-over is formed (solid pathway arrow) and then is rearranged (open pathway arrow) to restore parallel res-site synapsis. The substrate has 3 (-) interdomainal supercoils, and each round of recombination introduces a (+) interdomainal node. Accordingly, the immediate product of a single round of recombination has 3 (-) nodes and 1 (+) node. If released from the protein, this DNA can unfold to a structure with just 2 (-) nodes, the singly interlinked catenane. If the product is not released, successive rounds of recombination can occur, yielding more complex structures in which successive products are alternately knots and catenanes. The second round product is a 4-noded [2 (+) and 2 (-)] knot and the third is a 5-noded [3 (+) and 2 (-)] figure-8 catenane, as shown. Both of these also retain a (-) supercoil because none of their nodes can be cancelled by unfolding. The two (-) substrate interdomainal nodes shown near each res site could be due to solenoidal wraps of each site around the resolvase. The third (-) node between the res sites would be fixed by enzyme geometry or perhaps by the negative superhelicity of the substrate.

Biochemistry: Wasserman and Cozzarelli

7 (ref. 11; A. Stasiak, personal communication). Neither structure is expected from a simple, single round of recombination. The fact that the figure-8 catenane always has an entrapped positive supercoil (11) is particularly surprising in light of the negative superhelicity of the substrate. In our model, these structures arise from an infrequent iteration of the basic mechanism for forming singly linked catenanes. The upper row in Fig. 7 depicts three successive strandexchange events of DNA that is imagined bound to resolvase; the lower row is a redrawing of substrate and products in standard form. Once the first crossing-over takes place, resolvase normally dissociates from the DNA, releasing the singly interlinked catenane that is inert to further reaction (9). We postulate that occasionally the protein acts processively, failing to dissociate and carrying out further crossovers that generate, successively, the 4-noded knot and the figure-8 catenane. The interdomainal linkage change in recombination, defined by the difference between successive products, is in this model a single positive node. A positive linkage change is equivalent to a loss of a negative supercoil; it therefore would be energetically favored. The synapsed substrate in Fig. 7 has two res sites aligned in parallel and three interdomainal supercoils between these sites. It has been shown elsewhere (5) that for recombination of directly repeated sites, a + 1 mechanism requires parallelsite synapsis and an odd number of interdomainal supercoils. To generate the observed resolvase products-the singly linked catenane, the 4-noded knot, and the figure-8 catenane-there must be three interdomainal supercoils and they must be negative in sign. The linkage value of the singly linked catenanes did not enter into this derivation but is clearly predicted to be -3 + 1 or -2. Our finding that this is indeed true supports our model for recombination. The accommodation of exactly three interdomainal supercoils at synapsis can be ensured only in very limited ways. The simplest may be pictured as solenoidal wrapping of DNA around the protein, as occurs with nucleosomes (23) and as postulated for Int protein (24, 25). If, at each res site, resolvase molecules are encircled by one or more DNA wraps, one (but only one) negative interdomainal supercoil can be formed at each site, as is diagrammed in Fig. 7. The third negative interdomainal node can be constrained between the two solenoids by the structure of the resolvaseDNA complex. It should be emphasized that although the data are in excellent agreement with our model for synapsis and recombi-. nation, other, albeit more complex, mechanisms also can explain our observations. However, the model we present can be tested readily, since further iterative recombination events should yield more complex structures of specific geometry. Int protein also may entrap negative interdomainal supercoils and introduce a single interdomainal node in the course of recombination (24, 26). Both the methodology and the basic mechanism presented here therefore may have widespread applicability.

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We thank Robley Williams for sharing advice and facilities for electron microscopy, Mark Navre for assistance in generating the predicted denaturation map, and Elisabeth Lindheim for help in preparing the manuscript. We also thank Andrzej Stasiak for helpful discussions and Kevin McEntee for supplying RecA protein. S.A.W. is a fellow of the Miller Institute for Basic Research in Science. This work was supported by National Institutes of Health Grants GM31655 and GM31657.

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