THEJOURNAL BIOLOGICAL OF CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 30, Issue of October 25, pp. 20567-20573,1991 Printed in U.S.A.

Supercoiled DNA-directed Knotting by T4 Topoisomerase*

(Received for publication, April 25, 1991)

Steven A. WassermanSQ and Nicholas R. Cozzarellill

From the $Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235 and the YDepartment of Molecular Biology, University of California, Berkeley, California 94720

The mechanism by which the type 2 topoisomerase pass through the transient double-stranded break, and then from bacteriophage mediatesknottingof negatively rejoins the broken strand. Theform of the knot is determined T4 supercoiled DNA was deducedfroman analysis of not by the enzyme, but by the random folds and twists of the product topology. The knotted products were nicked DNA that become locked in place by strand passage. The and then subjected to electrophoresis in order to separesulting catalogue of products containseach entry of a mathrate species on the basis of the minimum number of ematicians knot table. crossingsin the knotted form. Knots defined numwith In light of the substantial insight into topoisomerase I bers of crossings were purified and the configuration action gained from studies of its knotting reactions (15-18), of these crossings determined in the electron microwe set out to investigate the knotting of DNA by a type 2 scope by the RecA coating method. The product knots enzyme from T4. Type 2 topoisomerases pass entire duplexes were exclusively of the twist form, in which aninterwound regionis entrapped by single interlock of two through each other and usually require ATP. In contrast to a looped ends. The interwound region was of negative topoisomerase I, high concentrations of T4 topoisomerase can sign in >98% of the molecules examined, whereas the efficiently knot covalently closed DNA, provided that the single interlock was equally likely to be positive or substrate is supercoiled and no ATP is added to the reaction added, the T4 topoisomerase carries out multiple negative. These results are interpreted in terms of a (6). If ATP is rounds of reaction, relaxing the substrate and untying any model for knot formation in which random strand passage mediated the by topoisomerase links bent or knots formed. Thus knotting by the T4 enzyme reflects a branched portions of a superhelix that has a specific limited reaction with a supercoiled substrate, resulting, as we interwoundgeometry.Superhelix interwinding and demonstrate here, in a unique family of products. Our studies reveal a fundamental similarity between the DNA contacts stabilized by excess enzyme molecules explain the very high frequency of knotting. type 1 and type 2 reactions; both enzymes knot DNAby carrying out random strand passage reactions. Both enzymes also augment greatly the frequency of knotting by what we interpret as bridging of segments of the DNA chain. Striking a Topoisomerases, by carrying out DNA strand passage re- dissimilarities in product distribution arise, nonetheless, due actions, fulfill a vital role in the replication and segregation to conformational differences between relaxed and superof double helical DNA (1-4). In vitro, topoisomerases readily coiled DNA substrates. Moreover, a detailed analysis of the knot and unknot as as catenate and decatenate well DNA (5- product array from the T4 topoisomerase reaction serves to 11).In uiuo, however, knotted and catenated DNAs accumu- solidify our picture of the structure of supercoiled DNA in late to significant levels only in cells deficient for topoisom- solution as a branchedinterwound superhelix. erase activity (2-4, 12). Although the active segregation of chromosomes might be postulated to drive decatenation in EXPERIMENTAL PROCEDURES the cell, the same hypothesis could not explain the near DNA and Enzymes-Plasmids pBNW3.8d, pRR51, and pA2 have absence of knots in wild-type cells, nor the rapid unlinking of been described (19-21). T4 topoisomerase partially purified from an catenated plasmids generated by recombination (13, 14). extract of phage-infected cells by DNA cellulose and hydroxyapatite Rather, itseems likely that theintracellular state of the DNA chromatography was a gift of Ken Kreuzer (Duke University, Duris the predominant influence on the outcome of topoisomer- ham, NC). It was purified to homogeneity by chromatography on ase-mediated strand passage events, particularly for intra- Sephacryl S-300 as previously described (22). Knotting Reactions-Knotting molecular processes. In order to investigate the influence of taining 50 m Tris (pH 7.6), 60 was carried out in a mixture conM M m KC1, 10 m MgCl,,20 pg/ml M DNA structure on the action of topoisomerases, we have DNA, and 40 pg/ml bovine serum albumin (22). The solution was therefore undertaken a study DNA knotting. of prewarmed to 32 C prior to addition of 1.75 pg ofT4 topoisomerase/ Topoisomerase-mediated knotting of duplex DNA in vitro pgof DNA. After a 4-min incubation at 32 C, the reaction was has been well characterized for the Escherichia coli topoisom- quenched by addition of EDTA to 10 mM. Following extraction of erase I (15, 16). The reaction, which requires a nicked or the mixture twice with phenol, the volume of the quenched reaction by extraction gapped substrate, is a simple strand passage reaction. Topoi- product was then reduced 2-foldthen extractedwith sec-butyl alcohol. The concentrated solution was twice with ether and somerase I breaks one strand of the duplex opposite a preex- the DNA precipitated with sodium acetate and ethanol. The precipiisting interruption in the other, allows a duplex segment to tated DNA was resuspended to 0.1 reaction volume in TE buffer (10

* This work was supported by a grant from the Lucille P. Markey

Charitable Trust (to S. A. W.) and by grants from the National Institutes of Health (to N. R. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. ! Lucille P. Markey scholar. j

m Tris, pH 8.0, and 1m EDTA). M M Nicking Reactions-After knotting, supercoils were removed from DNA by limited nicking (23) in a reaction containing 50 m Tris M (pH 7.61, 6 mMMgC12, 100 pg/ml each DNA and ethidium bromide, and 1.9 pg/ml DNase I. Electrophoretic analysis indicated that under these conditions, a 30-min reaction at 30 C yielded approximately 90% nicked, 5% linear, and 5% supercoiled DNA. The reaction was quenched by addition of EDTA to 10 m and NaCl to 150 mM. The M

20567

20568

T4 Topoisomerase Knots
A.
n .-

mixture was then extracted twice with phenol, the volume reduced 4fold by extraction with sec-butyl alcohol, and the concentrated solution extracted twice with ether. Gel Electrophoresis and Quuntitation-Nicked knotted DNA samples (1pg for analytical gels and 40 pg for preparative reactions) were subjected to agarose gel electrophoresis buffered with Tris-acetate containing 0.03% SDS' or with Tris-borate (89 m Tris, 89 m M M borate, 2.5 m EDTA, pH 8.3) (2, 21). Following staining with 0.5 M pg/ml ethidium bromide, the banding pattern was recorded on Polaroid film andthe negative scanned with a Molecular Dynamics densitometer. Purification of Knotted DNA-Alternative methods (24) were used for the purification of knotted DNA with equivalent results. In one, samples were subjected to electrophoresis in a 0.7% agarose gel in Tris-borate for 38.5 h a t 60 V. The gel was stained and a trough cut behind the trefoil species. The trough was filled with hydroxyapatite (Bio-Gel HTP) andelectrophoresis carried out with reverse polarity for 2.25 h a t 60 V. The hydroxyapatite was then packed into 0.5-ml columns, and each column was washed with a buffer of 10 m M M potassium phosphate, pH 6.8, and 1 m EDTA. The DNA was then eluted with 400 m buffer and extracted sequentially with phenol, M sec-butyl alcohol, and ether. In the other procedure, samples were subjected to electrophoresis in a 0.75% agarose Tris-acetate gel for 43 h a t 42 V. The gel was washed twice in water to remove SDS and then stained. Agarose slices containing knotted species were cut from the gel and dissolved in 10 m phosphate buffer saturated with potassium iodide. The M DNA samples were then loaded onto 0.5-ml columns of hydroxyapatite (Bio-Gel HTP) equilibrated with 10 m buffer. The DNA was M eluted as above, extracted with phenol, and dialyzed twice against T E at room temperature. Electron Microscow-Nicked, knotted DNAs were denatured by glyoxal treatment a t 62 "Cfor 90 min (25). The single-stranded DNA was desalted and coated with RecA in the presence of 5 m ATP and M 5 m magnesium acetate, as described previously (26). The DNAM protein complexes werethen fixed with glutaraldehyde and tungsten shadowed for electron microscopy (25).Criteria for tracing knotsand scoring nodes were as reported previously (15).

Node -0

0.

i-3
-4

r-

-5
-6 -7

-a
-9

- 10

FIG. 1. Electrophoretic analysis of DNA knots formed by T4 topoisomerase. A, pBNW3.8d was knotted with T4 topoisomerase and nicked with DNase I to remove supercoils. It was then subjected to electrophoresis with markers in a 0.7% agarose gel in Tris-borate a t 70 V for 19 h. Lane I,80 ng of supercoiled (sc) substrate. Lane 2, 65 ng of linear ( I ) substrate generated with BarnHI endonuclease. Lane 3, 900 ng of nicked knots. Lanes 4 and 5, 1-pg marker ladders of catenanes generated by Int reaction with partially relaxed substrates. Nicked circles (n) andlinearized substrate ( I ) in lanes 35 are products of DNase I treatments, as is the free recombinant circle ( r ) in lanes 4 and 5. B, knotted and nicked pA2 subjected to electrophoresis in a 0.75% agarose gel in Tris-acetate with SDS for 43 h a t 42 V.

plasmids was broad, ranging from 3-noded knots to knotted products with 18 or more crossings.The spacing of the ladder rungs remained one node. These electrophoretic analyses indicated that, like E. coli topoisomerase I, the T4 type 2 enzyme could tie knots with variable numbers of crossings of odd or even number. However, electrophoretic separation according to the minimum RESULTS number of duplex crossings could not indicate whether the T4 enzyme producedall of the knots seen with topoisomerase To explore the mechanism of the T4topoisomerase knotting reaction, we first characterized the products by gel elec- I, since the number of crossings is an incomplete descriptor trophoresis. A supercoiled 3800-bp plasmid, pBNW3.8d, was of all but the 4-noded knot. There is only a single knot with knotted with purified T4 topoisomerase and singly nicked 4 nodes. There are, however, two enantiomeric knots with 3 with DNase I; nicking removes supercoiling but leaves knot- nodes and eight knot isomers with 6 nodes. To determine ting intact. The DNA was then subjected to gel electrophoresis which particular isomers arose in knottingreactions with the and stained with ethidium bromide (Fig. l4.Under the T4 topoisomerase, we undertook an electron microscopic .) conditions used, mobility in the gel was proportional to the analysis of the reaction products. In order to visualize overlying and underlying DNA segminimum number of crossing, or nodes, in the knotted form. Four features of the knotting reaction were evident from ments at knot crossings, we thickened the DNA by coating it the electrophoretic analysis. 1) Topoisomerase-generated with RecA. Since RecA reacts more readily with single- than knots were generated with both odd and even numbers of double-stranded DNA, gel-purified knots were denatured to crossings, forming a node ladder with steps-of-one spacing separate the knotted single strand of each duplex from the (Fig. IA, lune 3). By comparison, catenanes resulting from nicked strand. This does not affect the topology of the knot. the same substrate by recombination mediated by the X inte- The single-stranded DNA was then reacted with RecA, adgrase (Int) have even numbers of crossings exclusively (Fig. sorbed to carbon-coated grids, shadowed, and examined by l.4, lanes 4 and 5).2) The top rung of the T4topoisomerase electron microscopy. We began our analysis with the 3-noded knots or trefoils. knot ladder was composedof trefoils, knots with 3 crossings. Trefoils can be of only two forms, distinguished by whether The trefoil (Fig. 2, a and b) is the simplest knot, since knot crossings are always integral and knots cannot be tied with the three, irreducible nodes are of positive or negative sign fewer than 3 crossings. 3) The amount of a given product (Fig. 2, a and b). The sign convention is explained in the species declined with node number (Fig. L4, lane 3). 4) Knot legend to Fig. 2. The trefoils formed by T4 topoisomerase formation in the T4 topoisomerase reaction required super- were overwhelminglyof one enantiometric form; 85 out of 87 coiled substrate, since nicked or relaxed pBNW3.8d yielded trefoils examined were negative in sign; only two were positive. Examples are shown in Fig. 3. less than 1% knotted product (data notshown). The strongly biased array of product trefoils formed by the Larger supercoiled substrates, pRR51 (5950bp) and pA2 T4 topoisomerase is in sharp contrast theracemic mixture to (6400 bp), yielded an even higher percentage of knotted products, 50-75% in a standard reaction. Fig. 1B shows the PA' of (+) and (-) trefoils from an E. coli topoisomerase I knotting pattern. The distribution of knotted species for these larger reaction (15). Our gel analysis revealed, however,that theT4 enzyme, like topoisomerase I, generated the 4-noded knot, The abbreviations used are: SDS, sodium dodecyl sulfate; bp, base which contains 2 (+) as well as 2 (-) nodes. Furthermore, the pair(s). T4 topoisomerase is known to relax both positive and negative

T 4 Topoisomerase Knots
A

20569

FIG. 2. Topology of DNA knots. Projection drawings of knotted molecules are shown. Knots are classified as tonumber and configuration of the irreducible number of nodes, or crossings. Knots a and b have 3 nodes, knot c has 4 nodes, knots d and g have 5 nodes, and knots e, f, and h have 6 nodes. If two molecules have an equal number of nodes, but one cannot be stretched to superimpose on the otheror its mirror image, the two knots are distinct and diastereomeric. If one knot can only fit the mirror image of the other, the two are enantiomers distinguished by node signs. Node sign, shown for a single node in the top row of knots, is determined separately for each crossing. Arrowheads denote an arbitrary orientation along the entire DNA axis. A node is negative in sign if the overlying arrow can be aligned with the underlying one by a clockwise rotation of less than 180". The node is positive if a counterclockwise rotation results in alignment. Knots a and b are enantiomers. Knots e, j , and h are diastereomers. Knots can also be grouped into families of related forms. Knots a-e are members of the twist family, which have an interwound region with 1, 2,3.. n nodes and a single interlock made up of two nodes. The twist family is subdivided further according to whether there is an odd (a, b, d ) or even ( c , e) number of nodes. For the former, the sign of all the nodes is the same; for the latter, the interlocking nodes have the sign opposite to that in the interwound region. Knot g is a member of the torus family. Knots of this family can be drawn without intersection on the surface of a torus, a doughnut-shaped solid, and are the most regular in structure of all knots. a, trefoil or 3-noded knot with 3 (-) nodes. b, trefoil with 3 (+) nodes. c, twist knot with 2 (-) and 2 (+) nodes (achiral). d, twist knot with 5 (-) nodes. e, twist knot with 4 (-) and 2 (+) nodes. f, a granny knot, a composite knot composed of two trefoils, each with 3 (-) nodes. g, torus knot with 5 (-) nodes. h, a 6-noded knot with 4 (-) and 2 (+) nodes that is not a twist knot.

FIG. 3. Electron microscopy of trefoil knots. Purified trefoil knots made by T4 topoisomerase were denatured, coated with RecA protein, adsorbed to grids, shadowed with tungsten, and photographed in the electron microscope. A, pRR51 knots. B, pRR51 (left) and pA2 (right) knots. Each micrograph is accompanied by a tracing showing the relative overlay of strands ateach crossing and by a redrawing of the knot with only the minimum number of crossings. Each knot has 3 irreducible (-) nodes.

supercoils (6) and can thus carry out binding and catalysis a t both (-) and (+) nodes. These facts, the topology of the 3and 4-noded knots, and a catalytic mechanism that is blind with regard to node sign can all be accommodated by a model postulating random strand passage events between portions of a negatively interwound supercoiled DNA substrate, as illustrated in Fig. 4 and detailed below.

If conformational fluctuations of the supercoiled substrate or topoisomerase-stabilized DNA bridges bring one loop of DNA across a second loop, a (+) node and a (-) node are formed. If the topoisomerase breaks one duplex at either the (+) or (-) node and allows the crossing duplex to pass through the gap before resealing the broken DNA, a knot of the twist family is produced (Fig. 4 . The (-) sign of the crossings in ) the twisted region of the knotreflects the (-) supercoiling of the plasmid substrate. In theprocess of strand passage supercoils have been converted from geometric forms that can be unfolded to topological forms that cannot. Two distinct classes of twist knots will arise depending on which of the overlap nodes is the site of reaction (Fig. 4 .If ) the topoisomerase carries out strandpassage at the(+) node, converting it to a (-) node, the knot will contain an odd number of nodes, all negative in sign. The 3- and 5-noded examples are shown, respectively, in Fig. 2, a and d. If the topoisomerase instead acts at the overlap node to convert (-) it into a (+) node, the knottedproduct will have a twist region containing an even number of (-) nodes and a linking region with two (+) nodes, as in the4-and 6-noded knots in Fig. 2, c and e.

20570

T4 Topoisonwrme Knots

FIG.4. Proposed mechanism for twist knot formation by T4 topoisomerase. The substrate for knotting by T4 topoisomerase consists of an interwound, negatively supercoiled DNA. As the superhelix bends, two regions (bold lines) cross. As shown, two nodes result, one (-) and one (+). All other nodes of the superhelix are (-), but their sign is omitted for clarity. Knotting occurs by topoisomerasemediated strand passage a t either the (+) node (upper pathway) or the (-) node (lower pathway). The product of the upper pathway is a twist knot with an odd number of (-) nodes, two in the linking region and therest inthe intervening twist region. There is atotal of 5 nodes in the example shown. The product of the lower pathway is a twist knot with 2 (+) linking nodes andan even number of intervening (-) nodes; there is a total of 4 nodes in the example shown. Note that unfolding removes any nodes not entrapped by knotting and that only a fraction of the original supercoiling contributes to the knot structure. For illustrative purposes, unfolding is shown in two stages. In the first, the DNA is nicked but the regions where the topoisomerase has acted remain juxtaposed; in the second, this constraint is removed and the DNA rearranged into a more standard form for knots. A quantitative treatment of knotting by type 2 topoisomerases (27) describes the relationship of enzyme mechanism, substrate supercoiling, and product knotting.

FIG.5. Electron microscopy of 5-noded knots. Electron micrographs of purified 5-noded knots of pA2,together with tracings and redrawings with the minimum number of crossings. Each knot has 5 irreducible (-) nodes.
TABLE I Product profile of knots made by T4 topoisomerose The reaction products were nicked to remove supercoils, displayed by gel electrophoresis, and quantified by densitometric scanning of the ethidium bromide-stained gel.
Plasmid substrate Name Product Knot Profile 3-noded 4-noded 5-noded
% of total product

Size
bp

6-noded

80 . 12.5 9.8 3800 pBNW3.8d 7.7 5.8 5.3 5950 pRR51 ND" 8.4 6400 11.1 DA' Not determined (species comigrated with linear).

7.4
6.4

9.9

and 4 (-) nodes (Fig. 2e). We noted, however, that the total amount of 6-noded species was greater than thatexpected by By this model, product trefoils are of (-) sign not because extrapolation from the amountof knots with fewer crossings. the enzyme must invert a node to form a knot, but (+) because The 3-noded, 4-noded, and 5-noded knots form a series of amount inversion of a (-) node will form a knot with an even number decreasing abundance (Table I). In contrast, the total of nodes in itsfully unfolded form. Rare (+) trefoils could be of 6-noded knots from the two larger substrates, for which a formed by the chance entrapment of a single (+) node by the second 6-noded species is more evidentin gel profiles, is greater than the amount 5-noded knots. of enzyme and strandpassage a t (-) node (27). The relative overabundance of 6-noded species suggested This model leads to the strong prediction that the knot8 formed by T4 topoisomerase should be restricted not only in that theremight be a pathwayfor forming 6-noded knots that node sign, but also in knot form. Only twist knots are expected is inaccessible to knots with 3, 4, or 5 crossings. Mathemati6-noded species can to arise by the mechanism shown in Fig. 4. For knots with an cally there is indeed such a distinction. A odd number of crossings, all crossings should be (-) in sign; be formed by knotting of a circle at two separate points. for knots of even node number, two nodes should be of (+) Furthermore, since the trefoil is thesimplest knot, a6-noded species composed of two trefoils is the simplest possible sign and the rest negative. Among 5-noded knots, the proposed mechanism predicts composite knot. Since the T4 topoisomerase was present in that the T4topoisomerase will produce only one of the four large excess, reaction could readily have occurred at more than one site on each substrate molecule. possible forms. The expected knot is a twist form with 5 (-) The expected compound knot is the granny knot composed nodes (Fig. 2 4 , whereas the twist knot with 5 (+) nodes and the torus knots with 5 (-) nodes (Fig. 2g) or 5 (+) nodes of 2 (-) trefoils whose form is illustratedin Fig.2f. The should not appear. When 5-noded knots generated by T4 granny knot and the 6-noded twist knot are diastereomers gel topoisomerase were examined, all 39 molecules were twist whose compaction in the could be distinct enough to result in differential mobility (26). Compound species are also exknots with 5 (-) nodes (Fig. 5). None of the three other possible 5-noded knots, all of which are formed by topoisom- pected for knots with greater than 6 crossings. We did not, however, analyze more highly knotted species, because both erase I, were observed. Although the analysis of the 5-noded knots offered substan- the amount knotted product and theresolving power ofthe of tial support for the supercoiled DNA-directed knotting model gel decline with the number of knot crossings. described above, an anomaly in the electrophoretic gel profile To test whether the two 6-noded species were indeed of the prompted us to extend our analysis to the 6-noded knots. predicted configurations, the topology of the combined 6Close examination of the electrophoretic ladders of T4 topo- noded species was examined directly by the RecA coating isomerase knots revealed two knotted species in the region of method, Out of 22 molecules, 10 molecules were 6-noded twist the gel where 6-noded molecules migrate (Fig. 1B). simple knots with 4 (-) and 2 (+) nodes; the remaining 12 molecules A reaction of the type illustrated in Fig. 4 should give rise to were compound 6-noded knots, granny knots, comprised of only a single type of 6-noded knot, a twist knot with 2 (+) two (-) trefoils (Fig. 6). Given that there are eight different

T4 Topoisomerase Knots

20571

2, a, c-e). Only two exceptional knots of (+) sign, both 3noded, were found among 148 examined. Such molecules probably arise during those rare circumstances when the high concentration of bound topoisomerase captures a (+) node in the otherwise negatively supercoiled DNA substrate (27). The knots formed by the T4 topoisomerase differ from those formed by the Int recombinase, which are exclusively (+) torus knotswith an odd number of nodes (28), and those formed during processive recombination by Tn3 resolvase, which have only an even number of nodes and can be neither torus nor twist (26). Coincidentally, however, experiments in which the T4 topoisomerase knots served as topological standIll Ill ards demonstrated that theproducts of processive recombination by the Gin invertase constitute the same subset of knots asis produced by the T4enzyme (29). Paradoxically, the non-random nature of the T4topoisomeraseproducts provides substantial support for a random strand passage mechanism for the enzyme. Our proposed mechanism, in which substrate configuration plays a critical FIG.6. Electron microscopy of 6-noded knots. Electron mi- role in determining knot structure, is illustrated in Fig. 4 for crographs of purified 6-noded knots of pA2, together with tracings and a single catalytic cycle. Of 148 molecules examined, 146 can redrawings with the minimum number of crossings. The molecule on be viewed as twist knots with (-) nodes in the interwound the left is a twist knot with 4 (-) and 2 (+) nodes. The molecule on the right is a granny, the compound knot composed of two trefoils, region (Table 11). This result can only be readily understood by assuming that the sign of the twist region reflects the each with 3 (-) nodes. supercoiling of the substrate. There is in fact evidence from electron microscopy, the structure of the Int products, and TABLE I1 computer simulationof DNA structure that the linking deficit Topology of knots tied by T4topoisomerase The predicted knot topology is that derived from the scheme shown of DNA is expressed in theform of interwound (plectonemic) rather than solenoidal supercoils (28, 30-32). The current in Fig. 4. results confirm this important conclusion for DNA in soluNumber of tion. Number Predicted knot node predicted knots Predicted knot of nodes composition" found/total knots form shown Knotted plasmid DNAs have also been analyzed from a scored mutant E. coli strain in which a deletion of the topoisomerase 3 3 (-) (twist) Figs. 2a and3 85/87 I gene and a compensatory mutation in the gyrase B gene 4 2 (-), 2 (+) (twist) NDb Fig. 2c lead to theaccumulation of knotted DNAs at a level 10 times 5 5 (-) (twist) Figs. 2d and 5 39/39 that of wild-type strains (12, 33). Those knots analyzed by 6 4 (-), 2 (+) (twist) or 12/12 Figs. 2e and 6 the RecA coating method appear, like the T4 enzyme prodFigs. 2f and 6 loll0 3 (-), 3 (-) (granny) ucts, to be (-) twistknots, butthere is an unexplained Total 146/148 preponderance of knots with an odd number of nodes. InKnot type is indicated in parentheses. creased knotting has also been detected in wild-type strains Not determined (only one possible structure). upon addition of high concentrations of the DNAgyrase possible 6-noded knots (4), yet only the two predicted forms inhibitor, norfloxacin;' the structure of these knots is not were observed, we conclude that these knotted products in- known. The production of (-) twist knots in the mutant deed result from random catalysisby T4 topoisomerase acting strain suggests a mechanism of knot formation similar to that proposed here for the invitro reaction with T4 topoisomerase on anordered DNA structure. and further indicates that DNA inside of E. coli cells is a (-) interwound superhelix. DISCUSSION Knotting Frequency and Specificity-As pointed out origiKnotting Mechanism-When analyzed by gel electropho- nally by Liu et al. (6), theefficiency of knotting (up to 75%) resis, the products of E. coli topoisomerase I (15) and T4 by T4 topoisomerase in vitro is far greater than expected on topoisomerase knottingreactions (6) appear quite similar. the basis of theoretical calculations. The probability of knotEach reaction yields knotted products in which the trefoil is ting can be estimated for molecules modeled as a series of the most abundant species and themolecules form a steps-of- connected but freely jointed (Kuhn) segments of sufficient one electrophoretic ladder. Nonetheless, the knotted products length to account for the bending stiffness of the DNA. Each of T4 topoisomerase reaction are clearly distinguishable from such Kuhn segment is twice the persistence length. Using the the knots tied by topoisomerase I. Whereas topoisomerase I accepted valve for the length of the Kuhn segment of free produces every knot theoretically possible ( 1 3 , we have dem- DNA (1000 A) and for the effective diameter of DNA (36), onstrated here that the T4topoisomerase reaction gives rise we calculate that at equilibrium only about 1-2% of relaxed to a unique subset of the products seen withtopoisomerase I. DNA would be knotted for plasmids in the size range (3800The results are summarized in Table11. 6400 bp) we used. The 3-, 4-,5-, and 6-noded knots produced by T4 topoisomThese calculations fail to take into account the presence of erase are all either simple twist knots or composites of two excess topoisomerase molecules and the supercoiling of the twist knots. The odd-noded simple twist knots contained only substrate. It has been suggested that a restriction on the (-) nodes; the even-noded simple knots also had 2 (+) nodes. volume of the DNA chain imparted by both supercoiling and Despite the topological sign difference the simple knots shared excess bound protein forces a more tortuous path for the an overall geometry: in each there is a (-) interwound region of variable length held in place by a single interlock (see Fig. D. Adams and N. R. Cozzarelli, unpublished results.

20572

T4 Topoisomerase Knots
that (-1 supercoiling supplies the interwound part of the knot. Our conclusion then is that the high frequency of knotting requires a structural contributionfrom both plectonemic supercoiling and protein cross-linking. Given that bending of the superhelix to bring nearby points on the DNA into contact should be energetically disfavored, one would expect knots with many nodes to predominate among the products, contrary to what is observed. Rather it seems likely that the branching of interwound DNA (30) provides a ready means for facilitating contact between disparatepointswithout requiring extensive bending of the superhelix, as shown in Fig. 4. The equation derived in the appendix that relatessubstrate supercoil structure to the number of nodes in the knots produced by a type-2 topoisomerase confirms the reduction in knotcomplexity by branching. Moreover, the predicted (5.9) and measured (5.4)average number of knot nodes for pBNW3.8d are in very good agreement (see Appendix; Ref. 40). In summary, the topology of the T4topoisomerase knots is explained by the branched (-) plectonemic supercoiling of the substrate and random strand passages by the enzyme. The frequency of knotting is greatly increased over that calculated from random passage in relaxed molecules byboth supercoiling and topoisomerase-induced crosslinking of the substrate.
REFERENCES
1. Cairns, J. (1963) J. Mol. Biol. 6 , 208-213 2. Sundin, O., and Varshavsky, A. (1980) Cell 2 1 , 103-114 3. DiNardo, S., Voelkel, K., and Sternglanz, R. (1984) Proc. Natl. Acad. Sci. U. S. A. 81,2616-2620 4. Wasserman, S. A., and Cozzarelli, N. R. (1986) Science 2 3 2 , 951-960 5. Liu, L. F., Liu, C., and Alberts, B. M. (1979) Nature 2 8 1 , 456461 6. Liu, L. F., Liu, C., and Alberta, B. M. (1980) Cell 19,697-707 7. Tse, Y., and Wang, J. (1980) Cell 22,269-276 8. Brown, P.O., and Cozzarelli, N. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,843-847 9. Hsieh, T.(1983) J.Biol. Chem. 258,8413-8420 10. Hirose, S., Tabuchi, H., and Yoshinaga, K. (1988) J. Biol. Chem. 263,3805-3810 and 11. Kreuzer, K. N., Cozzarelli, N. R. (1980) Cell 20,245-254 12. Shishido, K., Komiyama, N., and Ikawa, S. (1987) J. Mol. Biol. 195,215-218 13. Bliska, J. B., and Cozzarelli, N. R. (1987) J. Mol. Biol. 194,205218 14. Bliska, J. B., Benjamin, H. W., and Cozzarelli, N. R. (1991) J. Biol. Chem. 266,2041-2047 15. Dean, F. B., Stasiak, A., Koller, T., and Cozzarelli, N. R. (1985) J. Biol. Chem. 260,4975-4983 16. Dean, F. B., and Cozzarelli, N. R. (1985) J. Bwl. Chem. 260, 4984-4994 17. Liu, L. F., Depew, R. E., and Wang, J. C. (1976) J. Mol. Biol. 106,439-452 18. Wang, J. C., and Liu, L. F. (1979) in Molecular Genetics (Taylor, J. H., ed) Part 3, pp. 65-88, Academic Press, New York 19. Wasserman, S. A., White, J. H., and Cozzarelli, N.R. (1988) Nature 334,448-450 20. Reed, R. R. (1981) Cell 2 5 , 713-719 21. Benjamin, H. W., Matzuk, M. M., Krasnow, M. A., and Cozzarelli, N. R. (1985) Cell 4 0 , 147-158 22. Kreuzer, K. N., and Jongeneel, C. V. (1983) Methods Enzymol. 100,144-160 23. Greenfield, L., Simpson, L., and Kaplan, D. (1975) Biochim. Biophys. Acta 407,365-375 24. Smith, H. 0. (1980) Methods Enzymol. 66,371-380 25. Wasserman, S. A., and Cozzarelli, N. R. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,1079-1083 26. Wasserman, S. A., Dungan, J. M., and Cozzarelli, N.R. (1985) Science 229,171-174 27. Cozzarelli, N.R., Krasnow, M. A., Gerrard, S. P., and White, J. H. (1984) Cold Spring Harbor Symp. Quunt. Bwl. 49,383-400

chains. Thisis equivalent to a reduction in theeffective Kuhn length andcould, in turn, explain the observed high frequency of knotting (6). Indeed, Monte Carlo calculations show that the probability of knotting increases greatly with the number of Kuhn segments (34), reflecting the greater flexibility of a DNA composed of an increased number of segments. However, we calculate that the effective Kuhn length would have to be reduced to an implausibly low value of less than two turns of the double helix to explain our pBNW3.8d results. Furthermore, if supercoiling and excess protein were simply equivalent to increasing the effective number of Kuhn segments,knots of all topological types wouldbe produced. Instead only a particular subclass of knot topoisomers are observed. Frank-Kamenetskii and Vologodskii (35) proposed an alternative mechanism for the efficient knotting by T4 topoisomerase. Knotting frequency varies inversely with effective DNA diameter (36), and they suggested that excess protein provides an attractive potential between DNA segments and thereby a smaller effective DNA diameter. An attractive potential does indeed increase greatly the calculated frequency of knotting (35). Their model, however,also does not account for the specificity of product knot structure. In searching to explain both the efficiency and specificity of the T4 topoisomerase knotting reaction we first consider the consequences of high amounts of T4 topoisomerase. E. coli topoisomerase I is known to play a stoichiometric role in its knotting reaction; knotting requires a 20-fold greater level of topoisomerase I than is necessary for strand passage (15). High levels of topoisomerase are also needed for knotting by the T4, Drosophila, Bombyx mori, and HeLa type 2 enzymes (6,9,10); we routinely use a stoichiometry of 15-20 molecules of T4 topoisomerase (Mr= 260,000) per plasmid. Since a topoisomerase must bind two separate DNAs in order to pass them through each other, it should act as a bidentate ligand andthus cross-link, or bridge, disparate points along the DNA axis. Indeed, the type 2 topoisomerase of Drosophila binds preferentially at crossovers (38), and in the absence of ATP the T4 topoisomerase efficiently brings together two or more segments of DNA (37). Furthermore, cross-linking promotes knotting, as demonstratedby the fact that a bidentate DNA binding protein that introduces only a single cross-link dramatically increases knotting by catalytic amounts of T4 topoisomerase in the presence of ATP (39). In terms of polymer statistics, crosslinking by excess topoisomerase can be considered as introducing a strong local attractive potential. However, under our conditions, excess T4 topoisomerase is insufficient by itself to bring about knotting in the absence of supercoiling; we do not observe knotting with relaxed DNA even at a stoichiometry of 22 to 1. Excess amounts of a Drosophila type 2 topoisomerase can knot nicked DNA in the presence of ATP (9); we predict that such knots would be random in form. Preliminary calculations based on Monte Carlo simulations with a 3.5-kilobase pair DNA molecule indicate that supercoiling should increase the frequency of knotting of naked DNA roughly 7-f01d.~ The knots produced by simulation were all of the (-) twist form, exactly as found experimentally. Nevertheless, the level of knotting seen in these simulations is still an order of magnitude below that observed in uitro. Thus, supercoiling alone cannot explain the high degree of knotting. Moreover, the requirements for a high amount of topoisomerase and theabsence of ATP argue persuasively for some stoichiometric role of the protein. Just as persuasively, the (-) twist topology of the product knots clearly indicates
S. Levene and N. R. Cozzarelli, unpublished results.

T4 Topoisomerase Knots
28. Spengler, S. J., Stasiak, A., and Cozzarelli, N. R. (1985) Cell 4 2 , 325-334 29. Kanaar, R., Klippel, A., Shekhtman, E., Dungan, J. M., Kahmann, R., and Cozzarelli, N.R. (1990) Cell 6 2 , 353-366 30. Boles, T.C., White, J. H., and Cozzarelli, N. R. (1990) J. Mol. Biol. 213,931-951 31. Klenin, K. V., Vologodskii, A. V., Anshelevich, V. V., Dykhne, A. M., and Frank-Kamenetskii, M.D. (1991) J. Mol. Bwl. 2 1 7 , 413-419 32. Adrian, M., ten Heggeler-Bordier, B., Wahli, W., Stasiak, A. Z., Stasiak, A., and Dubochet, J. (1990) EMBO J. 9,4551-4554 33. Shishido, K., Ishii, S., and Komiyama, N. (1989) Nucleic Acids. Res. 17,9749-9759 34. Frank-Kamenetskii, M. D., Lukashin, A. V., and Vologodskii, A.

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V. (1975) Nature 2 5 8 , 398-402 35. Frank-Kamenetskii, M. D., and Vologodskii, A. V. (1981) Sou. Phys. USP. 24,679-696 36. Klenin, K. V., Vologodskii, A. V., Anshelevich, V. V.,Dykhne, A. M., and Frank-Kamenetskii, M. D. (1988) J. Biomol. Struct. Dynarn. 5,1173-1185 37. Kreuzer, K. N., and Huang, W. M. (1983) in Bacteriophage T4 (Matthews, C. K., Kutther, E. M., Mosig, G., and Berget, P. B., eds) American Society for Microbiology, Washington, D. C. 38. Zechiedrich, E. L., and Osheroff, N. (1990) EMBO J. 9, 45554562 39. Mukherjee, S., Erickson, H., and Bastia, D. (1988) Proc. Natl. Acad. Sci. U. A . 86,6287-6291 S. 40. White, J. H. (1991) J. Biol. C h r n . 2 6 6 , 20574-20575